An in vitro evaluation of the antibacterial and anticancer properties of the antimicrobial peptide nisin Z A Lewies orcid.org/ 0000-0002-5624-1386 Thesis submitted for the degree Doctor of Philosophy in Pharmaceutics at the North-West University Promoter: Prof LH du Plessis Graduation May 2018 Student number: 20577966 “We must not forget that when radium was discovered no one knew that it would prove useful in hospitals. The work was one of pure science. And this is a proof that scientific work must not be considered from the point of view of the direct usefulness of it. It must be done for itself, for the beauty of science, and then there is always the chance that a scientific discovery may become like the radium a benefit for mankind. ” ― Marie Curie― Acknowledgements I am grateful to many individuals who gave their time, expertise, support and assistance in making this study possible. To each and every one who contributed in one way or another, my heartfelt appreciation. I regret that I am not able to thank everyone in this space. I wish to thank the following individuals and institutions who played a significant role in the completion of this thesis;  My promoter, Prof. Lissinda H. Du Plessis, for her valuable assistance, encouragement and the tremendous amount of trust in giving me the freedom to follow my own path.  Dr. Johannes F. Wentzel, for his very valuable assistance, input and encouragement throughout this study.  My co-authors, Prof. Carlos Bezuidenhout, Dr. Anine Jordaan and Ms. Haley C. Miller, for their valuable input  For personal finances I thank the National Research Foundation (NRF) of South Africa and the North-West University Potchefstroom Campus  I would also like to express my heartfelt appreciation to the Biochemistry Department of the North-West University Potchefstroom Campus for allowing me to use some of their facilities in the completion of this study.  Thanks to Handary (Brussels, Belgium), for the kind donation of the Ultra-pure nisin Z used in this study. I am also indebted to my support system, the people who were always there, the people who I met along the way and the combination of who will form part of my journey forward. Thanks to my family, and especially my parents, my sisters and brother, for all of their support, unconditional love and the sacrifices they have made in order to give me the opportunity to excel in life. I would also like to thank my grandmother (the wisest woman I know), for her unconditional love, support and great deal of interest towards not just this study but every aspect of my life. To my scientific family, Jaco and Annemarie Wentzel, Abel Bronkhorst, Vida Ungerer, Chris Badenhorst, Jean Du Toit, Lizelle Zandberg and Rozanne Harmse, I love you guys! Thank you for putting up with me through the hard times and celebrating the good times with me. A special thanks to Jaco Wentzel, for the enormous amounts of patience, believe, encouragement (and coffee) and support. Finally, my Heavenly Father who blessed me with wonderful opportunities and the strength and perseverance to endure the hard times. Preface The present thesis is theoretical and empirical investigation on the in vitro antibacterial and anticancer activities of the antimicrobial peptide nisin Z. This study is guided by the conviction that studies focusing on further elucidating the safety profile and multi-functionality as well as molecular mechanisms of this antimicrobial peptide, may aid in its adaption from food preservative to potent and effective therapeutic agent for human use. This thesis is compiled in article-format according to the guidelines set by the North-West University, and consists of three published articles, one submitted manuscript, a book chapter that has been accepted for publication (Appendix B) and one scientific poster (Appendix C). I acted as lead author in all of the papers, a detailed statement of contribution can be found at the end of each paper. Each article, chapter or manuscript was inserted in the thesis exactly as published or submitted and therefore complies with the requirements set by the different journals or publishers. Documentation regarding permission from journals to use published articles in this thesis is provided in Appendix H. Permission from authors to include all aforementioned articles in this thesis is provided in the following section. Additional articles in which I participated as co-author, which share points of contact with this study but do not form part of the thesis, are presented in Appendix E. The content and structure of this thesis is summarized in Chapter 1 (section 1.4). Author contribution and permission statements I, Angélique Lewies, am the main researcher responsible for the proposal, planning and execution of this study, along with (i) extensive review of the relevant literature, (ii) assessment, optimization and standardization of the bulk of the experimental protocol and methods, (iii) collection, analysis, interpretation and presentation of data, (iv) design, planning and writing of research articles, (v) presenter of conference related content, and (vi) writing of all sections of this thesis. Prof. Lissinda H. du Plessis Promoter responsible for guidance; intellectual input and evaluation of research outputs. Dr. Johannes F. Wentzel Colleague and co-author responsible for guidance, expert advice and technical assistance on bacterial related experiments, cytotoxicity- and flow cytometry assays. Preface Dr. Anine Jordaan Developed method and performed the transmission electron microscopy analysis of lipid nanoparticle formulations, results represented in paper III found in Chapter 3 of this thesis. Prof. Carlos Bezuidenhout Expert guidance with anti-bacterial experiments and critical review of paper III found Chapter 3 of this thesis Ms. Haley C. Miller (maiden Van Dyk) Assistance with the design, execution, data analysis and interpretation of the bioenergetics analyses performed on the Seahorse XFe96 Extracellular analyser, the results of which are presented paper IV found in Chapter 4 of this thesis. Statement by co-authors I hereby confirm that I approve the publication of the aforementioned manuscript(s), and that my role related to the completion of this thesis, An in vitro evaluation of the antibacterial and anticancer properties of the antimicrobial peptide nisin Z, is representative of my contribution. I also give my consent that the PhD student, Angélique Lewies, may include the manuscript(s) as part of her thesis. Table of contents Page List of figures i List of tables iii Abstract iv Keywords vi Opsomming vii Sleutel woorde ix Chapter 1: Introduction 1.1. Background and problem identification 1 1.2. Hypothesis 3 1.3. Aims and objectives 4 1.4. Structure of thesis 4 1.5. References 6 Chapter 2: Literature overview 2.1. Introduction 8 2.2. Antimicrobial peptides 8 2.3. The lantibiotic nisin 10 2.3.1. Nisin structure and mechanism of antibacterial activity 10 2.3.2. Bacterial spectra and pharmaceutical application of nisin 13 2.3.3. Limitations for the use of nisin as an antibacterial agent 14 2.4. Cancer treatment and antibacterial peptides 17 2.4.1. Nisin effect on cancer cells 17 2.4.2. Bioenergetics and reactive oxygen species generation in cancer cells as targets for novel anticancer therapies 18 2.5. References 20 2.6. Paper I:The potential use of natural and structural analogues of antimicrobial peptides in the fight against neglected tropical diseases 21 2.7. Paper II: Antimicrobial peptides the Achilles heel for antibiotic resistance? 64 Chapter 3: Interaction of the antimicrobial peptide nisin Z with conventional antibiotics and the use of nanostructured lipid carriers to enhance antibacterial activity (Paper III) 91 Graphical abstract and summary 91 Chapter 4: The antimicrobial peptide nisin Z induces selective toxicity and apoptotic cell death in cultured melanoma cells (paper IV) 103 Graphical abstract and summary 103 Chapter 5: The potential of nisin Z to increase the cytotoxicity and selectivity of conventional chemotherapeutic agents 5.1. Introduction 118 5.2. Materials and methods 120 5.2.1. Cell culturing conditions 120 5.2.2. MTT (3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide) assay 121 5.2.3. Evaluating the synergistic interactions between nisin Z and the chemotherapeutic agents 121 Table of contents 5.2.4. Data analysis 122 5.3. Results and discussion 122 5.4. Conclusion 131 5.5. References 132 Chapter 6: Summary, conclusion and future prospects 6.1. Exploring the multi-functionality of antimicrobial peptides as novel therapeutics 133 6.2. The antibacterial activity of nisin Z (Chapter 3, paper III) 134 6.3. The anticancer activities of nisin Z (Chapter 4, paper IV and chapter 5) 135 6.4. Important conclusions that were drawn from this study 137 6.5. Future prospects and recommendations 139 6.6. References 140 Reference list 141 Appendix A: Validation of the modified BCA protein assay for the quantification of nisin Z 153 Appendix B: Book chapter 167 Appendix C: Conference poster presentation 189 Appendix D: Certificate of analysis ultra-pure nisin Z 192 Appendix E: Additional publications 194 Appendix F: Proof of ethical training 208 Appendix G: Language editing certificate 210 Appendix H: Permission for use of copyright material 212 i List of figures Page Chapter 2: Literature overview Figure 2.1: Different binding positions of vancomycin and nisin to lipid II in bacterial cell walls 11 Figure 2.2: Structure of nisin Z 12 Figure 2.3: The mode of pore formation of nisin Z in cellular membranes 13 Figure 2.4: Comparision of lipid-based nanoparticles 16 Figure 2.5: Metabolism of non-malignant cells compared to that of cancer cells. 19 Figure 2.6: Role of reactive oxygen species (ROS) in normal homeostasis and pathophysiology 20 Chapter 3: Chapter 3: Interaction of the antimicrobial peptide nisin Z with conventional antibiotics and the use of nanostructured lipid carriers to enhance antibacterial activity (Paper III) Graphical abstract 91 Figure 3.1: Representative example of a modified broth micro-dilution plate of S. aureus treated with a 1:1 combination of novobiocin and nisin Z 97 Figure 3.2: MIC of nisin Z towards E.coli at increasing concentrations of EDTA 98 Figure 3.3: Toxicity assay for AMPs in HaCat cells 99 Figure 3.4: (A) TEM image of optimal SLN formulation indicating morphology, (B) TEM images of NLCs indicating (i) the size distribution and (ii) the morphology. (C) Release of nisin Z from NLCs compared to free nisin Z release at 37ºC and pH 7.4 (PBS) over a period of 24 h 100 Chapter 4: The antimicrobial peptide nisin Z induces selective toxicity and apoptotic cell death in cultured melanoma cells (paper IV) Graphical abstract 103 Figure 4.1: Cytotoxicity results for melanoma (A375) and non-malignant keratinocyte (HaCat) cells following nisin Z exposure 109 Figure 4.2: Representative flow cytometric dot-plots indicating the population sizes of apoptotic and necrotic (A and B) non-malignant keratinocyte (HaCat) and (C and D) melanoma (A375) cells after exposure to 50–200 μM of nisin Z for 24 h. 110 Figure 4.3: Intracellular ROS accumulation in DCFH-DA stained melanoma (A375) cells. 111 Figure 4.4: Mitochondrial stress respiratory flux profiles for melanoma (A375) cells exposed to nisin Z, as determined with the Seahorse Extracellular Flux Analyser and twelve consecutive measurements of the oxygen consumption rate (OCR). 112 Figure 4.5: Glycolysis stress test profiles for melanoma (A375) cells exposed to nisin Z, as determined with the Seahorse Extracellular Flux Analyser with twelve consecutive measurements of extracellular acidification rate (ECAR) 113 Figure 4.6: Mitochondrial membrane potential of melanoma cells (A375) exposed to different nisin Z concentrations. 114 Figure 4.7: A375 cell invasion and proliferation assays 115 Chapter 5: Chapter 5: The potential of nisin Z to increase the cytotoxicity and selectivity of conventional chemotherapeutic agents Figure 5.1: Toxicity of 5-fluoruracil (FU) and 5-FU + nisin Z 125 List of figures ii Figure 5.2: Toxicity of hydroxy urea and hydroxy urea + nisin Z 126 Figure 5.3: Toxicity of methotrexate and methotrexate + nisin Z 127 Figure 5.4: Toxicity of etoposide and etoposide + nisin Z 128 Figure 5.5: Toxicity of imatinib and imatinib + nisin Z 129 Figure 5.6: Toxicity results for mono-treatment and combinations of chemo- therapeutic agents (50 µM) and nisin Z (150 µM) as determined with the MTT assay 130 iii List of tables Page Chapter 3: Chapter 3: Interaction of the antimicrobial peptide nisin Z with conventional antibiotics and the use of nanostructured lipid carriers to enhance antibacterial activity (Paper III) Table 3.1: MIC values and ƩFIC values for antibiotic:antibacterial peptide combinations. 98 Table 3.2: Characterisation of solid lipid nanoparticles formulated with different amounts of Span 80 99 Table 3.3: Characterization of solid lipid nanoparticles and nanostructured lipid carrier formulations. 100 Table 3.4: MIC values for unloaded and nisin Z loaded NLC formulations 101 Chapter 5: Chapter 5: The potential of nisin Z to increase the cytotoxicity and selectivity of conventional chemotherapeutic agents Table 5.1: Mechanism of action of selected chemotherapeutic agents 120 Table 5.2: Toxicity results for cells exposed to 150 µM nisin Z. 122 Table 5.3: Effect of the exposure of melanoma (A375) cells to 150 µM nisin Z on reactive oxygen species generation, mitochondrial membrane potential and bioenergetics compared to unexposed cells 124 Table 5.4: Synergistic interactions between nisin Z and etoposide in melanoma cells 130 iv Abstract The rise in antibiotic resistance and the lack of the development of new antibiotics poses a considerable threat to human health. This is especially of concern in individuals who are immune-compromised (due to immunosuppressive diseases or chemotherapy). There is a desperate need for intervention aimed at strengthening our current arsenal of antibiotics or developing new antibiotics to prevent high death rates due to infections with antibiotic resistant superbugs. In spite of the higher susceptibility to bacterial infections in cancer patients undergoing chemotherapy, resistance to conventional chemotherapy agents also poses a threat to the successful treatment of cancer. Antimicrobial peptides (AMPs) are multifunctional and several peptides have both antibacterial and anticancer activities, as well as displaying immune-modulatory properties. Therefore, AMPs may be considered alternatives to antibiotics/chemotherapy agents or as adjuvants to conventional antibiotics/chemotherapy agents. In this thesis, the antibacterial as well as anticancer activities of the Generally Regarded as Safe (GRAS) status AMP, nisin Z, were evaluated. The antibacterial activity was assessed with regard to the interaction of nisin Z with conventional antibiotics on Staphylococcus aureus, S. epidermidis and Escherichia coli. Additionally, the use of biodegradable lipid nanoparticles has been shown to enhance the antibacterial activity of antibiotics and AMPs. Therefore, the effectiveness of nanostructured lipid carriers (NLCs) for the entrapment of nisin Z was also assessed. The anticancer activity of nisin Z was evaluated against cultured melanoma cells. Reprogramming of cellular metabolism is now considered one of the hallmarks of cancer. Most malignant cells present with altered energy metabolism which is associated with elevated reactive oxygen species (ROS) generation. This is also evident for melanoma, the leading cause of skin cancer related deaths. Altered mechanisms affecting mitochondrial bioenergetics pose attractive targets for novel anticancer therapies. In this study, the anti-melanoma potential of nisin Z was evaluated in vitro. The underlying anticancer mechanism of nisin Z with regard to the ability of this AMP to induce ROS production, apoptosis, disrupt the energy metabolism (glycolysis and mitochondrial respiration) and inhibit cell proliferation and invasion of melanoma cells was investigated. Likewise, the ability of nisin Z to enhance the cytotoxicity and selectivity of conventional chemotherapeutic agents was also investigated. Finally, synergistic interactions between nisin Z and conventional chemotherapeutic agents were examined. Abstract v Results indicated that nisin Z exhibited additive interactions with numerous conventional antibiotics. Notable synergism was observed for novobiocin-nisin Z combinations. The addition of the non-antibiotics adjuvant ethylenediaminetetraacetic acid (EDTA) significantly improved the antimicrobial activity of free nisin Z towards E.coli. NLCs containing nisin Z were effective against Gram positive species at physiological pH, with an increase in effectiveness in the presence of EDTA. Results indicate that nisin Z may be advantageous as an adjuvant in antimicrobial chemotherapy, while contributing in the battle against antibiotic resistance. NLCs have the potential to enhance the antibacterial activity of nisin Z towards Gram-positive bacterial species associated with skin infections. The minimum inhibitory concentrations (MICs) and half maximal inhibitory concentrations (IC50) were used as a measure of the toxicity and selectivity of nisin Z to bacterial and mammalian cells, respectively. Based on the results from this study, nisin Z displays selective toxicity to bacterial and cancer cells, compared to non-malignant cells. Furthermore, nisin Z was shown to negatively affect the energy metabolism (glycolysis and mitochondrial respiration) of melanoma cells, increase ROS production and cause apoptosis. Results also indicate that nisin Z can decrease the invasion and proliferation of melanoma cells demonstrating its potential use against metastasis associated with melanoma. In the current study it was found that combinations of nisin Z with 5-fluoruracil, hydroxy urea and etoposide were able to enhance the cytotoxicity of these conventional chemotherapeutic agents to melanoma cells. The etoposide-nisin Z combination also displayed a synergistic interaction. In conclusion nisin Z with its GRAS status, in addition to displaying direct antibacterial and anticancer properties, shows great potential to be used as an adjuvant with conventional antibiotics and chemotherapy agents. vi Keywords Adjuvant therapy; Antibiotic resistance; Antimicrobial peptides; Apoptosis; Bioenergetics, Melanoma; Nisin Z; Nanostructured lipid carriers; Reactive oxygen species (ROS); Synergism. vii Opsomming Die toename in antibiotiese weerstandbiedendheid en ʼn tekort aan die ontwikkeling van nuwe antibiotikums hou ʼn ernstige bedreiging in vir menslike gesondheid. Dit is veral kommerwekkend in individue met afgetakelde immuniteite as gevolg van siektetoestande wat die immuun stelsel onderdruk of chemoterapeutiese behandelinge. Daar is ʼn desperate behoefte vir daadwerklike aksie met die doel om die huidige arsenaal van antibiotikums te versterk om sodoende toekomstige mediese krisisse af te weer. Afgesien van die geneigdheid van kankerpasiënte wat chemoterapie ondergaan om bakteriese infeksies op te doen, is kanker weerstand teen konvensionele chemoterapeutiese middels ook ʼn ernstige bedreiging vir die suksesvolle behandeling van kanker. Verskeie antimikrobiese peptiede (AMPs) besit nie net antibakteriële- en antikanker-aktiwiteite nie, maar ook immuun- modulerende eienskappe. AMPs kan as moontlike alternatiewe oorweeg word vir konvensionele antibiotikums en chemoterapeutiese middels. In hierdie tesis is die antibakteriële- en antikanker-aktiwiteit geëvalueer van die algemeen as veilig geagte (GRAS) AMP, nisien Z. Die antibakteriële-aktiwiteit van nisien Z is teen konvensionele antibiotikums getoets op drie verskillende bakteriese variante, Staphylococcus aureus, S. epidermidis en Escherichia coli. Bykomend is die gebruik van biodegradeerbare lipied-nanopartikels om die aktiwiteit van antimikrobiese middels te verbeter ook ondersoek. Die effektiwiteit van nano-gestruktureerde lipieddraers (NLCs) om nisien Z op te neem is ook geëvalueer. Bykomend is die antikanker-aktiwiteit van nisien Z teen melanoma selle getoets. Herprogramering van sellulêre metabolisme word tans oorweeg as een van die kenmerke van kanker; en meeste kankeragtige selle het aangepaste energie-metabolismes wat gewoonlik geassosieer word met verhoogte reaktiewe suurstof spesie (ROS) generasie. Verhoogte ROS produksie word ook waargeneem in melanoma, die hoof oorsaak van velkanker-verwante sterftes. Veranderinge in die mitochondriale bioenergetiese meganismes van kankerselle is aantreklike teikens vir antikanker-terapieë. Die anti-melanoma potensiaal van nisien Z is geëvalueer in hierdie studie. Die onderliggende antikanker meganismes van nisien Z wat aanleiding gee tot ROS produksie, apoptosis, die onderbreking van die sekulêre energie metabolisme (glukolise en mitochondriale respirasie) en die inhibering van sel-verdeling en metastasis van melanoma selle is ook ondersoek. Bykomend is nisien Z se vermoë om sitotoksisiteit en selektiwiteit van konvensionele chemoterapeutiese middels te verbeter geëvalueer. . Opsomming viii Resultate het aangedui dat nisien Z bykomende interaksies het met verskeie konvensionele antibiotikums. Veral van belang was die waargenome sinergisme van die novobiocin-nisien Z kombinasies. Die byvoeging van die nie-antibiotiese bevorderingsmiddel, etielenedianientetraasetiese suur (EDTA) het die antimikrobiese aktiwiteit van vrye nisien Z teenoor E. coli beduidend verbeter. NLCs wat nisien Z bevat is effektief teen Gram positiewe spesies by fisiologiese pH met ʼn toename in effektiwiteit in die teenwoordigheid van EDTA. Die resultate dui daarop dat nisien Z die potensiaal het om as bevordingsmiddel op te tree in antimikrobiese chemoterapie en moontlik die verligting kan bring in die stryd teen antibiotiese weerstandbiedendheid. Daar is bewys dat NLC die antimikrobiese aktiwiteit verbeter teenoor Gram-positiewe bakteriële spesies wat geassosieer is met vel infeksies. Die minimum inhiberende konsentrasies (MIC) en die half maksimale inhibisie konsentrasies (IK50) was gebruik as ʼn aanduiding van die toksisiteit en selektiwiteit vir bakteriese en soogdier selle. Nisien Z is selektief toksies teenoor bakteriële- en kankerselle, in vergelyking met nie-kankerselle. Dit is ook aangetoon dat hierdie AMP die energie meganismes (glukolise en mitochondriale respirasie) van melanoma selle negatief affekteer, ROS generasie bevorder en lei tot apoptosis. Resultate het aangedui dat nisien Z die selverdeling van melanoma selle inperk, wat die AMP se moontlike potensiale aanwending teen metastatiese melanoma demonstreer. Addisioneel, is dit gewys dat kombinasies van nisien Z met 5-fluorurasil, hidroksie-urea en etoposied in staat is om die sitotoksisiteit van hierdie chemoterapeutiese middels teenoor melanoma selle te verhoog. Die etoposied-nisien Z kombinasies het ook sinergistiese interaksies getoon. In gevolgtrekking; die algemeen as veilig geagte AMP, nisien Z, besit goeie antibakteriese- en antikanker-eienskappe; en besit bykomend goeie potensiaal vir die toepassing as bevorderingsmiddel met konvensionele antibiotikums en chemoterapeutiese middels. ix Sleutel woorde Kombinasieterapie, Antibiotikum weerstand, Antimikrobiese peptiede, Apoptose, Bioenergetika, Melanoom, Nisien Z, Nano-gestruktureerde lipieddraers, Reaktiewe suurstof spesies, Sinergisme 1 Chapter 1: Introduction 1.1. Background and problem identification The discovery and subsequent development of antibiotics can be considered one of the major breakthroughs in modern medicine. One of the most important clinical outcomes of antibiotic use includes decreased mortality rates caused by common bacterial infections. Furthermore, improved surgical approaches are obtained as antibiotics can be given prophylactically preoperatively to reduce incidences of surgical site infections or to treat infections that arise as a consequence of surgery (Webb et al., 2006, Cartmill et al., 2009, Kawakita and Landy, 2017). In cancer patients undergoing chemotherapy, bacterial infections are also one of the major complications that arise as a consequence of the weakened immune system (Gudiol and Carratala, 2014). Antibiotics are therefore of cardinal importance during cancer treatment, as the use of antibiotics in combination with cancer treatment strategies can contribute to the survival of patients by enabling the use of more aggressive therapies. Therefore, both the rise in antibiotic resistance and the lack of development of new antibiotics poses a considerable threat to human health. Alarmingly, it is estimated that if no intervention is made with regard to strengthening our current arsenal of antibiotics, or with developing new antibiotics; antibiotic resistant superbugs might kill one person every three seconds by 2050 (O’Neill, 2016). Antimicrobial peptides (AMPs) are produced by all known living species and are considered natural antibiotics which play an important role in the innate immunity (Hancock and Diamond, 2000). An emerging trend in AMP research is that the multifunctional nature of AMPs is being studied. Due to their direct killing action of both Gram-positive and -negative bacteria and their role in modulating the host immunity, they are ideal candidates to be developed as antimicrobial agents to be used alone or in combination with current antibiotics for treating bacterial infections (Wright, 2016). Also, as opposed to current antibiotics, AMPs are multifunctional, and have been shown to display anticancer activities (Schweizer, 2009). Hence, AMPs can furthermore be considered for treatments in combination with current chemotherapy treatments to not only address the issues relating to bacterial infections, but to also possibly increase the effectiveness of conventional chemotherapeutic agents. Chemotherapy resistance also poses a threat to the effective treatment of cancer (Luqmani, 2005); therefore adjuvants that are able to produce synergistic interactions with conventional chemotherapeutic agents without increasing the toxicity to non-malignant cells, should be investigated. Chapter 1: Introduction 2 AMPs are therefore ideal candidates to be developed as agents for the treatment of bacterial infections and also cancer. However, although studies that focus on the toxicity of AMPs are gaining interest, only a few have been considered for use due to toxicity issues that may arise as a result of their use (Marr et al., 2006a). Nisin, a 3.5 kDa AMP produced by the non- pathogenic bacteria Lactococcus lactis, has Generally Regarded as Safe (GRAS) status and is approved for human consumption (Müller-Auffermann et al., 2015a). Nisin has been approved by both the Federal Drug Administration (FDA) and World Health Organisation (WHO) for use as a food preservative and is currently being used in more than 48 countries for this purpose (Cotter et al., 2005, Jones et al., 2005). Of great importance is the fact that despite being used for almost 50 years little incidence of stable or transmissible resistance has been reported for nisin (Gravesen et al., 2002, Willey and Van der Donk, 2007, Fernandez et al., 2008). The GRAS status and also general lack of resistance to nisin makes it an ideal candidate to be developed as an antimicrobial agent for human use. Nisin has activity against Gram-positive bacteria. However, it lacks activity against most Gram- negative bacteria, primarily due to the fact that access to the cytoplasmic membrane is blocked by the outer membrane of these bacteria. To overcome this, chelating agents can be used together with nisin to chelate divalent cations and destabilize the outer membrane, ultimately leading to the permeabilization of the outer bacterial membrane. If the integrity of the outer membrane is compromised, nisin can move unchallenged to the inner membrane of Gram-negative bacteria and exert its antimicrobial action (Natrajan and Sheldon, 2000). An abundance of studies have focused on the adjuvant potential (producing synergistic interactions) of nisin in combination with conventional antibiotics for the treatment of Gram- positive infections (Giacometti et al., 2000, Dosler and Gerceker, 2012, Mataraci and Dosler, 2012), and more recently Gram-negative bacteria (Naghmouchi et al., 2012, Naghmouchi et al., 2013, Rishi et al., 2014). However, most of these studies focus on using the nisin A (low content containing 2.5 % nisin) variant. Although nisin A and nisin Z display similar antimicrobial activity, nisin Z has a higher rate of diffusion in agar studies and enhanced activity at neutral pH (de Vos et al., 1993). The low solubility and low stability of nisin at physiological pH makes clinical application thereof difficult. Previously the use of biodegradable solid lipid nanoparticles (SLNs) has been shown to enhance the activity of nisin at pH 7.4 (Prombutara et al., 2012). Nanostructured lipid carriers (NLCs) are second- generation SLNs that are considered to be more ideal for the entrapment of peptides (Martins et al., 2007). The use of NLCs to enhance the antimicrobial efficacy of nisin has, however, not yet been investigated. AMPs, and especially bacteriocins, hold great potential for being used as anticancer agents due to their selectivity towards cancer cells (Kaur and Kaur, 2015). Cancer cells present with Chapter 1: Introduction 3 altered energy metabolism (DeBerardinis and Chandel, 2016) and high levels of reactive oxygen species (ROS) generation (Trachootham et al., 2009). Both the altered energy metabolism and elevated ROS present targets for novel anticancer therapies which are selectively toxic to malignant cells. Nisin has been shown to display anticancer activities through the induction of apoptosis and inhibition of cell proliferation (Joo et al., 2012, Kamarajan et al., 2015). Some AMPs have been shown to induce ROS production that triggers apoptosis in Candida albicans and also in cancer cells (Cruz-Chamorro et al., 2006, Park and Lee, 2010, Hwang et al., 2011, Sharma and Srivastava, 2014). However, this mechanism has not yet been proven for nisin in cancer cells. Although it has been shown that nisin affects the expression of genes involved in energy and nutrient pathways in head and neck squamous cell carcinoma (HNSCC) (Joo et al., 2012), its effect on the energy metabolism of cancer cells has not yet been investigated. As mentioned earlier, AMPs which also display anticancer activity could also be used in combination with conventional chemotherapeutic agents. These combinations may lead to the enhanced effectiveness of these agents, prevent recurrence of cancer following treatment and possibly reduce instances of chemotherapy resistance (Gaspar et al., 2013, Swithenbank and Morgan, 2017). Although an abundance of studies has focused on the use of nisin as an adjuvant for antibiotics, studies focusing on its use as an adjuvant in combination with conventional chemotherapy agents are lacking. 1.2. Hypotheses The following hypotheses were investigated in this study: i. Nisin Z can be used as an adjuvant* with conventional antibiotics against Gram- positive and negative bacteria. The antimicrobial activity of nisin Z towards Gram- negative bacteria can be enhanced by using the chelating agent ethylenediaminetetraacetic acid (EDTA) and also through entrapment in NLCs. ii. Nisin Z holds the potential of not only inducing apoptosis and of preventing cell proliferation in cancer cells, but also of affecting the bioenergetics (glycolysis and mitochondrial respiration) and leading to an increase in ROS production which is associated with apoptosis. iii. Nisin Z holds the potential to be used as adjuvant* with conventional chemotherapy agents. iv. Nisin Z is a multi-functional peptide which does not display toxicity to non-malignant (“healthy”) cells, which can be considered an antibacterial peptide due to its activity Chapter 1: Introduction 4 against Gram-positive bacteria and as an anticancer peptide due to its activity towards cancer cells. * To produce synergistic interactions 1.3. Aim and objectives The aim of this study was to investigate the in vitro antibacterial and anticancer properties of the AMP nisin Z (ultra-pure containing 95 % (w/w) nisin). The objectives of this study were: i. To evaluate the potential of nisin Z to be used as an adjuvant for conventional antibiotics in Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and Gram-negative (Escherichia coli) bacteria (Chapter 3). ii. To evaluate the potential of NLCs to enhance the entrapment efficiency of nisin Z compared to that of SLNs (Chapter 3). iii. To evaluate the potential of EDTA and NLCs to enhance the antibacterial efficacy of nisin Z toward both Gram-positive and negative bacterial species (Chapter 3). iv. To evaluate the selectivity of nisin Z to melanoma (A375) cells- and bacterial cells compared to non-malignant human keratinocyte (HaCat) cells. This was done by evaluating the minimum inhibitory concentration (MIC) of nisin Z for Gram-positive and -negative bacteria and the half maximal inhibitory concentration (IC50) values for A375 and HaCat cells, respectively (Chapters 3 and 4). v. To evaluate the mechanism associated with the anticancer properties of nisin Z in melanoma regarding its effect on the mode of cell death (apoptosis vs necrosis), bioenergetics, ROS production, cell proliferation and its potential to prevent metastasis (Chapter 4). vi. To evaluate the potential of nisin Z to enhance the cytotoxicity and selectivity of conventional chemotherapeutic agents, and to produce synergistic interactions with conventional chemotherapeutic agents (Chapter 5). 1.4. Structure of thesis This thesis compromises of six chapters and appendices which (excluding the current chapter) are summarised as follows: Chapter 1: Introduction 5 Chapter 2: Literature overview This chapter consists of an in-depth review of the relevant literature on nisin and other aspects relevant to this study. Two review articles are also presented in the said chapter to (i) highlight the multi-functionality of AMPs and (ii) highlight the potential use of AMPs to address the issue relating to antibiotic resistance.  Paper I: Lewies, A., Wentzel, J.F., Jacobs, G. and Du Plessis, L.H. 2015. The potential use of natural and structural analogues of antimicrobial peptides in the fight against neglected tropical diseases. Molecules. 20(8):15392-433  Paper II: Lewies, A., Du Plessis, L.H and Wentzel, J.F. 2018. Antimicrobial peptides: the Achilles heel to antibiotic resistance? (Manuscript submitted to European Journal of Pharmaceutical Sciences) Chapter 3: Interactions of the antimicrobial peptide nisin Z with conventional antibiotics and the use of nanostructured lipid carriers to enhance antimicrobial activity This chapter consists of a paper describing the interactions of nisin Z with conventional antibiotics as well as the use of NLCs and EDTA to enhance the antimicrobial activity of nisin Z to Gram-positive and negative bacteria.  Paper III: Lewies, A., Wentzel, J.F., Jordaan, A., Bezuidenhout, C. and Du Plessis, L.H. 2017. Interactions of the antimicrobial peptide nisin Z with conventional antibiotics and the use of nanostructured lipid carriers to enhance antimicrobial activity. International Journal of Pharmaceutics. 526:244-53 Chapter 4: The antimicrobial peptide nisin Z induces selective toxicity and apoptotic cell death in cultured melanoma cells This chapter consists of a paper describing the selective cytotoxicity nisin Z to cultured cancer (melanoma) cells and investigates the effect of nisin Z on ROS production and apoptosis, the bioenergetics (glycolysis and mitochondrial respiration), and invasion and proliferation of melanoma cells. Chapter 1: Introduction 6  Paper IV: Lewies, A., Wentzel, J.F., Van Dyk, H.C. and Du Plessis, L.H. 2018. The antimicrobial peptide nisin Z induces selective toxicity and apoptotic cell death in cultured melanoma cells. Biochimie. 144:28-40 Chapter 5: The potential of nisin Z to increase the cytotoxicity and selectivity of conventional chemotherapeutic agents This chapter explains that nisin Z can be used in combination with conventional chemotherapy agents so as to increase the cytotoxicity and selectivity of these conventional chemotherapeutic agents. Possible synergistic interactions of nisin Z with conventional chemotherapeutic agents were also investigated. These results have not yet been published. However, some results from this chapter are presented in an invited book chapter submitted to InTechOpen Cytotoxicity ISBN 978-953-51-5869-1 (Appendix B).  Book Chapter: Lewies, A., Du Plessis, L.H. and Wentzel, J.F. 2017. The cytotoxic, antimicrobial and anticancer properties of the antimicrobial peptide nisin Z alone and in combination with conventional treatments. Book chapter accepted for publication** InTechOpen Cytotoxicity ISBN 978-953-51-5869-1 **Book to be published February 2018. Chapter 6: Summary, conclusion and future prospects This chapter describes the conclusions drawn from this study. Recommendations are also made for future studies. Appendices The validation of the analytical method for determining the entrapment of nisin Z in the lipid nanoparticles, invited book chapter, conference output (Poster presentation at the 7th European Molecular Biology Organization (EMBO) meeting held in Mannheim Germany September 2016), Certificate of Analysis for ultra-pure nisin Z, additional co-authored research articles and the certificate for language editing of this thesis, are included as appendices at the end of the thesis. 1.5. References The references used in this section are included in the final reference list at the end of this thesis. The Harvard reference style is used throughout the thesis in accordance with the Chapter 1: Introduction 7 guidelines of the NWU. However, the specific reference style as specified for each article’s guidelines to authors is used where applicable. 8 Chapter 2: Literature overview 2.1. Introduction The discovery and development of antibiotics have undoubtedly changed the face of modern medicine. However, a rise in antibiotic resistance poses a global threat to public health. Although bacteria have been adapting to their environments for millions of years and antibiotic resistance can be seen as a natural part of evolution, modern artificial pressures such as the misuse of these drugs in humans and livestock have been accelerating the rate of antibiotic resistance in bacteria (Rodriguez-Rojas et al., 2013). Due to the lack of the development of new antibiotics and the ever-increasing rise in antibiotic resistance, it is estimated that by 2050 antibiotic-resistant superbugs will lead to the death of millions of people annually and pose a greater threat to human health than cancer (O’Neill, 2016). This is not surprising when taking into account that normal procedures such as caesarean sections during birth may lead to the acquirement of multi-drug resistant bacterial infections. Also, in immunocompromised patients such as cancer patients, bacterial infections could lead to higher mortality rates (Gudiol and Carratala, 2014, O’Neill, 2016, WHO, 2016). Therefore, research focusing on the development of alternatives to current antibiotics is gaining interest. Antimicrobial peptides (AMPs) are promising candidates in this regard (Hancock and Sahl, 2006, Fox, 2013). Compared to current antibiotics which have a narrow spectrum of activity, AMPs have broad-spectrum antibacterial activity and also display antifungal-, antiviral-, anti-parasitic- (Jenssen et al., 2006) and selective anticancer- (Cruz- Chamorro et al., 2006) activities. Despite the effect of antibiotic resistance on cancer treatment, cancer cells also tend to rapidly develop chemotherapy resistance (Soengas and Lowe, 2003, Luqmani, 2005, Wellbrock, 2014). Furthermore, some conventional chemotherapeutic agents display non-specific toxicity toward non-tumorigenic cells. Due to the fact that AMPs have selective anticancer activities and immune-modulatory capabilities (Hancock and Diamond, 2000, Kaur and Kaur, 2015) the therapeutic potential of AMPs as alternatives/adjuvants to current chemotherapeutic drugs should also be investigated, especially with regard to the mode of action in cancer cells. 2.2. Antimicrobial peptides AMPs are produced by all known living species and are considered to be natural antibiotics. AMPs commonly consist of 12-100 peptide residues, are positively charged and amphipathic. Chapter 2: Literature overview 9 There is, however, little sequence homology among AMPs and they have a broad range of secondary structures. These include α-helix, β-sheet and coiled/extended structures (Jenssen et al., 2006). AMPs have three main mechanisms of action; (i) electrostatic or hydrophobic interactions with the negatively charged bacterial membranes leading to the permeabilization of these membranes; (ii) interaction with internal targets such as DNA, RNA and enzymes and (ii) the modulation of the innate immunity (Yeaman and Yount, 2003). The different classes of AMPs and mechanisms of actions are discussed in more detail in an invited review (Lewies et al., 2015) published in Molecules, as part of a topical collection focusing on Natural Products as Leads or Drugs against Neglected Tropical Diseases. This article can be found at the end of this literature overview (paper I). The aim of this review was to discuss the potential of selected AMPs (both naturally occurring and structural analogues of natural AMPs) to successfully treat a variety of neglected tropical diseases (NTDs). These NTDs include those caused by bacteria (leprosy/Hansen disease and trachoma), protozoa (Chagas disease, human African trypanosomiasis and leishmaniasis), helminths (taeniasis and onchocerciasis) and viruses (dengue viral disease and rabies). This review highlights the multi-functionality of AMPs. Also, as part of this study, a review article has been completed and submitted to European Journal of Pharmaceutical Sciences on the use of antimicrobial peptides (AMPs) in the fight against antibiotic resistance titled Antimicrobial peptide: the Achilles heel to antibiotic resistance?, which can be found at the end of this literature overview (paper II). This review concludes that AMPs are not only promising candidates as alternatives to current antibiotics due to their direct killing activity but can also act as adjuvants with conventional antibiotics to obtain synergistic interactions. Moreover, due to the immune-modulatory effects of AMPs, they can be employed to address issues relating to bacterial infections for which antibiotics have not proven to be successful, including septicemia and infections in individuals who are immune-compromised and therefore cannot provide immune support for antibiotic therapy. The potential of ribosomally synthesised AMPs above non-ribosomally synthesised AMPs and conventional antibiotics is furthermore highlighted. Ribosomally synthesised, gene-encoded AMPs are evolutionarily conserved parts of the innate immune system and are also referred to as host defence peptides (Hancock and Diamond, 2000). These AMPs are produced by plants, insects and animals. However, these AMPs are not limited to multicellular organisms. Bacteria utilize similar AMPs known as bacteriocins to obtain a competitive advantage over other micro-organisms in their habitat (Cotter et al., 2005). The focus of this study was on the bacteriocin nisin. Chapter 2: Literature overview 10 2.3. The lantibiotic nisin Bacteriocins are small, heat-stable, ribosomally synthesised AMPs produced by bacteria, and are promising candidates as an alternative to conventional antibiotics (Ahmad et al., 2017, Behrens et al., 2017). Lantibiotics are a subgroup of bacteriocins produced by Gram- positive bacteria, which are post-translationally modified and contain the unusual amino acids lanthionine, β-methyl lanthionine and dehydrated amino acids (Yang et al., 2014). Perhaps the best-known of these is the lantibiotic nisin, which was approved by the World Health Organisation (WHO) in 1969 and the US Federal Food and Drug Administration (FDA) in 1988 for use as a food preservative, and has promising potential for clinical application with its Generally Regarded As Safe (GRAS) status (Jones et al., 2005, Shin et al., 2016). 2.3.1. Nisin structure and mechanism of antibacterial action Nisin was identified in 1928, the same year as penicillin (Rogers and Whittier, 1928). Nisin is produced by the non-pathogenic bacteria Lactococcus lactis. It is a 3.5 kDa polycyclic peptide, which has 34 amino acid residues including the uncommon amino acid residues didehydroaminobutyric acid, didehydroalanine, lanthionine and methyllanthionine, (Mulders et al., 1991, Kleanhammer et al., 1993). The post-translationally introduced thioether bridges, which form the lanthionine rings, provide a degree of protection against proteolytic degradation (Bosma et al., 2011). Nisin A and nisin Z are two naturally occurring variants of nisin. These two variants are structurally similar, but differ by a single amino acid at position 27. Asparagine (Asn) is found in nisin Z, whereas this amino acid is replaced by a histidine (His) in nisin A (Mulders et al., 1991). Both of these variants have similar antimicrobial activity. However, at neutral pH nisin Z is more soluble and has a higher rate of diffusion than nisin A (De Vos et al., 1993). Commercial preparations of nisin A (containing 2.5 % pure nisin) are sold as Nisaplin or Chrisin and are used for various food applications (Martínez et al., 2016). A commercial preparation of nisin Z (Novasin) has also received GRAS status (FDA, 2001). Nisin exhibits a dual mode of action by binding to lipid II, an important cell-wall synthesis precursor, on the bacterial membrane of Gram-positive bacteria. The nisin-lipid II complex then leads to inhibition of cell wall biosynthesis and the formation of pores in the cell membrane (Pag and Sahl, 2002). The glycopeptide antibiotic vancomycin is considered one of the last line treatments against Gram-positive antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) (Liu et al., 2011, Tarai et al., 2013). Chapter 2: Literature overview 11 Vancomycin similarly binds to lipid II and inhibits cell wall biosynthesis. However, vancomycin binds to the D-Ala-D-Ala moiety of the lipid II pentapeptide (Figure 2.1) and bacterial strains that contain the vanA-type gene cluster are resistant to vancomycin by mutating the terminal D-Ala to D-Lactate in the lipid II pentapeptide. O O H2C HO OH OH HN CO CH3 O CHH3C C O D-Ala NH HC O CH3 O P O O- O P O -O O Nisin Vancomycin -D-Glu D-Lys D-Ala D-Ala Figure 2.1: Different binding positions of vancomycin and nisin to lipid II in bacterial cell walls. Vancomycin binds to the D-Ala-D-Ala moiety of the lipid II pentapeptide, whereas nisin binds to the pyrophosphate moiety of lipid II. Adapted from (Hsu et al., 2004) Clinical variants of MRSA containing the vanA-type gene cluster, which displays resistance to vancomycin, have been identified (Perichon and Courvalin, 2009). Nisin remains active against the vanA-type resistant strains due to the fact that it binds in a different way to lipid II (Hsu et al., 2004). Lanthionine, is composed of two alanine residues which are cross-linked on their carbon atom by a thioether linkage, the posttranslational addition of the thioether bridge forms the lanthionine rings (Horinouchi et al., 2010).The two lanthionine rings at the N-terminus of nisin form the lipid II binding motif as illustrated in Figure 2.2. This binding Chapter 2: Literature overview 12 motif binds to the pyrophosphate moiety of lipid II. The C-terminus then interacts with the membrane and nisin is inserted across the membrane leading to pore formation (Hsu et al., 2004, Paiva et al., 2011) (Figure 2.3). Figure 2.2: Structure of nisin Z. The lipid binding motif is formed by the two lanthionine rings at the N-terminus. The three lanthionine rings at the C-terminus are important for pore-formation associated with binding to lipid II. The residues in red have a positive net charge, those in blue are hydrophobic. Dha, dihydroalanine; Dhb, dihydrobutyrine; S, thioether bridge; Ala-S-Ala, lanthionine and Abu-S-Ala, methyllanthionine. Adapted from (Peschel and Sahl, 2006a, Paiva et al., 2011) Over and above the dual mode of action exerted by nisin through binding to lipid II, nisin also has three other mechanisms of action, namely: (1) inhibition of bacterial spore outgrowth; (2) pore formation that is independent of lipid II binding and (3) the activation of autolytic enzymes resulting in cell-wall degradation (Pag and Sahl, 2002) as shown in Figure 2.3. Antibacterial agents that have multiple modes of action are especially of interest as it is considered difficult for bacteria to develop resistance to all these mechanisms simultaneously. In the case of nisin, this has been proven to be true as there is very little evidence of stable and transmissible resistance occurring in food products treated with nisin, despite the fact that nisin has been used as a food preservative for almost 50 years (Gravesen et al., 2002, Willey and van der Donk, 2007, Fernandez et al., 2008). Chapter 2: Literature overview 13 Figure 2.3: The mode of pore formation of nisin Z in cellular membranes. (A) Lipid II independent pore formation, (B) Binding to lipid II through the N-terminus of nisin; the nisin-lipid II complex then leads to inhibition of cell wall biosynthesis and the formation of pores in the cell membrane. The positively charged C-terminus is then inserted into the negatively charged membrane to form the pore. Where; G, N-Acetylglucosamine (GlcNAc);M, N-Acetylmuramic acid (MurNAc), PPi, Pyrophosphate. Adapted from (Pag and Sahl, 2002) 2.3.2. Bacterial spectra and pharmaceutical applications of nisin Due to its ability to inhibit the growth of/kill Gram-positive bacteria including food-borne pathogens such as Staphylococcus aureus, Listeria monocytogenes and Clostridium botulinum, nisin is used as a preservative (European food additive list number E234) in over 48 countries to protect food from spoilage (Cotter et al., 2005, Jones et al., 2005). Nisin moreover has antibacterial activity against the clinically important pathogens such as vancomycin-resistant Enterococci (VRE), Streptococcus pneumonia and MRSA (Goldstein et al., 1998, Brumfitt et al., 2002, Dosler and Gerceker, 2011). Compared to vancomycin and metronidazole, nisin displayed enhanced bactericidal activity against clinical isolates of Chapter 2: Literature overview 14 Clostridium difficile (Bartoloni et al., 2004). Nisin is also active against the vegetative cells and spores of a variety of Clostridium and Bacillus strains (Gut et al., 2011, Le Lay et al., 2016). Several studies have demonstrated synergism between nisin and antibiotics against Gram-positive bacteria. Nisin displayed synergism with colistin and clarithromycin against Pseudomonas aeruginosa (Giacometti et al., 2000), with streptomycin, penicillin, rifampicin and lincomycin against P. fluorescens and antibiotic-resistant variants (Naghmouchi et al., 2012), as well as with daptomycin, teicoplanin and ciprofloxacin against MRSA biofilms (Mataraci and Dosler, 2012). In a study by Dosler and Gerceker, nisin-antibiotic combinations were shown to have synergistic interactions against clinical isolates of methicillin-susceptible S. aureus (MSSA), MRSA and Enterococcus faecalis. A major finding from their study was that a high incidence of synergistic interactions occurred with a nisin- ampicillin combination against MSSA and nisin-daptomycin combination against E. faecalis strains (Dosler and Gerceker, 2012). When nisin is combined with penicillin, chloramphenicol or ciprofloxacin it can significantly reduce the biofilm formation of E. faecalis (Tong et al., 2014). Mastitis-causing Staphylococcus strains tend to develop resistance toward antibiotics or develop biofilms (Gill et al., 2005, Melchior et al., 2006, Oliveira et al., 2006). Nisin has been successfully applied as a sanitizer against mastitis causing Staphylococcus and Streptococcus species in lactating cows even when these species are antibiotic resistant (Cao et al., 2007, Wu et al., 2007). Three nisin based products were developed for the treatment of bovine mastitis, namely Ambicin N® (Applied Microbiology, Inc., New York) and Mast Out® as well as Wipe Out® Dairy wipes (ImmuCel Corporation, Maine, USA) (Cotter et al., 2005, Pieterse and Todorov, 2010). In vivo nisin has also been shown to be an effective alternative to antibiotics in the treatment of staphylococcal mastitis during lactation in pregnant women (Fernandez et al., 2008). Although the bacterium strain which produces nisin (L. lactis ) is found in some Probiotic supplements (ProbioticsDB.com, 2017), there are currently no medically approved nisin products/treatments for humans. 2.3.3. Limitations for the use of nisin as an antibacterial agent Nisin holds the potential for use against multi-drug resistant Gram-positive bacteria, as an adjuvant with conventional antibiotics, has low levels of cytotoxicity and negligible levels of resistance under routine use. However, one of the major obstacles for the therapeutic use of nisin as an antibacterial agent is its narrow spectrum of antibacterial activity. Most Gram- negative bacteria are not susceptible to nisin. The main reason for this is due to the outer membrane of Gram-negative bacteria which limits the interaction of nisin with lipid II. The Chapter 2: Literature overview 15 activity of nisin is also pH dependent; at pH 2.5 (the pH at which nisin is the most stable) it has the ability to act against the Gram-negative bacterium Helicobacter pylori, which causes gastric ulcers (Lubelski et al., 2008). However, at physiological pH nisin has reduced solubility and reduced stability, which are considered additional major obstacles for the therapeutic application of nisin (Field et al., 2015). The activity of nisin towards Gram-negative bacteria can be enhanced by the addition of chelating agents such as EDTA or citrate (Long and Phillips, 2003, Prudêncio et al., 2015). The addition of these chelating agents leads to the destabilization of the outer membrane of Gram-negative bacteria, ultimately leading to the permeabilization of the outer membrane. Due to the fact that the integrity of the outer membrane is compromised, nisin can then move freely to the inner membrane and exert its antibacterial activity. The use of Tween®80 together with nisin can also enhance its activity towards Gram-negative bacteria, as was found when incorporating nisin, EDTA and Tween®80 into polymer films for the treatment of Salmonella typhimurium (Natrajan and Sheldon, 2000). Other strategies that can be used to enhance the antibacterial activities and stability of antibiotics and antimicrobial peptides are the use of nanoparticles. The use of metal nanoparticles to enhance the activity of antimicrobial agents is popular (Shimanovich and Gedanken, 2016). However, the focus has shifted from the more toxic, inorganic nanoparticle to using biodegradable nanoparticles. Lipid nanoparticles are attractive candidates in this regard. Niosomes (non-ionic surfactant-based nano lipid vesicles) into which nisin and EDTA were simultaneously incorporated have been evaluated for their ability to enhance the activity of nisin towards Gram-positive bacteria (S. aureus) and Gram- negative bacteria (E. coli) (Kopermsub et al., 2011). Simultaneous incorporation of nisin and EDTA into niosomes increased the inhibitory effect of the niosome formulations on S. aureus but not against E.coli. In a study by Prombutara and co-workers the encapsulation of nisin into solid lipid nanoparticles (SLNs) was able to extend the antimicrobial activity of nisin against Listeria monocytogenes and Lactobacillus plantarum at pH 7.4 (Prombutara et al., 2012). SLNs were introduced in the 1990s as an alternative to conventional colloidal delivery systems such as nano-emulsions (Lucks and Muller, 1993). SLNs have submicron sizes of 50 - 1000 nm (Martins et al., 2007) and resemble nano-emulsions in which the inner liquid lipids are replaced by lipids that are solid at room- and body temperature; and stabilised with an emulsifying layer in an aqueous dispersion (Figure 2.4.). Compared to other conventional colloidal delivery systems such as nano-emulsions, SLNs display long-term chemical and Chapter 2: Literature overview 16 physical stability on storage (Mehnert and Mader, 2001, Muller et al., 2002). However, due to the hydrophobic nature of the lipid matrix, SLNs are more suited for the encapsulation of lipophilic compounds although the use of SLNs for the encapsulation of hydrophilic compounds has been reported, by using a double-emulsion (water-oil-water) formulation technique (Gallarate et al., 2009, Zhen et al., 2010). Another disadvantage of SLNs includes low drug loading capacities due to the formation of a perfect lipid crystal matrix (Wissing et al., 2004). Fig 2.4: Comparison of lipid-based nanoparticles. (A) Nano-emulsion, liquid lipid core enclosed by lipid monolayers; (B) Solid lipid nanoparticle, solid lipid core enclosed by lipid monolayer and (C) Nanostructured lipid carrier, a mixture of solid and liquid lipid enclosed by a lipid monolayer. To overcome the disadvantages associated with SLNs, nanostructured lipid carriers (NLCs) were developed. NLCs consist of a mixture of solid and liquid lipids and therefore have a higher drug-loading capacity and stability (Müller et al., 2016) (Figure 2.4). NLCs also offer a novel approach for the formulation of peptides and proteins with poor aqueous solubility (Martins et al., 2007). It should be noted that a number of AMPs have been considered for clinical development and that most AMPs are considered for topical application (Fox, 2013). NLCs are optimal for dermal application (Müller et al., 2016). The effectiveness of NLCs for use with nisin has not yet been investigated. However, the AMP LL37 has been successfully incorporated into NLCs and displayed antimicrobial activity towards E. coli, showing promise for enhanced wound healing when applied topically (Garcia-Orue et al., 2016). Finally, adjuvant therapy with conventional antibiotics can be considered to enhance the activity of both nisin and the combined antibiotic to Gram-negative bacteria. Nisin:β-lactam antibiotic combinations were evaluated for the treatment of clinical isolates of Salmonella enterica serovar Typhimurium, and synergism was observed for nisin-ampicillin, nisin- cefotaxime and nisin-ceftriaxone combinations (Rishi et al., 2014). Chapter 2: Literature overview 17 2.4. Cancer treatment and antimicrobial peptides Cancer patients receiving treatment such as radiation, chemotherapy, surgery or transplantation of bone marrow/blood stem cells are at risk of bacterial infections due to lowered immunity. Therefore, bacterial infections are one of the most frequent complications in cancer patients (Wisplinghoff et al., 2003). The use of antibiotics in combination with cancer treatment strategies may contribute to the survival of these patients by enabling the use of more aggressive therapies. The development of antibiotic resistance also poses a great threat to cancer patients and could lead to a higher mortality rate due to infections caused by multi-drug resistant bacteria (Gudiol and Carratala, 2014). Due to the immune- modulatory effect of AMPs, they may be considered adjuvants to antibiotics for the treatment of bacterial infections in immunocompromised patients such as those who suffer from cancer (Hancock, 2015, Wright, 2016). Despite the effect of antibiotic resistance on cancer treatment, the toxicity associated with some conventional chemotherapeutic agents as well as the development of chemotherapy resistance, also call for the development of novel anticancer therapies. AMPs, and especially bacteriocins, display selectivity to cancer cells (Kaur and Kaur, 2015). These AMPs are therefore ideal potential candidates as alternatives to current chemotherapeutic agents or to be used as adjuvants with conventional chemotherapeutic agents to lower the therapeutic doses needed. 2.4.1. The effect of nisin on cancer cells Two studies have previously investigated the anti-tumour potential of nisin in vitro and in vivo for head and neck squamous cell carcinoma (HNSCC) (Joo et al., 2012, Kamarajan et al., 2015). The study by Joo and co-workers indicated that low content nisin A was able to selectively induce apoptosis and cell cycle arrest, and reduce cell proliferation in HNSCC cells, compared to primary keratinocytes in vitro. In vivo nisin treatment reduced the overall tumour burden compared to non-nisin-treated groups, in a floor-of-mouth oral cancer xenograft mouse model. Also, to examine the mechanism by which nisin facilitates its anti- proliferative and pro-apoptotic effects on HNSCC cells, the effect of nisin-treatment on the expression of 39 000 genes were examined by using Affymetrix gene arrays. The expression of multiple genes was altered, including those in the cell cycle and apoptotic pathways, energy and nutrient pathways, membrane physiology, signal transduction and protein binding pathways and ion transport. The CHAC1 gene, an apoptosis mediator and cation transport regulator, was the most highly up-regulated gene. This study was the first to indicate that the antibacterial food preservative nisin could effectively reduce and prevent Chapter 2: Literature overview 18 tumorigenesis both in vitro and in vivo (Joo et al., 2012). More recently a study by Kamarajan and co-workers indicated that nisin Z has great potential as an alternative cancer therapy. Nisin Z was able to selectively induce apoptosis through a calpain-dependent pathway in HNSCC cells, while also decreasing clonogenic capacity, orasphere formation and cell proliferation. In vivo, HNSCC tumorigenesis in mice was reduced following nisin Z treatment, and survival was extended following long-term treatment with nisin Z. Also, mice treated with nisin Z displayed normal organ histology with no evidence of fibrosis, necrosis or inflammation (Kamarajan et al., 2015). The ability of nisin to increase the activity of the chemotherapeutic drug, doxorubicin, was also investigated in vivo by Preet and co-workers. The combination of nisin and doxorubicin was able to cause a greater reduction in the tumour volumes in dimethylbenz (a) anthracene induced skin carcinogenesis in mice, as opposed to nisin and doxorubicin alone. An in situ apoptotic assay performed on skin tissue/tumours indicated a significant increase in apoptosis in groups treated with both nisin and doxorubicin, in contrast with groups treated with nisin and doxorubicin alone (Preet et al., 2015). 2.4.2. Bioenergetics and reactive oxygen species generation in cancer cells as targets for novel anticancer therapies Otto Warburg was the first to link metabolism and cancer, describing the over-reliance of cancer cells on aerobic glycolysis (Warburg, 1956b). Originally it was hypothesised that cancer cells are forced to rely more on glycolysis to fulfil their energy demand due to impaired mitochondrial oxidative phosphorylation (OXPHOS) (Warburg, 1956a). However, today the reprogramming of cellular metabolism is considered one of the six hallmarks of cancer (Ward and Thompson, 2012), and it is evident that cancer cells increase both their glycolysis and mitochondrion glucose oxidation simultaneously compared to their surrounding tissue (DeBerardinis and Chandel, 2016). The increased bioenergetics play a role in tumour progression through the biosynthesis of molecules (nucleic acids and lipids) that are necessary for proliferation and growth. Furthermore, lactate that is preferentially formed from pyruvate during glycolysis in cancer cells contributes to the invasion and metastasis of cancer cells (Figure 2.5.) (Jozwiak et al., 2014, DeBerardinis and Chandel, 2016). Therapies that target not only the glycolytic metabolism but also the mitochondrion of cancer cells are gaining interest (Armstrong, 2006, Constance and Lim, 2012, Wen et al., 2013). Chapter 2: Literature overview 19 Figure 2.5: Metabolism of non-malignant cells compared to that of cancer cells. Non-malignant cells are more dependent on aerobic metabolism for energy, and in the absence of oxygen, these cells rely more on anaerobic glycolysis. Cancer cells increase both their glycolysis and mitochondrion glucose oxidation simultaneously compared to their surrounding tissue regardless of whether or not oxygen is available. Lactate formed by glycolysis contributes to the invasion/metastasis of cancer cells, whereas intermediates (nucleic acids and fatty acids) that are formed as a result of the elevated metabolism contribute to the proliferations and growth of cancer cells. Where; PPP, pentose phosphate pathway; TCA, citric acid cycle; OXPHOS, oxidative phosphorylation. Adapted from (Jozwiak et al., 2014, DeBerardinis and Chandel, 2016) Reactive oxygen species (ROS) fufil an important role in the maintenance of cellular and tissue homeostasis, however, high levels of ROS overwhelm the cells’ antioxidant capacity and leads to oxidative stress. High levels of oxidative stress are associated with the pathophysiology of many human diseases, which include cancer (Kryston et al., 2011, Ziech et al., 2011, Bolisetty and Jaimes, 2013). Therefore, as can be expected, cancer cells have a higher level of ROS than normal cells. ROS overproduction has been shown to be present in breast, liver, prostate, colon, pancreatic, melanoma, bladder and ovarian cancers (Afanas’ev, 2011). These elevated ROS promote many aspects of tumour development and progression by regulating certain signalling pathways (Liou and Storz, 2010). The elevated ROS levels can also serve as a target for cancer therapies, as a disproportional increase in ROS can induce cell cycle arrest, senescence and apoptosis (Trachootham et al., 2009) (Figure 2.6). Chapter 2: Literature overview 20 Figure 2.6: Role of reactive oxygen species (ROS) in normal homeostasis and pathophysiology. Low levels of ROS are necessary for normal signalling as well as proliferation and differentiation of cells, an increase in ROS in non-malignant (“healthy”) cells leads to carcinogenesis. This higher ROS level in cancer cells can serve as a target for cancer treatments that are able to selectively increase the ROS production past the threshold for survival of cancer cells, leading to cell cycle arrest, senescence and apoptosis. In view of overcoming chemotherapy resistance in cancer cells, anticancer agents that are able to target and disrupt glycolysis and/or mitochondrial respiration leading to ATP depletion while simultaneously increasing ROS past the threshold for cancer cell survival, are especially of interest (Indran et al., 2011). The primary mode of action of most AMPs seems to be pore formation. The cationic, amphiphilic nature of AMPs allow them to interact with and penetrate cell membranes by pore formation. This ultimately leads to cell death through the leakage of cytoplasmic essential components (summary of pore-forming mechanisms is reviewed in paper I). Some AMPs are also able to interact with internal targets leading to the inhibition of nucleic acid and protein synthesis, inhibition of enzymatic activity and the inhibition of cell wall synthesis. Some studies have indicated that AMPs have the ability to induce increased ROS production and apoptosis. For example, AMPs have been shown to induce increased ROS, which triggers apoptosis in Candida albicans (Park and Lee, 2010, Hwang et al., 2011, Sharma and Srivastava, 2014), while the AMP magainin was shown to induce ROS production and apoptosis which were associated with altered mitochondrial function in the human promyelocytic leukaemia (HL-60) cell line (Cruz- Chamorro et al., 2006). Although nisin has been shown to induce apoptosis in cancer cells, it still needs to be evaluated whether nisin affects the bioenergetics and ROS production of cancer cells while simultaneously inducing apoptosis. 2.5. References The references used in this section are included in the final reference list at the end of this thesis Chapter 2: Literature overview 21 Paper I: The potential use of natural and structural analogues of antimicrobial peptides in the fight against neglected tropical diseases. Angélique Lewies. Johannes. F. Wentzel, Garmi Jacobs and Lissinda. H. Du Plessis. Published in: Molecules (2015), Volume 20, pp 15392-15433. doi:10.3390/molecules200815392 Chapter 2: Literature overview 22 Chapter 2: Literature overview 23 Chapter 2: Literature overview 24 Chapter 2: Literature overview 25 Chapter 2: Literature overview 26 Chapter 2: Literature overview 27 Chapter 2: Literature overview 28 Chapter 2: Literature overview 29 Chapter 2: Literature overview 30 Chapter 2: Literature overview 31 Chapter 2: Literature overview 32 Chapter 2: Literature overview 33 Chapter 2: Literature overview 34 Chapter 2: Literature overview 35 Chapter 2: Literature overview 36 Chapter 2: Literature overview 37 Chapter 2: Literature overview 38 Chapter 2: Literature overview 39 Chapter 2: Literature overview 40 Chapter 2: Literature overview 41 Chapter 2: Literature overview 42 Chapter 2: Literature overview 43 Chapter 2: Literature overview 44 Chapter 2: Literature overview 45 Chapter 2: Literature overview 46 Chapter 2: Literature overview 47 Chapter 2: Literature overview 48 Chapter 2: Literature overview 49 Chapter 2: Literature overview 50 Chapter 2: Literature overview 51 Chapter 2: Literature overview 52 Chapter 2: Literature overview 53 Chapter 2: Literature overview 54 Chapter 2: Literature overview 55 Chapter 2: Literature overview 56 Chapter 2: Literature overview 57 Chapter 2: Literature overview 58 Chapter 2: Literature overview 59 Chapter 2: Literature overview 60 Chapter 2: Literature overview 61 Chapter 2: Literature overview 62 Chapter 2: Literature overview 63 Chapter 2: Literature overview 64 Paper II: Antimicrobial peptides: The Achilles heel to antibiotic resistance? Angélique Lewies, Lissinda. H. Du Plessis and Johannes. F. Wentzel This manuscript is submitted to the journal European Journal of Pharmaceutical Sciences and is written according to the guidelines set by the journal which can be found at https://www.elsevier.com/journals/european-journal-of-pharmaceutical-sciences/0928- 0987/guide-for-authors The tables and graphs are placed in text for ease of reading. Chapter 2: Literature overview 65 Antimicrobial peptides: The Achilles heel to antibiotic resistance? Angélique Lewies1, 2, Lissinda H. Du Plessis1, Johannes F. Wentzel1 1Centre of Excellence for Pharmaceutical Sciences (PHARMACEN), North-West University, Potchefstroom, 2520, South Africa. 2Centre of Excellence for Nutrition (CEN), North-West University, Potchefstroom, 2520, South Africa. Abstract Antimicrobial resistance to antibiotics is an imminent threat to the effective treatment of bacterial infections and alternative antibiotic strategies are urgently required. The golden epoch of antibiotics is coming to an end and the development of new therapeutic agents to combat bacterial infections should be prioritized. This article will review the potential of antimicrobial peptides (AMPs) to combat the threat of antimicrobial resistance. The modern- day antimicrobial resistance dilemma is briefly discussed followed by a review of the potential of AMPs to be used alone or in combination with current antibiotics in order to enhance anti-bacterial properties of antibiotics while also potentially combatting resistance. This article reiterates that many AMPs exhibit direct microbial killing activity and also play an integral role in the innate immune system. These properties make AMPs attractive alternative antimicrobial agents. Furthermore, AMPs are promising candidates to be used as adjuvants in combination with current antibiotics in order to combat antibiotic resistance Combinations of AMPs and antibiotics are less likely to develop resistance or transmit cross- resistance. The further identification and therapeutic development of AMPs and antibiotic- AMP combinations are strongly recommended. Keywords: Adjuvant therapy, Antibiotic resistance, Antimicrobial peptides, Innate immunity, Synergism, Chapter 2: Literature overview 66 1. Introduction Scenario: January 2053. As the sun rises over Boston Massachusetts, the light ominously glistens on multiple quarantine tent-covered homes. It is flu season in the United States but ironically influenza is not the primary concern. An outbreak of omni-antibiotic-resistant pneumonia is ravaging the country. Similar to the 1918 pandemic that devastated this region more than 130 years ago, influenza infection goes hand-in-hand with increased susceptibility to secondary bacterial infections. With no effective antibiotic agent available for the treatment of pneumonia, infected people are quarantined in their homes to minimize the transmission of this disease. Alas, the United States is not the only region affected by antimicrobial resistance. An epidemic of typhoid fever is sweeping large parts of Africa and Asia resulting in the deaths of millions a year. The multidrug-resistant H58-typhoid lineage has become the dominating typhoid strains worldwide with frequent outbreaks also now occurring in South America. Apart from the re-emergence of previously treatable diseases, antibiotic-resistant microorganisms have become sought-after bioweapons. Several European countries have become victims of bioterrorism agents such as the Shiga toxin producing Escherichia coli serotype, O157:H7. Besides the obvious adverse health threats these agents pose, treating patients or neutralizing these agents from the affected environments have proven difficult without effective antibiotic proxies. In this post-antibiotic era we have entered a second dark age, an era where mankind is yet again plagued by microbial diseases. This is not the plot of a Hollywood movie, but a dramatized, grim picture painted by a recent report on the economic impact of antibiotic resistance (O’Neill, 2016). This report projects that by 2050 antibiotic resistance could potentially lead to 10 million deaths per annum. Additionally, the economic impact of antibiotic resistance will be enormous, estimated at around 100 trillion US dollars amounting to a decrease of 2% to 3.5% in the gross domestic product. Currently, healthcare environments have become a breeding ground for antimicrobial resistance. Methicillin-resistant Staphylococcus aureus (MRSA) especially, presents a substantial risk to patients in hospitals, clinics, and inmates in correctional facilities, where individuals with weakened immune systems and open wounds are in close proximity and are more likely to contract this infection than the general public. Community-acquired pneumonia is a common illness, especially among children, the elderly and people who smoke. Pneumonia is more common in the winter months and often occurs in association with influenza virus infections. Evidence exists that suggests that the majority of deaths during the 1918 influenza pandemic were not the primary result of the influenza Chapter 2: Literature overview 67 virus – rather that most victims succumbed to secondary bacterial pneumonia co-infections (Brundage and Shanks, 2008; Morens et al., 2008). This is disconcerting when taking into account the growing frequency of resistance to penicillin and other antibiotics among pneumococci (Garau, 2002; Ho et al., 2009). As we prepare for possible future epidemics, antibiotic resistance is posing a serious threat to the effective treatment of respiratory tract infections, including community-acquired pneumonia. Salmonella enterica serovar Typhimurium is the leading cause of human typhoid fever (Parry et al., 2002). Typhoid fever has plagued the human race for millennia. One of the earliest documented cases is believed to be responsible for ending Athenian dominance in ancient Greece after the Greek statesman, Pericles, succumbed to this fever along with one-third of the population of Athens in 430 BC (Papagrigorakis et al., 2006). In modern times, Typhoid is still a common disease and a report from 2010 estimated that 26.9 million cases of typhoid fever occur annually on a global scale (Buckle et al., 2012). Infections are also rarely fatal, mainly due to the availability of effective treatment with antibiotics. Alarmingly though, phylogeographical analyses indicate a major and continuing clonal replacement of resident Salmonella Typhimurium with the drug-resistant H58 haplotypes (Wong et al., 2015). Multi- drug resistant outbreaks of Typhoid are increasingly being reported, whereas this disease was previously absent and the H58 clad seems to fuel the epidemiological transformation of this disease (Wong et al., 2015; Yan et al., 2016). This may be one of the chief reasons why the WHO considers Salmonella to be a high-priority pathogen for the development of new antibiotics (WHO, 2017b). The majority of E. coli strains are not pathogenic, but virulent strains can be the causative agent of gastroenteritis, neonatal meningitis as well as urinary tract infections. The virulent strain O157:H7, in particular, may cause serious illness or even death in the very young, the elderly and the immuno-compromised (Nataro and Kaper, 1998). Interestingly, this strain is classified as a bioterrorist agent as it is capable of producing the Shiga toxin which causes the premature destruction of the red-blood cells, eventually leading to hemolytic-uremic syndrome (Lim et al., 2010). E. coli infections are usually treated with rehydration and antibiotics (fluoroquinolones, azithromycin, and rifaximin), but effectively treating these infections are becoming increasingly challenging as these strains become more resistant to frontline antibiotics. E. coli is also considered by the WHO as a critical priority pathogen for the development of new antibiotics (WHO, 2017b). Neisseria gonorrhea is a gram-negative diplococcus able to infect human mucosal membranes, resulting in the sexually transmitted disease gonorrhea. This ancient pathogen Chapter 2: Literature overview 68 seems to have evolved to exclusively infect humans and has been the causative agent of gonorrhea for thousands of years. Unemo and Shafer noted that a possible early reference to this disease can be found in the Old Testament of the Christian Bible: “When any man has a bodily discharge, the discharge is unclean” (Leviticus 15:1–3) (Unemo and Shafer, 2014). In modern times, gonorrhea is one of the most prevalent sexually transmitted bacterial infections and the WHO estimates that approximately 78 million people are annually infected with gonorrhea globally. Also, the WHO Global Gonococcal Antimicrobial Surveillance Programme (WHO GASP), which monitors the trends in drug resistance in gonorrhea, indicated that there is widespread resistance to ciprofloxacin, azithromycin and emergence of resistance to the current last resort treatment; extended-spectrum cephalosporins (cefixime or ceftriaxone) (WHO, 2017a). The H041 N. gonorrhoeae strain is highly resistant to the extended-spectrum cephalosporin, ceftriaxone, one of the last remaining first-line treatments (Ohnishi et al., 2011). If current antimicrobial resistant trends continue, this ancient disease may again become untreatable. Another contributing factor to the antibiotic resistance crisis is the fact that the development of new antibiotics has virtually come to a standstill. Between the years 1980-2000, more than 50 new antimicrobial drug applications were approved, however, during the last decade less than 15 have been approved according to the Centers for Disease Control and Prevention (CDC, 2013). Even if novel antibiotics are developed, deployment will likely be highly restricted in the first few years with the aim of maintaining the agent’s effectiveness, resulting in low initial returns on investment as well as very little incentive to invest further in research and development. Microbial resistance against antibiotic agents is a solemn threat to the effective treatment of various infectious diseases. The golden epoch of antibiotics is coming to a close and we are now entering the post-antibiotic era. This increasing rise in antibiotic resistance among bacterial species has forced research into the development of new therapeutic agents to combat bacterial infections. 2. Antibiotic resistance: a modern-day dilemma with an ancient origin The discovery of penicillin in 1928 by Alexander Flemming and subsequent development of a wide range of antibiotics have transformed modern health care. Common bacterial infections such as tuberculosis and pneumonia could be effectively treated and were no longer death sentences. Apart from drastically reducing morbidity and mortality, antibiotics also formed the foundation of many of the greatest advancement in medical science and surgery of the last century. To date, antibiotics have been developed against almost all illness-causing bacteria and have been made readily available globally. Unfortunately, the Chapter 2: Literature overview 69 successful deployment of antibiotics has resulted in these drugs being used more as a financial commodity rather than a valuable community resource that should be rationally managed. This has led to the accelerated development of antimicrobial resistance among many bacteria. However, resistance to antibiotic substances is by no means a modern-day manifestation, as microorganisms have been adapting to meet the challenges of their environments for millions of years. Bacteria are inheriting resistant genes from previous generations, while also being one of a few organisms capable of obtaining genetic material from other species through a process known as horizontal gene transfer (Read and Woods, 2014). The mutation-inducing SOS response of bacteria to certain genetic stressors also gives these organisms an evolutionary counter to antimicrobials (Cirz et al., 2005). With the introduction of antibiotics, the evolution of resistance mechanisms has been artificially accelerated. Within two decades of deployment, penicillin was ineffective against most S. aureus infections and another antibiotic, methicillin, was developed and deployed. MRSA was observed in Europe and the USA within 3 years after introducing this antibiotic (Davies and Davies, 2010). To counter the staphylococcal onslaught, fluoroquinolones were introduced. However, antimicrobial resistance to this agent also promptly arose (Blumberg et al., 1991). Glycopeptides are currently reserved as last-resort treatments and its deployment is carefully monitored by health professionals. Disturbingly, multiple MRSA strains have shown reduced glycopeptide susceptibility (Appelbaum, 2007). Arguably even more alarming is the recent emergence of bacterial strains resistant to the so-called “last line” antibiotics. Plasmid-mediated polymyxin (colistin) resistant E. coli has been reported in China (Liu et al., 2016) while in August of 2016, a carbapenem-resistant Klebsiella pneumonia strain was isolated in Nevada, USA, resistant to all available antibiotics (Chen et al., 2017). Scientists also reported the existence of drug-resistant bacteria off beaches in Rio de Janeiro that hosted Olympic swimming events (Reuters, 2016). Today, the impact of antimicrobial resistance can be felt in many areas of everyday life, from standard medical procedures to the Olympics. The inability to effectively treat common bacterial infections is starting to affect our broader society and alternatives to antibiotics are desperately needed. Antimicrobial peptides (AMPs) are considered promising candidates as alternatives to current antibiotics in the treatment and prevention of microbial infections (Fox, 2013; Hancock and Lehrer, 1998; Hancock and Sahl, 2006). Chapter 2: Literature overview 70 3. A brief history of antimicrobial peptides AMPs, which are produced by all known living species, can be considered natural antibiotics. In prokaryotes, the existence of AMPs has been known since 1939, when gramicidin was isolated from the bacteria Bacillus brevis (Dubos, 1939). Some sources attribute the discovery of AMPs in eukaryotes to pioneering work done by Alexander Flemming in the 1920s when he discovered lysozyme, which is considered the first instance of a peptide with antibacterial activity (Flemming, 1922; Wang et al., 2016). Although the bactericidal activity of lysozyme occurs through the enzymatic lytic destruction of the cell wall (Vocadlo et al., 2001), it can be classified as an AMP due to its second mode of action, namely the non- enzymatic bactericidal activity which leads to membrane permeabilization and pore formation (Elmogy et al., 2015). The discovery of lysozyme also played a pivotal role in our understanding of modern innate immunity (Gallo, 2013). The discovery of AMPs contributed to answering the question as to why plants and insects, which lack adaptive immune systems, have the ability to fight off microbial infections. Purothionin, discovered by Balls and colleagues in 1942, was one of the first plant-derived AMPs to be isolated from the wheat endosperm, Triticum aestivum (Balls et al., 1942). This discovery was driven by the observation that wheat flour contains a substance that was lethal to bread yeast in 1896 (Jago and Jago, 1926). In 1941 it was established that bee venom from the European honey bee, Apis mellifera, also had antibacterial properties. In 1967, this antimicrobial activity was attributed to a peptide component of bee venom known as melittin (Fennell et al., 1967). In 1940 gramicidin was the first AMP to be tested clinically but was found not suitable for systemic application due to its high toxicity (Van Epps, 2006). However, these studies subsequently led to renewed interest in the antibiotic penicillin. The mass production and deployment of penicillin ushered in the golden era of antibiotics, which led to less attention being given to AMPs and the role they play in the immune defense against antimicrobial infections. However, the rise in antibiotic resistance is driving research into the search for alternatives to current antibiotics. AMPs can be considered promising candidates to be used alone or in combination with current antibiotics, and are gaining renewed interest (Fox, 2013). To date, more than 2000 AMPs have been discovered as listed on the ADP3 database (Wang et al., 2017). It is also worth noting that research in the field of AMP discovery is thriving, with at least one new AMP being discovered every single year from 1985 to 2017 (Wang et al., 2016). AMPs not only exhibit broad spectrum antibacterial activity but many also display antifungal-, antiviral-, anti-parasitic- (Jenssen et al., 2006) and anticancer- (Schweizer, 2009) activities. Chapter 2: Literature overview 71 3.1. Antimicrobial peptides approved by FDA and used in a clinical setting to treat bacterial infections Although a number of AMPs have undergone clinical trials and several AMPs are in clinical development (Fox, 2013; Gordon et al., 2005; Hancock and Sahl, 2006), only a few have been successfully applied commercially. Three non-ribosomally synthesized AMPs colistin, gramicidin and daptomycin are currently being used as antibiotics. Due to the mentioned toxicity associated with gramicidin, it is presently only being used for the topical treatment of superficial infections (Van Epps, 2006). Colistin, produced by the Gram-positive bacterium Paenibacillus polymyxa, is considered a last-resort drug for treating Gram-negative superbugs (Li et al., 2006). Daptomycin, produced by the Gram-positive bacterium Streptomyces roseosporus, was approved by the FDA as an antibiotic in 2003 and has bactericidal activity against a variety of Gram-positive bacteria which include antibiotic- resistant strains such as MRSA, vancomycin-resistant enterococci and penicillin-resistant Streptococcus pneumonia (Steenbergen et al., 2005; Tran et al., 2015). Gene-encoded, ribosomally synthesized AMPs (also referred to as host defense peptides) are evolutionarily conserved components of the innate immune system of plants, insects, and animals (Hancock and Diamond, 2000; Midorikawa et al., 2003). These peptides serve as first line of defense against microbial infections. However, these AMPs are not limited to multicellular organisms and bacteria also utilize similar AMPs, bacteriocins, in order to obtain a competitive advantage over other micro-organisms in their habitat (Cotter et al., 2005). Perhaps the best known of these is the lantibiotic, nisin. Lantibiotics, produced by a wide range of Gram-positive bacteria to fend off other Gram-positive species, are a class of AMPs that contain the uncommon amino acids lanthionine or methyllanthionine (McAuliffe et al., 2001). Nisin is produced by the non-pathogenic, probiotic Lactococcus lactis. In 1969 nisin was approved by the World Health Organization (WHO) and the US Federal Food and Drug Administration (FDA) in 1988 for use as a food preservative and holds promising potential for clinical application with its Generally Regarded As Safe (GRAS) status (Shin et al., 2016). Nisin is used as a preservative (European food additive list number E234) in over 48 countries with a view to protect food from spoilage caused by pathogens such as Staphylococcus aureus, Listeria monocytogenes and Clostridium botulinum (Cotter et al., 2005; Jones et al., 2005). Nisin also has antibacterial activity against the clinically important pathogens such as vancomycin-resistant Enterococci, Streptococcus pneumonia and MRSA (Brumfitt et al., 2002; Dosler and Gerceker, 2011; Goldstein et al., 1998). In a study by Bartoloni and co-workers, it was found that nisin had better bactericidal activity, compared to Chapter 2: Literature overview 72 vancomycin and metronidazole, against clinical isolates of Clostridium difficile (Bartoloni et al., 2004). Nisin is also active against not only the vegetative cells but also the spores of a variety of Clostridium and Bacillus strains (Gut et al., 2011; Le Lay et al., 2016). Mastitis- causing Staphylococcus strains tend to develop resistance to antibiotics or to develop biofilms (Gill et al., 2005; Oliveira et al., 2006). Nisin has been successfully applied as a sanitizer against mastitis causing Staphylococcus and Streptococcus species in lactating cows even when these species are antibiotic resistant (Cao et al., 2007; Wu et al., 2007). Three nisin-based products were developed for the treatment of bovine mastitis, namely Ambicin N® (Applied Microbiology, Inc., New York) and Mast Out® as well as Wipe Out® Dairy wipes (ImmuCel Corporation, Maine, USA) (Cotter et al., 2005; Pieterse and Todorov, 2010). 3.2. Mode of action of AMPs The selectivity and potency of AMPs against microbes are determined by structural parameters, which mainly include net charge (cationic or anionic AMPs) and structural conformation (α-helical, linear/extended, β-sheet and cyclic AMPs) (Matsuzaki, 2009; Takahashi et al., 2010). AMPs exert their activity largely through three mechanisms. Firstly, interaction with the bacterial membrane through electrostatic or hydrophobic interactions, leading to the perturbation of the microbial membranes which occur in accordance with three proposed models, namely: (i) the barrel-stave model, (ii) the carpet model; and (iii) the toroidal model; secondly, the interaction of AMPs with internal targets, which can lead to the inhibition of DNA replication or translation, transcription or enzymes (Figure 1). Both these mechanisms have been discussed in more detail in a previous review article (Lewies et al., 2015). In the current review, more focus will be given to the third mechanism of action, namely the modulation of the immune system. At physiological concentrations, the antibacterial activity of some AMPs is antagonized by salt concentrations, monovalent and divalent ions and serum (Wu et al., 2008). Hence, AMPs are considered to be acting as natural effectors of the innate immune system and is involved in immune modulation and immune stimulation (Radek and Gallo, 2007). Chapter 2: Literature overview 73 Figure 1. Main antimicrobial mechanisms of AMPs during the infection of a mammalian host cell. The direct microbial action of AMPs involves the binding of these peptides to the microbial membrane/capsid, resulting in the infectious agent’s neutralization. There are three models proposed for this action, i) The barrel-stave model, where the AMPs embed themselves into the membrane to form pores; ii) The carpet model proposes that small portions of the membrane are removed by AMPs; and iii) The toroϊdal model is similar to the barrel-stave model, but with the exception that the AMPs are permanently bound to the phospholipids in the membrane. Internal functions of AMPs may include DNA replication/translation inhibition, transcription inhibition and enzyme inhibition. Many AMPs are also involved in immunoregulation. Some of these regulatory functions may include cell proliferation, cytokine regulation, chemotaxis of certain leukocyte classes, degranulation of mast cells, stimulation of phagocytosis and even modulation of gene expression. The innate immune response is initiated when the pathogen-associated molecular patterns (PAMPs) are recognized by the host pattern recognition receptors. Bacterial lipopolysaccharides (LPS), which are PAMPs associated with Gram-negative bacteria, fulfills a major role in septic shock syndrome caused by Gram-negative bacteria due to its ability to promote a strong induction of the innate immune system. Other PAMPs include Gram- positive lipoteichoic acid or peptidoglycan (PGN), which also stimulate pro-inflammatory responses, but not as pronounced as that associated with LPS (Cohen, 2002). AMPs can regulate the inflammatory response of the innate immune system by neutralizing LPS, preventing septic shock. LPS form aggregates which are in turn recognized by the host immune system (Mueller et al., 2005). Temporins, AMPs found in the skin secretions of frogs, disturb LPS aggregates which reduce the binding of LPS to LPS’ binding proteins (LBP), ultimately leading to decreased levels of secretion of tumour necrosis factor (TNF)-α by macrophages (Mangoni et al., 2008). The human cathelicidin peptide LL-37 is able to bind and neutralize LPS, thereby blocking the binding of LPS to CD14. This leads to the Chapter 2: Literature overview 74 disaggregation of LPS causing reduced binding to LBP and clearing LPS from the cell surfaces of macrophages, ultimately inhibiting the production of TNF-α and other pro- inflammatory cytokines (Rosenfeld et al., 2006). AMPs are additionally suggested to display regulatory control over pro-inflammatory gene expression, harmonizing the host innate immunity in response to infection. A study by Mookherjee and co-workers has shown that LL-37 selectively inhibits the expression of specific pro-inflammatory genes in the presence of LPS, hindering the LPS-induced transcription of TNF-α and other cytokines. No significant decrease was observed in the expression of LPS-induced genes that antagonize inflammation or chemokine genes that are typically considered pro-inflammatory (Mookherjee et al., 2006). AMPs also display chemokine-like activities. Several human AMPs (including α- and β-defensins as well as the cathelicidin LL-37) have been reported to be able to attract leukocytes to the site of infection or inflammation (Durr and Peschel, 2002). LL-37 is able to stimulate the production of IL-8, a chemokine for neutrophils and monocytes, which are then attracted to the site of infection (Tjabringa et al., 2003). In a study by Cirioni and co-workers the in vivo intravenous injection of LL-37 was able to protect rats against sepsis induced by Gram-negative bacteria significantly better compared to conventional antibiotics (Cirioni et al., 2006). The bacteriocin nisin is also known to interact with host immunity. Nisin has been shown to activate human neutrophils, stimulating neutrophil extracellular trap (NET) formation (Begde et al., 2011). Neutrophils neutralize bacteria primarily through phagocytosis. However, when phagocytosis is prevented, the formation of NETs is stimulated by IL-8, endotoxins such as LPS or the protein C kinase activator PMA, and bacteria are subsequently neutralized by NETs (Brinkmann and Zychlinsky, 2007). This observation could be troublesome as nisin is a bacterial-derived peptide and could possibly display endotoxin-like properties. However, it is more plausible that nisin can act as a chemokine stimulant; thus stimulating IL-8 production causing the formation of NETs. A study by Kindrachuk and co-workers supports this theory and has shown that, similarly to the human defense peptide LL-37, the nisin Z variant can selectively modulate the immune system through the suppression of LPS- induced TNF-α production and the induction of the synthesis of IL-8 and other chemokines. Nisin has little to no effect on most Gram-negative bacteria. However, it was also indicated that nisin Z might play a role in in vivo host immune response modulation, as nisin Z, when administered prophylactically in mice, had the ability to protect against Gram-negative (E. coli and S. enterica server Typhimurium) bacterial infections (Kindrachuk et al., 2013). In another study, nisin only had an immune-modulatory effect by decreasing both the TNF-α levels and the activation of nuclear factor (NF)-кB, when administered together with metal- chelating β-lactam antibiotics following infection of mice with S. enterica serovar Chapter 2: Literature overview 75 Typhimurium prior to nisin administration. Moreover, the effect of nisin/β-lactam antibiotic adjuvant therapy was more pronounced than when the antibiotics were administrated alone (Singh et al., 2014). Synthetic AMPs, which may have both direct bacterial killing activity and immune-modulatory activities, can be synthesized based on the structure of naturally occurring AMPs. Recently a synthetic AMP, clavanin-MO, was synthesized by Silva and co-workers based on the structure of naturally occurring clavanin-A, an AMP isolated from the haemocytes of the murine tunicate Styela clava. Clavanin-MO is nontoxic and depicted potent antimicrobial as well as immunomodulation activities in both in vitro and in vivo assays. In vivo results are especially of interest, as it was shown that the treatment of mice with clavanin-MO following infection with multi-drug resistant E. coli and MRSA caused an increase in migration of leukocytes to the site of infection, prolonged survival and almost completely eradicated infections 24 hours post infection. Furthermore, compared to the conventional β-lactam antibiotic imipenem, clavanin-MO displayed significantly higher bacterial killing activity against multi-drug resistant E. coli (Silva et al., 2016). 3.3. Applying AMPs to reduce antibiotic resistance Antibiotic adjuvant therapy can be employed to combat antibiotic resistance. Adjuvants can be divided into two classes; class I adjuvants, which affect bacteria by inhibiting active and intrinsic (passive) antibiotic resistance in bacteria and class II adjuvants, which enhance the ability of the host to neutralize bacteria (Wright, 2016). Many AMPs can be considered class I adjuvants that interact synergistically with antibiotics (Table 1) or as class II adjuvants due to the ability of AMPs to reinforce the host defense system through immunomodulation as discussed previously. Furthermore, septic shock caused by the release of LPS from the cell wall of Gram-negative bacteria, associated with several antibiotic treatments, also poses a major problem (Prins et al., 1995). Due to the ability of AMPs to neutralize LPS, they make attractive proxies for use in combination with antibiotic treatment to prevent septic shock. Chapter 2: Literature overview 76 Table 1: Synergism between antimicrobial peptides and antibiotics Antimicrobial peptide(s) Synergistic interaction with Organism Reference Cryptdin 2 Ampicillin Salmonella enterica serovar Typhimurium (Rishi et al., 2011) Arenicin-1 Ampicillin Erythromycin Chloramphenicol Staphylococcus aureus Staphylococcus epidermidis Pseudomonas aeruginosa Escherichia coli (Choi and Lee, 2012) Nisin Z Penicillin Streptomycin Leucomycin Rifampicin Pseudomonas fluorescens LRC-R73 Penicillin-resistant variant Streptomycin-resistant variant Lincomycin-resistant variant Rifampicin-resistant variant (Naghmouchi et al., 2012) Nisin* Indolicidin CAMA Daptomycin Teichoplanin Ciprofloxacin Methicillin resistant S. aureus biofilms (Mataraci and Dosler, 2012) Nisin* Ampicillin S. aureus # (Dosler and Gerceker, 2012) Daptomycin Enterococcus faecalis # Brevinin-2CE Levofloxacin Amoxicillin Chloramphenicol ESBL producing E. coli Methicillin resistant S. aureus # (Zhang et al., 2014) Chapter 2: Literature overview 77 Antimicrobial peptide(s) Synergistic interaction with Organism Reference Human beta defensin 3 LL-37 Tigecycline Moxifloxacin Piperacillin-tazobactam Meropenem Clostridium difficile (Nuding et al., 2014) Nisin* Ampicillin Cefotaxime Ceftriaxone Salmonella enterica serovar Typhimurium # (Rishi et al., 2014) LL-37 Azithromycin MDR Pseudomonas aeruginosa MDR Klebsiella pneumonia MDR Acinetobacter baumannii (Lin et al., 2015) Nisin Z Novobiocin Staphylococcus aureus Staphylococcus epidermidis (Lewies et al., 2017) ESBL; extended-spectrum β-lactamase, CAMA; cecropin (1-7)–melittin A (2-9) amid, MDR; Multi drug resistant. * Variant not specified, # Clinical isolates 3.4. Bacterial resistance and antimicrobial peptides AMPs are produced by all known living species (ranging from bacteria, fungi, and plants to invertebrates, non-mammalian vertebrates and mammals). Microbes are present in a wide variety of environments, including physiological systems where they have to fend off the hosts’ AMP-mediated defense to survive. It is therefore unrealistic to expect that there would be no resistance to AMPs (Yeaman and Yount, 2003). It should also be borne in mind that resistance mechanisms in bacteria are considered an evolutionary trait and it is therefore impossible to find a compound to which no degree of resistance will eventually emerge. Bacterial resistance mechanisms to AMPs have been reviewed extensively and will not be covered in depth in the current review. Instead, readers are referred to (Guilhelmelli et al., 2013; Yeaman and Yount, 2003). Chapter 2: Literature overview 78 Some resistance is associated with non-ribosomally synthesized AMPs, derived from bacteria, which are currently being employed as antibiotics including daptomycin and colistin. Although most strains of bacteria for which daptomycin are prescribed are still susceptible to this antibiotic, daptomycin resistant strains have been reported. This issue can be addressed by making use of β-lactam antibiotic-daptomycin combinations with the purpose of obtaining synergistic interactions (Tran et al., 2015). The polypeptide antibiotic, colistin, was first introduced in the 1950s but was rarely used due to its associated nephrotoxicity. However, with the emergence of multi-drug resistance Gram-negative infections, it was employed as the last line of defense against these infections (Li et al., 2006). Similar to antibiotics, AMPs are not immune to misuse in farming practices. A recent study from China detected the colistin resistant gene, MRC-1, in porcine E. coli which is mainly the result of unregulated overuse of this antibiotic in pig farming practices. The MRC- 1 gene can be transferred across strains through horizontal transfer and has already been detected in E. coli species found in humans (Liu et al., 2016). Therefore, the FDA has tightened restrictions on the use of colistin in both humans and animals in 2017, with colistin only to be given intravenously in humans or under the strict supervision of a veterinarian for animals (FDA, 2017a, b). The therapeutic potential of ribosomally synthesized AMPs is being recognized. Some synthetic analogues derived from these peptides (e.g. omiganan derived from indolicidin) are in clinical trials. However, none of them are currently approved for use as antibiotics (Fox, 2013). Resistance has been reported for this class of AMPs (Andersson et al., 2016; Perron et al., 2006). It should, however, be noted that most of these mechanisms have been studied in in vitro settings using a single AMP; hence it is difficult to compare these results with what would actually occur in vivo due to the fact that there are multiple AMPs present that could act synergistically. Some of these AMPs also have multiple modes of action. For example, nisin has five possible mechanisms of action involved in bacterial killing; (1) the inhibition of cell wall biosynthesis, (2) lipid II-dependent pore formation, (3) inhibition of bacterial spore outgrowth, (4) pore formation that is independent of lipid II binding, and (5) the activation of autolytic enzymes resulting in cell wall degradation (Pag and Sahl, 2002). It is considered difficult for bacteria to evolve resistance to all these mechanisms simultaneously. In the case of nisin, this has proven to be true, as there is very little evidence of stable and transmissible resistance occurring in food products treated with this AMP, despite the fact that nisin has been used as a food preservative for almost 50 years (Shin et al., 2015) Furthermore, there is an abundance of AMPs with different structural features and mechanisms of action. The fact that host defense AMPs have co-evolved with host defense Chapter 2: Literature overview 79 AMP-resistant mechanisms also offers some advantages as reviewed in (Peschel and Sahl, 2006). Studies into resistance mechanisms associated with certain structural features and ways of circumventing them are especially of interest in the fight against antibiotic resistance. For example, it is possible for host defense peptides to evade bacterial AMP- specific resistance, which depends on the recognition of AMP-specific sequence- or structural motifs, by inducing variations in the peptide sequence; hence producing multiple naturally occurring AMP variants (Peschel and Sahl, 2006). Insight into the relationship between structural features (net charge, secondary structure, and peptide sequence) and the susceptibility to resistance mechanisms could lead to the development of optimized synthetic AMPs, based on the structure of naturally occurring AMPs, with lowered toxicity, improved activity and a lower incidence of bacterial resistance. This approach could lead to the discovery of peptide derivatives with antimicrobial properties. In a recent study by Barreto-Santamaría and co-workers, this approach was used to elucidate the antibacterial activity of a synthetic peptide 34509, a peptide analogue of peptide 20268, which is in turn derived from the Plasmodium falciparum PfRif protein. Peptide 34509 was found to be cationic and having α-helical structural elements which are structural characteristics similar to those of AMPs (Barreto-Santamaria et al., 2016). Although the sequence of peptide 34509 has not been reported as an AMP, it is similar to the naturally occurring AMP latarcin 1 isolated from the poisonous spider Lachesana tarabaevi. Latarcin 1 has antibacterial activity, but is also toxic to eukaryotic cells (Barreto-Santamaria et al., 2016; Wang et al., 2016). Compared to peptide 20268 (which displays toxicity and has no bacterial activity), peptide 34509 was found to be non-toxic to eukaryotic cells and had activity to both Gram-positive and negative bacteria (Barreto-Santamaria et al., 2016). 3.5. Advantages of antimicrobial peptides above antibiotics Compared to conventional antibiotics, one of the major incentives for the use of AMPs is their diverse applications. Antibiotics usually have a narrow spectrum, acting only on bacteria, whereas AMPs have a broad spectrum of activity displaying antibacterial, antiviral, anti-parasitic and anticancer activities. The rate of acquired resistance is also lower for AMPs compared to antibiotics (Marr et al., 2006). Not only can AMPs be used as a single antimicrobial due to their direct killing actions or as adjuvants with conventional antibiotics to obtain synergistic interactions, but said AMPs can be employed to address issues relating to bacterial infections for which antibiotics have not been proven to be successful. These include septicemia (which has an estimated 30% death rate) and infections in individuals who are immune-comprised (due to immunosuppressive diseases or chemotherapy) who cannot provide immune support for antibiotic therapy (Hancock, 2015). AMPs possess the Chapter 2: Literature overview 80 potential of being applied as immune-modulatory/stimulatory compounds and are also able to neutralize endotoxins and prevent sepsis. This induction of AMPs can then stimulate the innate immunity and furthermore act synergistically with administered antibiotics in order to provide better clinical outcomes (Ottosson et al., 2016). AMPs appear to be ideal adjuvants to be used in combination with our current arsenal of antibiotics. Finally, ribosomally-synthesized AMPs hold better potential than conventional antibiotics and non-ribosomally synthesized AMPs (including gramicidin, polymyxin, bacitracin and glycopeptides) due to the fact that they are gene-encoded and therefore more susceptible to bioengineering strategies in an attempt to enhance their activities and possibly circumvent bacterial resistance. For example, nisin has activity against most Gram-positive bacteria but lacks activity against Gram-negative bacteria. Field and co-workers followed a bioengineering approach to produce nisin A serine 29 derivatives using a site-saturation mutagenesis approach. These derivatives displayed enhanced activity against MRSA and other Gram-positive bacterial species as well as Gram-negative food associated pathogens, which included, E. coli, Salmonella enterica serovar Typhimurium and Cronobacter sakazii (Field et al., 2012). 3.6. Obstacles associated with the therapeutic use of antimicrobial peptides Currently, one of the greatest obstacles for the clinical application of AMPs (especially ribosomally synthesized AMPs), is the cost of large-scale synthesis of these peptides. Sufficient progress has been made in the development of DNA recombinant methods for the cost-effective synthesis and purification of AMPs with increased yields for therapeutic application (Ali et al., 2014; Bommarius et al., 2010; Kong and Lu, 2014; Li, 2011). However, the commercial feasibility of these methods still needs to be evaluated. The burden of antibiotic resistance may soon outweigh the cost issue. For example, around the time that penicillin resistance started to emerge, nisin A was shown to be just as effective as penicillin in mice models infected with Streptococcus pyogenes, S. aureus, and Mycobacterium tuberculosis (Bavin et al., 1952). Initial studies concluded that nisin had low therapeutic potential due not only to its rapid clearance from the blood but also to the high cost associated with its production. However, the rise in antibiotic resistance and the economic burden associated with addressing this issue (e.g. 8 billion dollar cost associated with the treatment of MRSA) will most likely soon start to outweigh the issue related to the cost of the clinical development of AMPs as antimicrobials (Smith and Hillman, 2008; Yoneyama and Katsumata, 2006). Moreover, food grade nisin is currently being produced by Zhejiang Silver-Elephant Bio. Engineering Co. at a production capacity of 100 metric ton per year and Chapter 2: Literature overview 81 sold at 100 US dollars per kilogram. Although this formulation only contains 5 percent pure nisin, it implies that the development of stable biological production processes for pharmaceutical grade lantipeptides (and possibly other AMPs) is possible and only require more research efforts (Ongey and Neubauer, 2016). Due to the fact that AMPs are peptide-based drugs, bioavailability issues also need to be addressed. Studies therefore need to focus on the pharmacokinetics and -dynamics of AMPs to determine the dosage needed to obtain the required therapeutic outcome, as well as to avoid possible toxic side effects. It is of special importance that the administration of AMPs be regulated due to the fact that they form part of the host's natural innate immunity. Therefore the emergence of cross-resistance would prove to be detrimental (Bell and Gouyon, 2003). Cross-resistance between therapeutic AMPs and innate AMPs might occur during monotherapy with therapeutic AMPs. However, the combination of AMPs and AMPs with antibiotics has been shown to limit the evolution of resistance and cross-resistance as discussed in detail in (Fleitas and Franco, 2016). Foregoing the direct development of AMPs as antimicrobials and the high costs associated with this direct approach, structural and mechanistic studies of AMPs could also lead to the development of new classes of antibiotics. Peptidomimetics are being developed as a new chemical class of antibiotics; these are amphiphilic compounds that are modulated from the structural and biological properties of host defense peptides (Wright, 2016). 4. Other alternatives and ways to overcome antibiotic resistance Despite AMPs, several other antibacterial strategies hold the potential of relieving the burden on conventional antibiotics. Some of these alternatives include established technologies such as vaccines and antimicrobial nanoparticles, while other technologies have shown promise, but are not widely used in a clinical setting, e.g. Bacteriophages and antimicrobial surface coatings. Other, potentially effective, strategies exist such as rapid point-of-care diagnostics and regulatory policies (ECDC/EMEA, 2009; Nwokoro et al., 2016). However, these are complement strategies and do not form part of the scope of this review. Bacterial vaccines could considerably lift the current burden on antibiotics and obtain the added bonus of being a widely used technology in the fight against viral and bacterial infections. Additionally, as a well-established industry with a bacterial vaccine pipeline, several bacterial vaccines are already being routinely used in numerous healthcare systems (PhRMA, 2013). For example, a pneumococcal vaccine has been deployed worldwide in immunization programs and has significantly reduced Streptococcus pneumonia infections Chapter 2: Literature overview 82 (Dagan and Klugman, 2008). It is unlikely that vaccines will be able to cover all risk groups and will therefore not eradicate the need for antibiotics. Nonetheless, vaccines will most definitely reduce the use of antibiotics, in turn, delaying the acquirement of antimicrobial resistance. Bacteriophages are viruses that exclusively infect and replicate in bacteria. These viruses are among the most common and diverse entities in nature. Bacteriophage therapy has many potential applications in human medicine, including the use as an antibiotic agent. Phages are more specific than conventional antibiotics and usually harmless to the host organism and other bacterial species (Keen, 2012). While resistance to phages does occur, viruses are able to rapidly mutate, unlike antibiotics, and can therefore co-evolve to counter phage-resistance in bacteria (Matsuzaki et al., 2005). This technology has shown potential in clinical use in selected Eastern European countries. However, the West has been slow to adapt phage therapies (Nwokoro et al., 2016). Although the effectiveness of phage therapy is yet to be proven on large scale, with well-controlled Western trials, the overall body of evidence suggests that phage therapy is an effective alternative and should be researched further (Keen, 2012). Efflux pumps are membrane proteins involved in the extrusion of compounds across the cell membrane, without altering the structure of this substance (Van Bambeke et al., 2003). These transporters can fulfill an important role in conferring resistance to antibiotics by actively removing antibiotic compounds from the bacterial cell. Inhibiting efflux transporters targeting antibiotic compounds may provide an alternative toolset to combat antibiotic resistance (Willers et al., 2017). The use of efflux pump inhibitors as adjuvants in antibiotic treatments may potentially restore the activity of antibiotics against resistant bacteria. Despite the identification of numerous efflux pump inhibitors, limited clinical application studies have been performed. Additional effort is needed to bridge the gap between basic efflux pump inhibitors research and clinical application. Currently, no technology exists to render antibiotics redundant. However, alternative technologies do exist which can greatly reduce the burden on antibiotic use and greater effort should be spent in applying these technologies. 5. Conclusion In conclusion, although bacterial resistance to microbial agents can be considered a natural part of evolution, the mass production and use of antibiotics have accelerated the proses. Chapter 2: Literature overview 83 We are now facing a future in which we are descending into the pre-antibiotic age. Common bacterial infections will again become lethal. The development of new antimicrobial agents is not only time-consuming but also costly, leading to the lethargic development of new antibiotics and antimicrobial strategies that are not on par with the tempo of the antimicrobial resistance onslaught. It is clear that current alternative technologies, despite all their potential to relieve the pressure on antibiotics, will not be able to fill the void of new classes of antibiotics. Therefore, research should be aimed at finding a means to strengthen our current arsenal of antibiotics. AMPs are promising candidates in this regard due to their ability to directly kill bacteria, act synergistically with current antibiotics, modulate host immunity and at the same time enhance the hosts’ ability to kill bacteria and neutralize endotoxins. However, some limitations for the use of AMPs such as possible cross- resistance with AMPs that form part of the innate immunity, the high cost of production and toxicity issues that might arise due to their use, still need to be addressed. Therefore, studies should be aimed at addressing possible issues that might arise due to the cross-resistance between AMPs that form part of the host immunity and AMPs intended for therapeutic use. Combinations of AMPs or AMPs and antibiotics are less likely to develop resistance or transmit cross-resistance. Combinations of AMPs and antibiotics have also proven effective against bacterial infections listed by the WHO as critical and high priority pathogens for the development of new antibiotics (Table 1). This highlights the possibility that adjuvant therapy with AMPs and antibiotics could assist in addressing the antibiotic resistance crisis. However, the relevance of synergistic observations should be confirmed with the focus on clinical isolates of bacteria that are antibiotic resistant. Additionally, more research needs to be directed at finding economically feasible ways to produce large enough quantities of AMPs for therapeutic use. More in vitro and in vivo mechanistic studies are necessary in order to determine the mechanisms of action and possible resistance mechanisms that might arise from long-term exposure to AMPs. These results then need to be correlated with the structural properties of these AMPs. Studies such as these will guide the development of novel AMPs based on the structure of naturally occurring AMPs, with stronger therapeutic potential. Future studies that thoroughly examine the systemic peptide pharmacokinetics and pharmacodynamics also need to be undertaken. Through these studies the in vivo half-life and required dosing frequency can be rationally determined in order to minimize toxicity and optimize disease outcomes. To date, only a few non-ribosomally synthesized AMPs have been approved by the FDA as antibiotics. Colistin, considered as the last line of defense against multi-drug resistant Gram-negative bacteria has recently come under the spotlight due to the emergence of resistance which was induced because of its overuse in pig farming practices. Colistin was never considered for oral use, yet it is cheap and easily accessible; therefore it is used in animal feed to promote growth and prevent infection. Although the Chapter 2: Literature overview 84 FDA has now tightened control over the use of colistin in farming practices, they should have considered banning its use altogether. Human health should come before economic gain; hence, no antibiotics and antimicrobials considered for clinical development and therapeutic use in humans should be allowed for animal use or only be administered under the very strict control of veterinarians in case of life-threatening infections. Funding AL is grateful for financial assistance from the National Research Foundation (NRF) of South Africa (Grant number 94942). Opinions expressed and conclusions arrived at are those of the authors and are not to be attributed to the NRF. Declaration of interest Authors declare that they have no conflict of interest. References Ali, M.P., Yoshimatsu, K., Suzuki, T., Kato, T., Park, E.Y., 2014. Expression and purification of cyto-insectotoxin (Cit1a) using silkworm larvae targeting for an antimicrobial therapeutic agent. Applied microbiology and biotechnology 98, 6973-6982. Andersson, D.I., Hughes, D., Kubicek-Sutherland, J.Z., 2016. Mechanisms and consequences of bacterial resistance to antimicrobial peptides. Drug Resist Updat 26, 43-57. Appelbaum, P.C., 2007. Reduced glycopeptide susceptibility in methicillin-resistant Staphylococcus aureus (MRSA). International journal of antimicrobial agents 30, 398-408. Balls, A.K., Hale, W.S., Harris, T.H., 1942. Further observations on a crystalline wheat protein. 19 840-844. Barreto-Santamaria, A., Curtidor, H., Arevalo-Pinzon, G., Herrera, C., Suarez, D., Perez, W.H., Patarroyo, M.E., 2016. A New Synthetic Peptide Having Two Target of Antibacterial Action in E. coli ML35. Frontiers in microbiology 7, 2006. Bartoloni, A., Mantella, A., Goldstein, B.P., Dei, R., Benedetti, M., Sbaragli, S., Paradisi, F., 2004. In-vitro activity of nisin against clinical isolates of Clostridium difficile. Journal of chemotherapy 16, 119-121. Bavin, E.M., Beach, A.S., Falconer, R., Friedmann, R., 1952. Nisin in experimental tuberculosis. Lancet 1, 127-129. Begde, D., Bundale, S., Mashitha, P., Rudra, J., Nashikkar, N., Upadhyay, A., 2011. Immunomodulatory efficacy of nisin--a bacterial lantibiotic peptide. Journal of peptide science : an official publication of the European Peptide Society 17, 438-444. Bell, G., Gouyon, P.H., 2003. Arming the enemy: the evolution of resistance to self-proteins. Microbiology 149, 1367-1375. Blumberg, H.M., Rimland, D., Carroll, D.J., Terry, P., Wachsmuth, I.K., 1991. Rapid development of ciprofloxacin resistance in methicillin-susceptible and -resistant Staphylococcus aureus. The Journal of infectious diseases 163, 1279-1285. Bommarius, B., Jenssen, H., Elliott, M., Kindrachuk, J., Pasupuleti, M., Gieren, H., Jaeger, K.E., Hancock, R.E., Kalman, D., 2010. Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli. Peptides 31, 1957-1965. Brinkmann, V., Zychlinsky, A., 2007. Beneficial suicide: why neutrophils die to make NETs. Nature reviews. Microbiology 5, 577-582. Chapter 2: Literature overview 85 Brumfitt, W., Salton, M.R., Hamilton-Miller, J.M., 2002. Nisin, alone and combined with peptidoglycan-modulating antibiotics: activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. The Journal of antimicrobial chemotherapy 50, 731-734. Brundage, J.F., Shanks, G.D., 2008. Deaths from bacterial pneumonia during 1918-19 influenza pandemic. Emerging infectious diseases 14, 1193-1199. Buckle, G.C., Walker, C.L.F., Black, R.E., 2012. Typhoid fever and paratyphoid fever: Systematic review to estimate global morbidity and mortality for 2010. Journal of Global Health 2, 010401. Cao, L.T., Wu, J.Q., Xie, F., Hu, S.H., Mo, Y., 2007. Efficacy of nisin in treatment of clinical mastitis in lactating dairy cows. Journal of dairy science 90, 3980-3985. CDC, 2013. Antibiotic resistance threats in the United States. CDC, Atlanta. [Online] Available: https://www.cdc.gov/drugresistance/pdf/ar-threats-2013-508.pdf [Accessed 17 July 2017] Chen, L., Todd, R., Kiehlbauch, J., Walters, M., Kallen, A., 2017. Notes from the Field: Pan- Resistant New Delhi Metallo-Beta-Lactamase-Producing Klebsiella pneumoniae. MMWR Morb Mortal Wkly Rep 66. Choi, H., Lee, D.G., 2012. Synergistic effect of antimicrobial peptide arenicin-1 in combination with antibiotics against pathogenic bacteria. Research in microbiology 163, 479- 486. Cirioni, O., Giacometti, A., Ghiselli, R., Bergnach, C., Orlando, F., Silvestri, C., Mocchegiani, F., Licci, A., Skerlavaj, B., Rocchi, M., Saba, V., Zanetti, M., Scalise, G., 2006. LL-37 protects rats against lethal sepsis caused by gram-negative bacteria. Antimicrobial agents and chemotherapy 50, 1672-1679. Cirz, R.T., Chin, J.K., Andes, D.R., de Crecy-Lagard, V., Craig, W.A., Romesberg, F.E., 2005. Inhibition of mutation and combating the evolution of antibiotic resistance. PLoS biology 3, e176. Cohen, J., 2002. The immunopathogenesis of sepsis. Nature 420, 885-891. Cotter, P.D., Hill, C., Ross, R.P., 2005. Bacteriocins: developing innate immunity for food. Nature reviews. Microbiology 3, 777-788. Dagan, R., Klugman, K.P., 2008. Impact of conjugate pneumococcal vaccines on antibiotic resistance. The Lancet. Infectious diseases 8, 785-795. Davies, J., Davies, D., 2010. Origins and evolution of antibiotic resistance. Microbiology and molecular biology reviews : MMBR 74, 417-433. Dosler, S., Gerceker, A.A., 2011. In vitro activities of nisin alone or in combination with vancomycin and ciprofloxacin against methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains. Chemotherapy 57, 511-516. Dosler, S., Gerceker, A.A., 2012. In vitro activities of antimicrobial cationic peptides; melittin and nisin, alone or in combination with antibiotics against Gram-positive bacteria. Journal of chemotherapy 24, 137-143. Dubos, R.J., 1939. Studies on a Bactericidal Agent Extracted from a Soil Bacillus : I. Preparation of the Agent. Its Activity in Vitro. The Journal of experimental medicine 70, 1-10. Durr, M., Peschel, A., 2002. Chemokines meet defensins: the merging concepts of chemoattractants and antimicrobial peptides in host defense. Infection and immunity 70, 6515-6517. ECDC/EMEA. 2009. ECDC/EMEA Joint Working Group. Joint Technical Report: The bacterial challenge: time to react. [Online]. Stockholm: European Centre for Disease Prevention and Control. Available: https://www.ecdc.europa.eu/en/publications- data/ecdcemea-joint-technical-report-bacterial-challenge-time-react [Accessed 20 August 2017]. Elmogy, M., Bassal, T.T., Yousef, H.A., Dorrah, M.A., Mohamed, A.A., Duvic, B., 2015. Isolation, characterization, kinetics, and enzymatic and nonenzymatic microbicidal activities of a novel c-type lysozyme from plasma of Schistocerca gregaria (Orthoptera: Acrididae). Journal of insect science 15. Chapter 2: Literature overview 86 FDA. 2017a. CFR - Code of Federal Regulations Title 21 [Online]. Available: https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?fr=522.468 [Accessed 28 Septemeber 2017]. FDA. 2017b. Coly-Mycin® M Parenteral (Colistimethate for Injection, USP) [Online]. Available: https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/050108s033lbl.pdf [Accessed 28 September 2017]. Fennell, J.F., Shipman, W.H., Cole, L.J., 1967. Antibacterial action of a bee venom fraction (melittin) against a penicillin-resistant staphylococcus and other microorganisms. USNRDL- TR-67-101. Research and development technical report. United States. Naval Radiological Defense Laboratory, San Francisco, 1-13. Field, D., Begley, M., O'Connor, P.M., Daly, K.M., Hugenholtz, F., Cotter, P.D., Hill, C., Ross, R.P., 2012. Bioengineered nisin A derivatives with enhanced activity against both Gram positive and Gram negative pathogens. PloS one 7, e46884. Fleitas, O., Franco, O.L., 2016. Induced Bacterial Cross-Resistance toward Host Antimicrobial Peptides: A Worrying Phenomenon. Frontiers in microbiology 7, 381. Flemming, A., 1922. On a remarkable bacteriolytic element found in tissues and secretions Proceedings of the Royal Society of London B 93, 306 – 317. Fox, J.L., 2013. Antimicrobial peptides stage a comeback. Nature biotechnology 31, 379- 382. Gallo, R.L., 2013. The birth of innate immunity. Experimental dermatology 22, 517. Garau, J., 2002. Treatment of drug-resistant pneumococcal pneumonia. The Lancet. Infectious diseases 2, 404-415. Gill, S.R., Fouts, D.E., Archer, G.L., Mongodin, E.F., Deboy, R.T., Ravel, J., Paulsen, I.T., Kolonay, J.F., Brinkac, L., Beanan, M., Dodson, R.J., Daugherty, S.C., Madupu, R., Angiuoli, S.V., Durkin, A.S., Haft, D.H., Vamathevan, J., Khouri, H., Utterback, T., Lee, C., Dimitrov, G., Jiang, L., Qin, H., Weidman, J., Tran, K., Kang, K., Hance, I.R., Nelson, K.E., Fraser, C.M., 2005. Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm- producing methicillin-resistant Staphylococcus epidermidis strain. Journal of bacteriology 187, 2426-2438. Goldstein, B.P., Wei, J., Greenberg, K., Novick, R., 1998. Activity of nisin against Streptococcus pneumoniae, in vitro, and in a mouse infection model. The Journal of antimicrobial chemotherapy 42, 277-278. Gordon, Y.J., Romanowski, E.G., McDermott, A.M., 2005. A review of antimicrobial peptides and their therapeutic potential as anti-infective drugs. Current eye research 30, 505-515. Guilhelmelli, F., Vilela, N., Albuquerque, P., Derengowski Lda, S., Silva-Pereira, I., Kyaw, C.M., 2013. Antibiotic development challenges: the various mechanisms of action of antimicrobial peptides and of bacterial resistance. Frontiers in microbiology 4, 353. Gut, I.M., Blanke, S.R., van der Donk, W.A., 2011. Mechanism of inhibition of Bacillus anthracis spore outgrowth by the lantibiotic nisin. ACS chemical biology 6, 744-752. Hancock, R.E., 2015. Rethinking the Antibiotic Discovery Paradigm. EBioMedicine 2, 629- 630. Hancock, R.E., Diamond, G., 2000. The role of cationic antimicrobial peptides in innate host defences. Trends in microbiology 8, 402-410. Hancock, R.E., Lehrer, R., 1998. Cationic peptides: a new source of antibiotics. Trends in biotechnology 16, 82-88. Hancock, R.E., Sahl, H.G., 2006. Antimicrobial and host-defense peptides as new anti- infective therapeutic strategies. Nature biotechnology 24, 1551-1557. Ho, P.L., Cheng, V.C., Chu, C.M., 2009. Antibiotic resistance in community-acquired pneumonia caused by Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus, and Acinetobacter baumannii. Chest 136, 1119-1127. Jago, W., Jago, W., 1926. Toxic action of wheat flour to brewer’s yeast., in: Allen, W.P. (Ed.), Industrial Fermentations. The Chemical Catering Company, New York, pp. 128-167. Jenssen, H., Hamill, P., Hancock, R.E., 2006. Peptide antimicrobial agents. Clinical microbiology reviews 19, 491-511. Chapter 2: Literature overview 87 Jones, E., Salin, V., Williams, G.W. 2005. Nisin and the Market for Commercial Bacteriocins. TAMRC Consumer and Product Research Report No. CP-01-05. [Online]. Available: http://wwww.ageconsearch.umn.edu/bitstream/90779/2/CP%2001%2005%20Nisin%20Repo rt.pdf [Accessed 18 August 2015]. Keen, E.C., 2012. Phage therapy: concept to cure. Frontiers in microbiology 3, 238. Kindrachuk, J., Jenssen, H., Elliott, M., Nijnik, A., Magrangeas-Janot, L., Pasupuleti, M., Thorson, L., Ma, S., Easton, D.M., Bains, M., Finlay, B., Breukink, E.J., Georg-Sahl, H., Hancock, R.E., 2013. Manipulation of innate immunity by a bacterial secreted peptide: lantibiotic nisin Z is selectively immunomodulatory. Innate immunity 19, 315-327. Kong, W., Lu, T., 2014. Cloning and optimization of a nisin biosynthesis pathway for bacteriocin harvest. ACS synthetic biology 3, 439-445. Le Lay, C., Dridi, L., Bergeron, M.G., Ouellette, M., Fliss, I.L., 2016. Nisin is an effective inhibitor of Clostridium difficile vegetative cells and spore germination. Journal of medical microbiology 65, 169-175. Lewies, A., Wentzel, J.F., Jacobs, G., Du Plessis, L.H., 2015. The Potential Use of Natural and Structural Analogues of Antimicrobial Peptides in the Fight against Neglected Tropical Diseases. Molecules 20, 15392-15433. Lewies, A., Wentzel, J.F., Jordaan, A., Bezuidenhout, C., Du Plessis, L.H., 2017. Interactions of the antimicrobial peptide nisin Z with conventional antibiotics and the use of nanostructured lipid carriers to enhance antimicrobial activity. International journal of pharmaceutics 526, 244-253. Li, J., Nation, R.L., Turnidge, J.D., Milne, R.W., Coulthard, K., Rayner, C.R., Paterson, D.L., 2006. Colistin: the re-emerging antibiotic for multidrug-resistant Gram-negative bacterial infections. The Lancet. Infectious diseases 6, 589-601. Li, Y., 2011. Recombinant production of antimicrobial peptides in Escherichia coli: a review. Protein expression and purification 80, 260-267. Lim, J.Y., Hong, J.B., Sheng, H., Shringi, S., Kaul, R., Besser, T.E., Hovde, C.J., 2010. Phenotypic diversity of Escherichia coli O157:H7 strains associated with the plasmid O157. Journal of microbiology 48, 347-357. Lin, L., Nonejuie, P., Munguia, J., Hollands, A., Olson, J., Dam, Q., Kumaraswamy, M., Rivera, H., Jr., Corriden, R., Rohde, M., Hensler, M.E., Burkart, M.D., Pogliano, J., Sakoulas, G., Nizet, V., 2015. Azithromycin Synergizes with Cationic Antimicrobial Peptides to Exert Bactericidal and Therapeutic Activity Against Highly Multidrug-Resistant Gram-Negative Bacterial Pathogens. EBioMedicine 2, 690-698. Liu, Y.Y., Wang, Y., Walsh, T.R., Yi, L.X., Zhang, R., Spencer, J., Doi, Y., Tian, G., Dong, B., Huang, X., Yu, L.F., Gu, D., Ren, H., Chen, X., Lv, L., He, D., Zhou, H., Liang, Z., Liu, J.H., Shen, J., 2016. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. The Lancet. Infectious diseases 16, 161-168. Mangoni, M.L., Epand, R.F., Rosenfeld, Y., Peleg, A., Barra, D., Epand, R.M., Shai, Y., 2008. Lipopolysaccharide, a key molecule involved in the synergism between temporins in inhibiting bacterial growth and in endotoxin neutralization. The Journal of biological chemistry 283, 22907-22917. Marr, A.K., Gooderham, W.J., Hancock, R.E., 2006. Antibacterial peptides for therapeutic use: obstacles and realistic outlook. Current opinion in pharmacology 6, 468-472. Mataraci, E., Dosler, S., 2012. In vitro activities of antibiotics and antimicrobial cationic peptides alone and in combination against methicillin-resistant Staphylococcus aureus biofilms. Antimicrobial agents and chemotherapy 56, 6366-6371. Matsuzaki, K., 2009. Control of cell selectivity of antimicrobial peptides. Biochimica et biophysica acta 1788, 1687-1692. Matsuzaki, S., Rashel, M., Uchiyama, J., Sakurai, S., Ujihara, T., Kuroda, M., Ikeuchi, M., Tani, T., Fujieda, M., Wakiguchi, H., Imai, S., 2005. Bacteriophage therapy: a revitalized therapy against bacterial infectious diseases. Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy 11, 211-219. Chapter 2: Literature overview 88 McAuliffe, O., Ross, R.P., Hill, C., 2001. Lantibiotics: structure, biosynthesis and mode of action. FEMS microbiology reviews 25, 285-308. Midorikawa, K., Ouhara, K., Komatsuzawa, H., Kawai, T., Yamada, S., Fujiwara, T., Yamazaki, K., Sayama, K., Taubman, M.A., Kurihara, H., Hashimoto, K., Sugai, M., 2003. Staphylococcus aureus susceptibility to innate antimicrobial peptides, beta-defensins and CAP18, expressed by human keratinocytes. Infection and immunity 71, 3730-3739. Mookherjee, N., Brown, K.L., Bowdish, D.M., Doria, S., Falsafi, R., Hokamp, K., Roche, F.M., Mu, R., Doho, G.H., Pistolic, J., Powers, J.P., Bryan, J., Brinkman, F.S., Hancock, R.E., 2006. Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37. Journal of immunology 176, 2455-2464. Morens, D.M., Taubenberger, J.K., Fauci, A.S., 2008. Predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness. The Journal of infectious diseases 198, 962-970. Mueller, M., Lindner, B., Dedrick, R., Schromm, A.B., Seydel, U., 2005. Endotoxin: physical requirements for cell activation. Journal of endotoxin research 11, 299-303. Naghmouchi, K., Le Lay, C., Baah, J., Drider, D., 2012. Antibiotic and antimicrobial peptide combinations: synergistic inhibition of Pseudomonas fluorescens and antibiotic-resistant variants. Research in microbiology 163, 101-108. Nataro, J.P., Kaper, J.B., 1998. Diarrheagenic Escherichia coli. Clinical microbiology reviews 11, 142-201. Nuding, S., Frasch, T., Schaller, M., Stange, E.F., Zabel, L.T., 2014. Synergistic effects of antimicrobial peptides and antibiotics against Clostridium difficile. Antimicrobial agents and chemotherapy 58, 5719-5725. Nwokoro, E., Leach, R., Ardal, C., Baraldi, E., Ryan, K., Plahte, J., 2016. An assessment of the future impact of alternative technologies on antibiotics markets. Journal of pharmaceutical policy and practice 9, 34. O’Neill, J. 2016. The review on antimicrobial resistancs. Tackling drug-resistant infections globally: Final report and recommendations. [Online]. Available: https://www.amr- review.org/sites/default/files/160518_Final%20paper_with%20cover.pdf [Accessed 16 January 2017]. Ohnishi, M., Golparian, D., Shimuta, K., Saika, T., Hoshina, S., Iwasaku, K., Nakayama, S., Kitawaki, J., Unemo, M., 2011. Is Neisseria gonorrhoeae initiating a future era of untreatable gonorrhea?: detailed characterization of the first strain with high-level resistance to ceftriaxone. Antimicrobial agents and chemotherapy 55, 3538-3545. Oliveira, M., Bexiga, R., Nunes, S.F., Carneiro, C., Cavaco, L.M., Bernardo, F., Vilela, C.L., 2006. Biofilm-forming ability profiling of Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates. Veterinary microbiology 118, 133-140. Ongey, E.L., Neubauer, P., 2016. Lanthipeptides: chemical synthesis versus in vivo biosynthesis as tools for pharmaceutical production. Microbial cell factories 15, 97. Ottosson, H., Nylen, F., Sarker, P., Miraglia, E., Bergman, P., Gudmundsson, G.H., Raqib, R., Agerberth, B., Stromberg, R., 2016. Potent Inducers of Endogenous Antimicrobial Peptides for Host Directed Therapy of Infections. Scientific reports 6, 36692. Pag, U., Sahl, H.G., 2002. Multiple activities in lantibiotics--models for the design of novel antibiotics? Current pharmaceutical design 8, 815-833. Papagrigorakis, M.J., Yapijakis, C., Synodinos, P.N., Baziotopoulou-Valavani, E., 2006. DNA examination of ancient dental pulp incriminates typhoid fever as a probable cause of the Plague of Athens. International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases 10, 206-214. Parry, C.M., Hien, T.T., Dougan, G., White, N.J., Farrar, J.J., 2002. Typhoid fever. The New England journal of medicine 347, 1770-1782. Perron, G.G., Zasloff, M., Bell, G., 2006. Experimental evolution of resistance to an antimicrobial peptide. Proc Biol Sci 273, 251-256. Peschel, A., Sahl, H.G., 2006. The co-evolution of host cationic antimicrobial peptides and microbial resistance. Nature reviews. Microbiology 4, 529-536. Chapter 2: Literature overview 89 PhRMA, 2013. Medicines in development - vaccines. A report on the prevention and treatment of disease through vaccines. Pharmaceutical Research and Manufacturers of America, Washington DC. Pieterse, R., Todorov, S.D., 2010. Bacteriocins - exploring alternatives to antibiotics in mastitis treatment. Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] 41, 542-562. Prins, J.M., Kuijper, E.J., Mevissen, M.L., Speelman, P., van Deventer, S.J., 1995. Release of tumor necrosis factor alpha and interleukin 6 during antibiotic killing of Escherichia coli in whole blood: influence of antibiotic class, antibiotic concentration, and presence of septic serum. Infection and immunity 63, 2236-2242. Radek, K., Gallo, R., 2007. Antimicrobial peptides: natural effectors of the innate immune system. Seminars in immunopathology 29, 27-43. Read, A.F., Woods, R.J., 2014. Antibiotic resistance management. Evolution, medicine, and public health 2014, 147. Reuters. 2016. Studies find 'super bacteria' in Rio's Olympic venues, top beaches [Online]. Available: http://www.reuters.com/article/us-olympics-rio-superbacteria-exclusive- idUSKCN0YW2E8 [Accessed 23 August 2017]. Rishi, P., Preet, S., Bharrhan, S., Verma, I., 2011. In vitro and in vivo synergistic effects of cryptdin 2 and ampicillin against Salmonella. Antimicrobial agents and chemotherapy 55, 4176-4182. Rishi, P., Preet Singh, A., Garg, N., Rishi, M., 2014. Evaluation of nisin-beta-lactam antibiotics against clinical strains of Salmonella enterica serovar Typhi. The Journal of antibiotics 67, 807-811. Rosenfeld, Y., Papo, N., Shai, Y., 2006. Endotoxin (lipopolysaccharide) neutralization by innate immunity host-defense peptides. Peptide properties and plausible modes of action. The Journal of biological chemistry 281, 1636-1643. Schweizer, F., 2009. Cationic amphiphilic peptides with cancer-selective toxicity. European journal of pharmacology 625, 190-194. Shin, J.M., Ateia, I., Paulus, J.R., Liu, H., Fenno, J.C., Rickard, A.H., Kapila, Y.L., 2015. Antimicrobial nisin acts against saliva derived multi-species biofilms without cytotoxicity to human oral cells. Frontiers in microbiology 6. Shin, J.M., Gwak, J.W., Kamarajan, P., Fenno, J.C., Rickard, A.H., Kapila, Y.L., 2016. Biomedical applications of nisin. Journal of applied microbiology 120, 1449-1465. Silva, O.N., de la Fuente-Nunez, C., Haney, E.F., Fensterseifer, I.C., Ribeiro, S.M., Porto, W.F., Brown, P., Faria-Junior, C., Rezende, T.M., Moreno, S.E., Lu, T.K., Hancock, R.E., Franco, O.L., 2016. An anti-infective synthetic peptide with dual antimicrobial and immunomodulatory activities. Scientific reports 6, 35465. Singh, A.P., Preet, S., Rishi, P., 2014. Nisin/beta-lactam adjunct therapy against Salmonella enterica serovar Typhimurium: a mechanistic approach. The Journal of antimicrobial chemotherapy 69, 1877-1887. Smith, L., Hillman, J., 2008. Therapeutic potential of type A (I) lantibiotics, a group of cationic peptide antibiotics. Current opinion in microbiology 11, 401-408. Steenbergen, J.N., Alder, J., Thorne, G.M., Tally, F.P., 2005. Daptomycin: a lipopeptide antibiotic for the treatment of serious Gram-positive infections. The Journal of antimicrobial chemotherapy 55, 283-288. Takahashi, D., Shukla, S.K., Prakash, O., Zhang, G., 2010. Structural determinants of host defense peptides for antimicrobial activity and target cell selectivity. Biochimie 92, 1236- 1241. Tjabringa, G.S., Aarbiou, J., Ninaber, D.K., Drijfhout, J.W., Sorensen, O.E., Borregaard, N., Rabe, K.F., Hiemstra, P.S., 2003. The antimicrobial peptide LL-37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor. Journal of immunology 171, 6690-6696. Tran, T.T., Munita, J.M., Arias, C.A., 2015. Mechanisms of drug resistance: daptomycin resistance. Annals of the New York Academy of Sciences 1354, 32-53. Chapter 2: Literature overview 90 Unemo, M., Shafer, W.M., 2014. Antimicrobial resistance in Neisseria gonorrhoeae in the 21st century: past, evolution, and future. Clinical microbiology reviews 27, 587-613. Van Bambeke, F., Glupczynski, Y., Plesiat, P., Pechere, J.C., Tulkens, P.M., 2003. Antibiotic efflux pumps in prokaryotic cells: occurrence, impact on resistance and strategies for the future of antimicrobial therapy. The Journal of antimicrobial chemotherapy 51, 1055-1065. Van Epps, H.L., 2006. René Dubos: unearthing antibiotics. The Journal of experimental medicine 203, 259-259. Vocadlo, D.J., Davies, G.J., Laine, R., Withers, S.G., 2001. Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. Nature 412, 835-838. Wang, G., Li, X., Wang, Z., 2016. APD3: the antimicrobial peptide database as a tool for research and education. Nucleic acids research 44, D1087-1093. Wang, G., Li, X., Wang, Z. 2017. The Antimicrobial Peptide Database [Online]. Available: http://wwww.aps.unmc.edu/AP/main.php [Accessed 20 March 2017]. WHO. 2017a. Antibiotic-resistant gonorrhoea on the rise, new drugs needed [Online]. Available: http://www.who.int/mediacentre/news/releases/2017/Antibiotic-resistant- gonorrhoea/en/ [Accessed 12 October 2017]. WHO. 2017b. Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics [Online]. Available: http://www.who.int/medicines/publications/global-priority-list-antibiotic-resistant-bacteria/en/ [Accessed 12 October 2017]. Willers, C., Wentzel, J.F., du Plessis, L.H., Gouws, C., Hamman, J.H., 2017. Efflux as a mechanism of antimicrobial drug resistance in clinical relevant microorganisms: the role of efflux inhibitors. Expert Opin Ther Tar 21, 23-36. Wong, V.K., Baker, S., Pickard, D.J., Parkhill, J., Page, A.J., Feasey, N.A., Kingsley, R.A., Thomson, N.R., Keane, J.A., Weill, F.X., Edwards, D.J., Hawkey, J., Harris, S.R., Mather, A.E., Cain, A.K., Hadfield, J., Hart, P.J., Thieu, N.T., Klemm, E.J., Glinos, D.A., Breiman, R.F., Watson, C.H., Kariuki, S., Gordon, M.A., Heyderman, R.S., Okoro, C., Jacobs, J., Lunguya, O., Edmunds, W.J., Msefula, C., Chabalgoity, J.A., Kama, M., Jenkins, K., Dutta, S., Marks, F., Campos, J., Thompson, C., Obaro, S., MacLennan, C.A., Dolecek, C., Keddy, K.H., Smith, A.M., Parry, C.M., Karkey, A., Mulholland, E.K., Campbell, J.I., Dongol, S., Basnyat, B., Dufour, M., Bandaranayake, D., Naseri, T.T., Singh, S.P., Hatta, M., Newton, P., Onsare, R.S., Isaia, L., Dance, D., Davong, V., Thwaites, G., Wijedoru, L., Crump, J.A., De Pinna, E., Nair, S., Nilles, E.J., Thanh, D.P., Turner, P., Soeng, S., Valcanis, M., Powling, J., Dimovski, K., Hogg, G., Farrar, J., Holt, K.E., Dougan, G., 2015. Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events. Nature genetics 47, 632-639. Wright, G.D., 2016. Antibiotic Adjuvants: Rescuing Antibiotics from Resistance. Trends in microbiology 24, 862-871. Wu, G., Ding, J., Li, H., Li, L., Zhao, R., Shen, Z., Fan, X., Xi, T., 2008. Effects of cations and pH on antimicrobial activity of thanatin and s-thanatin against Escherichia coli ATCC25922 and B. subtilis ATCC 21332. Current microbiology 57, 552-557. Wu, J., Hu, S., Cao, L., 2007. Therapeutic effect of nisin Z on subclinical mastitis in lactating cows. Antimicrobial agents and chemotherapy 51, 3131-3135. Yan, M., Li, X., Liao, Q., Li, F., Zhang, J., Kan, B., 2016. The emergence and outbreak of multidrug-resistant typhoid fever in China. Emerging Microbes & Infections 5, e62. Yeaman, M.R., Yount, N.Y., 2003. Mechanisms of antimicrobial peptide action and resistance. Pharmacological reviews 55, 27-55. Yoneyama, H., Katsumata, R., 2006. Antibiotic resistance in bacteria and its future for novel antibiotic development. Bioscience, biotechnology, and biochemistry 70, 1060-1075. Zhang, Y., Liu, Y., Sun, Y., Liu, Q., Wang, X., Li, Z., Hao, J., 2014. In vitro synergistic activities of antimicrobial peptide brevinin-2CE with five kinds of antibiotics against multidrug- resistant clinical isolates. Current microbiology 68, 685-692. 91 Chapter 3: Interaction of the antimicrobial peptide nisin Z with conventional antibiotics and the use of nanostructured lipid carriers to enhance antibacterial activity (Paper III) Angélique Lewies, Johannes, F. Wentzel, Anine Jordaan, Carlos Bezuidenhout and Lissinda. H. Du Plessis Published in: International Journal of Pharmaceutics (2017), Volume 526, pp 244-253. https://doi.org/10.1016/j.ijpharm.2017.04.071. Graphical abstract of the article: In this article, the interaction of nisin Z with conventional antibiotics was evaluated and compared to the broad spectrum, but toxic antimicrobial peptide melittin in Gram-positive (Staphylococcus aureus, S. epidermidis) and Gram-negative (Escherichia coli) bacteria. Synergistic, additive and antagonistic interactions were evaluated. Synergistic and additive interactions were observed for nisin Z-antibiotic combinations. Although additive interactions were observed for melittin-antibiotic combinations, antagonism was also observed. Furthermore, nisin was not toxic to non-malignant cells even at concentrations above the minimum inhibitory concentrations (MICs) of the respective bacteria whereas melittin was toxic at concentrations below the MICs. The entrapment efficiency of nanostructured lipid carriers (NLCs) for nisin Z was compared to that of solid lipid nanoparticles (SLNs). Chapter 3: Paper III 92 The effectiveness of NLC formulations containing nisin Z towards the respective Gram- positive and negative bacteria (which are associated with skin infections) at physiological pH was also evaluated. NLCs displayed higher entrapment efficiency for nisin Z, and the NLC formulations containing nisin Z displayed activity towards S. aureus and S.epidermidis at physiological pH. The effectiveness of NLCs formulations containing nisin Z could be enhanced through the addition of ethylenediaminetetraacetic acid (EDTA). The validation of the modified BCA method as well as the chloroform extraction method to evaluate peptide entrapment of the lipid nanoparticles is given in Appendix A. Chapter 3: Paper III 93 Chapter 3: Paper III 94 Chapter 3: Paper III 95 Chapter 3: Paper III 96 Chapter 3: Paper III 97 Chapter 3: Paper III 98 Chapter 3: Paper III 99 Chapter 3: Paper III 100 Chapter 3: Paper III 101 Chapter 3: Paper III 102 103 Chapter 4: The antimicrobial peptide nisin Z induces selective toxicity and apoptotic cell death in cultured melanoma cells (Paper IV) Angélique Lewies, Johannes. F. Wentzel, Hayley C. Van Dyk and Lissinda H. Du Plessis. Published in: Biochimie (2018), Volume 144, pp 28-40 https://doi.org/10.1016/j.biochi.2017.10.009 Graphical abstract of the manuscript: Cytotoxicity results obtained from chapter 3 indicated that nisin Z was selective towards bacterial cells as no toxicity was observed in non-malignant cells even at concentrations above the minimum inhibitory concentrations (MICs) for the respective bacteria. In a previous study, it was found that following the exposure of head and neck squamous cell carcinoma (HNSCC) cancer cells to nisin the expression of multiple genes was altered, which included those in apoptotic and cell cycle pathways as well as energy and nutrient pathways (Joo et al., 2012). Another study found that nisin Z induced apoptosis and decreased proliferation in HNSCC cancer cells (Kamarajan et al., 2015). In this manuscript, it was evaluated whether nisin Z displays selective toxicity to human melanoma (A375) cells compared to non-malignant keratinocyte cells. . Chapter 4: Paper IV 104 The underlying mechanism of action of nisin Z in melanoma cells was also investigated with regards to the ability of nisin Z to induce apoptosis and reactive oxygen species (ROS) production, disrupt the energy metabolism (glycolysis and mitochondrial respiration) and inhibit cell proliferation and invasion of melanoma cells. This manuscript concludes that nisin Z not only induces selective toxicity to cancer cells, but also affects the bioenergetics (glycolysis and mitochondrial respiration), leads to the production of ROS and depolarisation of the mitochondrial membrane, and apoptosis in melanoma cells in vitro. Nisin Z was able to prevent the invasion and proliferation of melanoma cells as well. References The references used in this section are included in the final reference list at the end of this thesis Chapter 4: Paper IV 105 Chapter 4: Paper IV 106 Chapter 4: Paper IV 107 Chapter 4: Paper IV 108 Chapter 4: Paper IV 109 Chapter 4: Paper IV 110 Chapter 4: Paper IV 111 Chapter 4: Paper IV 112 Chapter 4: Paper IV 113 Chapter 4: Paper IV 114 Chapter 4: Paper IV 115 Chapter 4: Paper IV 116 Chapter 4: Paper IV 117 118 Chapter 5: The potential of nisin Z to increase the cytotoxicity and selectivity of conventional chemotherapeutic agents 5.1. Introduction Due to the toxicity associated with some conventional chemotherapeutic agents, as well as the development of chemotherapy resistance (Soengas and Lowe, 2003, Luqmani, 2005, Wellbrock, 2014), there is a need for the development of novel anticancer therapies. Furthermore, in the interest of overcoming chemotherapy resistance, the efficacy of chemotherapeutic agents can be enhanced by the co-administration of multi-functional agents to achieve synergistic interactions (Sylvester et al., 2011, Wei et al., 2013). Some AMPs, and especially bacteriocins, display selectivity towards cancer cells (Kaur and Kaur, 2015) and may be considered for its development as novel anticancer agents or to strengthen our current arsenal of chemotherapeutic agents by using it as an adjuvant. The bacteriocin nisin, which has been granted Generally Regarded as Safe (GRAS) status, is safe for human consumption. The Accepted Daily Intake (ADI) of nisin as determined by the FDA, prior to receiving GRAS status in 1988, is 2.94 mg/per day (0.049 mg/kg body weight/day) (Müller-Auffermann et al., 2015a). Studies have indicated that the no-observed- effect-level (NOEL) of nisin is considerably higher than anticipated by the FDA. Food Standards Australia New Zealand (FSANZ) indicated an ADI of 37.5 mg/per day (0.625 mg/kg body weight/day) (FSANZ, 2007), whereas DIMS Institute of Medical Science, Inc. concluded that nisin has an ADI of 134.83 mg/day (2.247. mg/kg body weight/day) (FAO and WHO, 2010). It is reported that different ADIs are proposed by the different studies as a result of the maximum dose used in each study, followed by the application of a 100-fold safety factor and not due to observed toxicity (FAO and WHO, 2010). In a study by Joo and co-workers, mice treated with nisin (low content nisin A variant) at a concentration of 150 mg/kg body weight/day (more than a 1000x higher than the FDA recommended ADI) for 3 weeks did not display signs of toxicity (Joo et al., 2012). In another study by Kamarajan and collaborators, mice could be treated with doses of ultra-pure nisin Z of 800 mg/kg body weight/day (more than 10 000x higher than the FDA recommended ADI) for 3 weeks without any signs of toxicity (Kamarajan et al., 2015). In both these studies nisin treatment was able to reduce head and neck squamous-cell carcinoma tumorigenesis in mice, while long-term (> 3 weeks) treatment with 800 mg/kg body weight/day of nisin Z extended the survival of mice. These studies show that the use of nisin is a potential cancer therapy. However, more studies on dosing determinations need to be undertaken. Chapter 5: Chemotherapeutic combinations 119 The metabolism as well as reactive oxygen species (ROS) levels of cancer cells are altered, and provide targets for novel anticancer agents (Liou and Storz, 2010, DeBerardinis and Chandel, 2016). Furthermore, it is suggested that anticancer agents that are able to target and disrupt glycolysis and/or mitochondrial respiration leading to ATP depletion, while simultaneously increasing ROS past the threshold for cancer cell survival and inducing apoptosis in cancer cells, should be investigated with the aim of addressing chemotherapy resistance in cancer (Indran et al., 2011). The anticancer activities (Joo et al., 2012, Kamarajan et al., 2015) as well as immune-modulatory properties (Begde et al., 2011, Kindrachuk et al., 2013, Singh et al., 2014) of nisin have been previously established. In the current study it was shown that nisin Z not only induces selective toxicity to cancer cells, but also affects the bioenergetics (glycolysis and mitochondrial respiration), leads to the production of ROS and depolarisation of the mitochondrial membrane, and apoptosis in melanoma cells in vitro as discussed in paper IV (Chapter 4) (Lewies et al., 2018). Results from the current study indicated that nisin Z was able to prevent the proliferation of melanoma cells similarly to what was reported previously for HNSCC cells (Kamarajan et al., 2015), and was also able to inhibit invasion of melanoma cells. An abundance of studies have investigated the use of nisin as adjuvant for conventional antibiotics (Giacometti et al., 2000, Dosler and Gerceker, 2012, Mataraci and Dosler, 2012, Naghmouchi et al., 2012, Rishi et al., 2014). In the current study it was additionally shown that nisin Z variant holds the potential of being used as adjuvant with novobiocin to obtain synergism for the treatment of Staphylococcus aureus and S. epidermidis infections (paper III, Chapter 3) (Lewies et al., 2017). However, few studies have focused on using nisin as an adjuvant for conventional chemotherapeutic agents. The ability of nisin to increase the activity of the chemotherapeutic drug doxorubicin was investigated in vivo by Preet and co- workers. Nisin, when used in combination with doxorubicin, enhanced the anticancer activities of doxorubicin. Apoptosis could be detected upon treatment of mice with induced skin carcinogenesis; there were also slight increases in the oxidative stress response to the adjuvant therapy. However, the exact mechanism by which nisin exerts its anticancer activities was not determined (Preet et al., 2015). In view of the observed effect of nisin Z on melanoma cells, the toxicity of nisin Z- chemotherapeutic agent combinations (Table 5.1) to melanoma cells was compared with the mono-treatment with these conventional chemotherapeutic agents. To assess the toxicity of nisin Z-chemotherapeutic combinations to non-malignant cells, non-tumorigenic human keratinocyte (HaCat) cells were included in toxicity assays. Chapter 5: Chemotherapeutic combinations 120 Table 5.1: Mechanism of action of selected chemotherapeutic agents Chemotherapeutic agent Biochemical activity Biological effects References Antimetabolites 5-Fluoruracil Pyrimidine nucleoside analogue Inhibition of thymidylate synthase Inhibition of RNA and DNA synthesis DNA damage (Longley et al., 2003, Bracci et al., 2014) Hydroxy urea Ribonucleotide reductase inhibitor Induce DNA damage and inhibits DNA synthesis (Tiwari, 2012) Methotrexate Folic acid antagonist Potent inhibitor of dihydrofolate reductase Inhibition of DNA and purine biosynthesis (Bertino et al., 1996, Tian and Cronstein, 2007) Toposiomerase inhibitors Etoposide Inducing DNA strand breaks by interfering with type II topoisomerase Cell cycle arrest and induction of apoptosis (Bracci et al., 2014) Imatinib Tyrosine kinase inhibitor Also shown to interfere with type I and II topiosemarese, inducing DNA strand breaks Inhibition of cell proliferation/cell cycle arrest and induces apoptosis (Baran et al., 2011, Iqbal and Iqbal, 2014) 5.2. Materials and methods Melanoma (A375) cells were purchased from the American Type Culture collection (ATCC® CRL-1619TM); the non-malignant human immortalised keratinocyte (HaCaT) cells were donated by the School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, South Africa. All reagents were of analytical grade and were purchased from Sigma-Aldrich Chemie GmbH (Schelldorf, Germany) unless stated otherwise. Nisin Z®+ Ultrapure Nisin Z (≥ 95% HPLC) was a kind donation from Handary (Brussels, Belgium). 5.2.1. Cell culturing conditions Cells were cultured under standard conditions in Dulbecco’s modified essential medium (DMEM; Hyclone, GE healthcare, South Logan, UT, USA) containing 10% foetal bovine Chapter 5: Chemotherapeutic combinations 121 serum (FBS), 1% penicillin/streptomycin, 2 mM L-Glutamine and 1% non-essential amino acids (Lonza, Basel, Switzerland). Cells were cultured at 37 °C in a humidified atmosphere of 5% CO2. For combination assays, cells were seeded at a density of 25 000 cells per well in 96 well plates. A 6 mM stock solution of Nisin Z in 0.01 N HCl (Associated Chemical Enterprises (PTY) LTD, Johannesburg, South Africa) was freshly prepared prior to each experiment. Stock solutions of all chemotherapeutic agents were prepared in dimethyl sulfoxide (DMSO). All experiments were performed in serum-free DMEM. 5.2.2. MTT (3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide) assay The effect of combinations of nisin Z with conventional chemotherapeutic agents (5- fluoruracil, metotrexate, etoposide, imatinib, hydroxy urea) on HaCat and A375 cells were determined by means of the 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay. For these assays, HaCat and A375 cells were seeded in 96-well plates (NEST, China) and incubated until cells were ~90% confluent. Cells were then exposed to increasing concentrations of the respective chemotherapeutic agents alone and combined with 150 µM nisin Z for 24 hours. Vehicle control groups were included, such that 0 µM control sample contains the same volume of DMSO or DMSO and HCl as the highest concentration of substance tested. Following exposure, growth medium was removed, cells carefully rinsed with 1 x phosphate buffered saline (PBS) and 100 µL fresh serum-free medium containing 0.5 mg/ml MTT solution added. Cells were subsequently incubated for 2 hours at 37 °C, after which the MTT was removed and replaced with 100 µL dimethyl sulfoxide (DMSO). After 1 hour of incubation at 37 °C, cell viability was determined using a microplate reader (SpectraMax® ParadigmTM Molecular Devices, Sunnyvale, CA, USA). Absorbance was measured at a wavelength of 560 nm and background at a wavelength of 630 nm with DMSO measured as a blank. Blank and background measurements were subtracted and cell viability was expressed as a percentage relative to the untreated control, which was set as 100 % viable. 5.2.3. Evaluating the synergistic interactions between nisin Z and the chemotherapeutic agents The coefficient of drug interaction (CDI) was used to evaluate synergistic interactions between the combinations of nisin Z and the chemotherapeutic agents (Chen et al., 2014, Li et al., 2016). The CDI was calculated using the following equation: 𝐶𝐷𝐼 = 𝐴𝐵 (𝐴+𝐵) Chapter 5: Chemotherapeutic combinations 122 Where, AB = relative cell viability of the combination, and A or B = relative cell viability of the single agent groups. Results are interpreted as CDI < 1 synergism, CDI = 1 additive effect and CDI > 1 antagonism (Li et al., 2016). 5.2.4. Data analysis Graphpad PrismTM version 5 (GraphPad Software Inc., Dan Diego, CA, USA) was used to analyse data. Statistical significance was determined with one-way analysis of variance (ANOVA) with Dunn’s multiple comparison tests for non-parametric data or the unpaired Student’s t-test where applicable. Data is presented as mean ± standard deviation (stdev) with significance set at p ≤ 0.05. 5.3. Results and discussion Previous results from this study have indicated that when cells were exposed to nisin Z selective toxicity to melanoma cells could be observed as opposed to non-malignant keratinocyte cells (paper IV, Chapter 4). The toxicity results of cells exposed to 150 µM nisin Z are summarised in Table 5.2. These results indicate that nisin Z treatment at 150 µM induced toxicity in melanoma cells. Membrane damage was also observed as detected with the LDH release assay. Table 5.2: Toxicity results for cells exposed to 150 µM nisin Z. ***p < 0.001 compared to controls (adapted from Lewies et al., 2018) Measurement Non-malignant keratinocyte (HaCat) cells Melanoma (A375) cells Percentage cell viability (MTT) 102.7±7.99 64.41±3.98*** Percentage LDH release 5.82±1.16 59.38±7.94*** Percentage apoptosis 10.08±3.16 39.93±2.67*** IC50 value 439 µM (±8.3) 188.5 µM (±8.7) Although the therapeutic window for nisin Z treatment is low (2.33), when comparing the IC50 value of the non-malignant keratinocytes to that of the melanoma cells (Deepa et al., 2012), the apoptosis results as well as the results from the MTT and LDH release assays support Chapter 5: Chemotherapeutic combinations 123 the notion that nisin Z displays selectivity towards cancerous cells. Additionally, it should be borne in mind that the solubility of nisin Z at neutral pH (DMEM pH 7.4) is reported to be ± 600 µg/ml (De et al., 2003). A previous study by Kamarajan and co-workers used nisin Z concentrations of 800 µg/ml but did not report any precipitation (Kamarajan et al., 2015). In their study a nisin Z stock solution was made in water and diluted into culture medium to achieve final concentrations of ≤ 800 µg/ml nisin Z. In the current study it was, however, found that nisin Z was poorly soluble in water and precipitation was observed. Consequently a 6 mM nisin Z stock solution was prepared in 0.01 N HCl and subsequently diluted to the desired concentrations in DMEM. Initially no precipitation was observed. However, after the treated culture plates were placed in the incubator at 37ºC, 5% CO2 and 95% humidity for several hours, precipitation occurred at nisin Z concentrations ≥ 250 µM (875 µg/ml). This could be accounted for by the fact that the CO2 caused slight increases in the pH of the DMEM. At nisin Z concentrations ≥ 250 µM cell death was observed in the non-malignant keratinocyte cells. Although the MTT data was used to calculate the IC50 values for both cell lines, the cell death could have occurred due to precipitation of nisin Z and not necessarily due to the toxic effect of nisin Z in non-malignant cells. Consequently, in all subsequent experiments nisin Z was used at concentrations ≤ 200 µM (700 µg/ml), and precipitation was not observed. The effect of nisin exposure on ROS production, mitochondrial membrane potential and bioenergetics (glycolysis and mitochondrial respiration) in melanoma cells was previously measured as part of this study. The results for cells exposed to 150 µM nisin Z are summarised in Table 5.3. These results and parameters have been discussed in detail in paper IV, Chapter 4 (Lewies et al., 2018). Briefly, the results indicate that nisin Z exposure led to a significant increase in ROS production (p<0.001) and mitochondrial membrane depolarization (p<0.001); and this could be linked to the observed apoptosis in the melanoma cells. Low levels of ROS can lead to apoptosis, whereas high levels of ROS are often associated with necrosis or caspase-independent apoptosis pathways (Hampton and Orrenius, 1997, Creagh and Cotter, 1999). Additionally, the collapse of the membrane potential leads to depolarization as a result of apoptosis or cell cycle arrest (Ly et al., 2003). The energy metabolism of melanoma cells was affected by the exposure to nisin Z as indicated in Table 5.3. Melanoma cells exposed to nisin Z have significantly lower (p<0.001) mitochondrial respiration under basal conditions. Mitochondrial respiration is composed of the oxygen consumption rate dedicated to ATP synthesis and oxygen consumption due to the natural proton leak across the inner mitochondrial membrane. Both these components were significantly decreased (p<0.001). It was shown that the functional capacity of the electron transport chain was decreased as was observed by the significant decreases in the Chapter 5: Chemotherapeutic combinations 124 spare respiratory capacity (p<0.001) and maximal respiratory capacity (p<0.001). Finally, the glycolytic capacity and reserve, limits the response cells can have to an acute increase in energy demand when the mitochondrial respiration is restricted. Nisin Z exposure led to a significant decrease in both the glycolytic response and reserve (p<0.001), indicating that nisin Z seems to inhibit the ability of melanoma cells to effectively utilize their glycolysis metabolism. Table 5.3: Effect of the exposure of melanoma (A375) cells to 150 µM nisin Z on reactive oxygen species generation, mitochondrial membrane potential and bioenergetics compared to unexposed cells ***p < 0.001 compared to control. OCR; oxygen consumption rate, ECAR; extracellular acidification rate (adapted from Lewies et al., 2018) Measurement 0 µM nisin Z 150 µM nisin Z ROS production (fold change vs. control) 1 6.19±0.62*** Percentage depolarization of mitochondrial membrane 21.57±1.41 49.59±2.49*** Mitochondrial respiration (OCR (pmol/min/arbitrary cell number) Basal respiration 1.54±0.15 0.61±0.07*** ATP production 1.21±0.10 0.51±0.06*** Proton leak 0.33±0.06 0.11±0.08*** Spare respiratory capacity 0.91±0.13 0.44±0.10*** Maximal respiration 2.45±0.21 1.05±0.16*** Glycolysis (ECAR (mpH/min/arbitrary cell number) Glycolytic capacity 0.87±0.26 0.49±0.06*** Glycolytic reserve 0.58±0.18 0.23±0.06*** Based on these findings the effect of the combination of 150 µM nisin Z with conventional chemotherapeutic agents with different modes of action (Table 5.1) on the toxicity in melanoma and non-malignant keratinocytes was evaluated. Chapter 5: Chemotherapeutic combinations 125 Figure 5.1: Toxicity of 5-fluoruracil (FU) and 5-FU + nisin Z in A) melanoma (A375) cells and B) non-malignant keratinocytes (HaCat) as determined with the MTT assay. Vehicle control groups were included and are represented by 0 µM. Results are expressed relative to the untreated controls which were set as being 100 percent viable. Bars represent the average and error bars the standard deviation, n = 4. *p < 0.05, **p < 0.01 and *** p < 0.001 for combination compared to chemotherapeutic agent alone. The combination of nisin Z with 5-Fluoruracil (FU) was able to increase the toxicity to melanoma cells over the entire concentration range tested compared to the mono-treatment of 5-FU (p < 0.05) (Figure 5.1 A), with no significant increase in toxicity to non-malignant keratinocytes (Figure 5.1 B). The 5-FU treatment begins to induce toxicity at 50 µM (p< 0.01 compared to the control), whereas the combination of 5-FU and nisin Z only initiates induction of toxicity at 200 µM (p < 0.001 compared to the control) in the non-malignant keratinocytes. Results indicate that the 5-FU-nisin Z combination has a higher level of toxicity at 25 µM, which is comparable to that of 5-FU mono-treatment at 400 µM. The anticancer activity of 5-FU may therefore be enhanced by combined treatment of nisin Z and 5-FU. The combination of nisin Z with hydroxy-urea was able to increase the toxicity to melanoma cells at hydroxy urea concentrations of 25 - 400 µM compared to the mono-treatment of hydroxy urea (p < 0.01) (Figure 5.2 A), with no significant increase in the toxicity to non- malignant keratinocytes (Figure 5.2 B). Results indicate that when used in combination with hydroxy urea, nisin Z holds the potential of increasing the anticancer activity of hydroxy urea. Chapter 5: Chemotherapeutic combinations 126 Figure 5.2: Toxicity of hydroxy urea and hydroxy urea + nisin Z in A) melanoma (A375) cells and B) non-malignant keratinocytes (HaCat) as determined with the MTT assay. Vehicle control groups were included and are represented by 0 µM. Results are expressed relative to the untreated controls which were set as being 100 percent viable. Bars represent the average and error bars the standard deviation, n = 4. *p < 0.05, **p < 0.01 and *** p < 0.001 for combination compared to chemotherapeutic agent alone. The combination of nisin Z with methotrexate had no effect on the activity of methotrexate on melanoma cells (Figure 5.3 A), whereas the nisin Z combination led to a significant decrease in the toxicity of methotrexate at concentrations ≥ 100 µM (p < 0.05 compared to mono-treatment) (Figure 5.3 B). Although, the toxicity of methotrexate could be decreased through combination therapy with nisin Z, the mono-treatment of methotrexate was effective at lower concentrations than the concentrations associated with toxicity. Chapter 5: Chemotherapeutic combinations 127 Figure 5.3: Toxicity of methotrexate and methotrexate + nisin Z in A) melanoma (A375) cells and B) non-malignant keratinocytes (HaCat) as determined with the MTT assay. Vehicle control groups were included and are represented by 0 µM. Results are expressed relative to the untreated controls which were set as being 100 percent viable. Bars represent the average and error bars the standard deviation, n = 4. *p < 0.05, and *** p < 0.001 for combination compared to chemotherapeutic agent alone. When combining etoposide with nisin Z, the activity towards melanoma cells was enhanced compared to mono-treatment across the entire concentration range (p < 0.001) (Figure 5.4 A), with no significant increase in toxicity to non-malignant keratinocytes (Figure 5.4 B). The combination of nisin Z with etoposide depicted a higher level of activity at the lowest concentration tested compared to the highest concentration for mono-treatment (p< 0.001). The anticancer activity of etoposide can therefore be significantly enhanced through the combination with nisin Z. Chapter 5: Chemotherapeutic combinations 128 Figure 5.4: Toxicity of etoposide and etoposide + nisin Z in A) melanoma (A375) cells and B) non-malignant keratinocytes (HaCat) as determined with the MTT assay. Vehicle control groups were included and are represented by 0 µM. Results are expressed relative to the untreated controls which were set as being 100 percent viable. Bars represent the average and error bars the standard deviation, n = 4. *** p < 0.001 for combination compared to chemotherapeutic agent alone. The combination of nisin Z with imatinib was not able to enhance the toxicity to melanoma cells (Figure 5.5 A), although the combination led to a decrease in the toxicity to normal cells at concentrations of 25 -50 µM compared to mono-treatment (p< 0.01) (Figure 5.5 B). However, when combining nisin Z with imatinib this decrease in toxicity was also observed in melanoma cells at 25 µM compared to mono-treatment (p < 0.01). Therefore, it seems that the combination of nisin Z with imatinib does not have the ability to increase the anticancer effectiveness of imatinib. Chapter 5: Chemotherapeutic combinations 129 Figure 5.5: Toxicity of imatinib and imatinib + nisin Z in A) melanoma (A375) cells and B) non- malignant keratinocytes (HaCat) as determined with the MTT assay. Vehicle control groups were included and are represented by 0 µM. Results are expressed relative to the untreated controls which were set as being 100 percent viable. Bars represent the average and error bars the standard deviation, n = 4. **p < 0.01 for combination compared to chemotherapeutic agent alone. The cell viability of melanoma cells following the mono-treatment of the respective chemotherapeutic agents (etoposide, 5-FU and hydroxy urea) at 50 µM was compared with that of the mono-treatment of nisin Z at 150 µM, followed by that of the combination (50 µM chemotherapeutic agent + 150 µM nisin Z). This was done to evaluate whether possible synergistic interactions occurred. Synergism occurs when the combined effects of the different components are greater than their individual effects. The cell viability of melanoma cells was significantly lower for all combinations compared to mono-treatment with the chemotherapeutic agent alone (p< 0.05) (Figure 5.6.). However, the only combination that displayed possible synergism was the combination of nisin Z with etoposide (Figure 5.6. A). Chapter 5: Chemotherapeutic combinations 130 Figure 5.6: Toxicity results for mono-treatment and combinations of chemo-therapeutic agents (50 µM) and nisin Z (150 µM) as determined with the MTT assay. Vehicle control groups were included and are represented by the control groups in the graphs. Results are expressed relative to the untreated controls which were set as being 100 percent viable. Bars represent the average and error bars the standard deviation, n = 4. **p < 0.01 and ***p < 0.001 for combination compared to chemotherapeutic agent alone. #p < 0.05 and ##p < 0.01 for combination compared to nisin Z alone. 5-FU; 5-Fluoruracil. To evaluate if synergism did in fact occur for the combination of nisin Z with etoposide, the CDI was calculated over the entire concentration range. Results presented in Table 5.4 indicated that etoposide-nisin Z combinations displayed synergism over the entire concentration range. Table 5.4: Synergistic interactions between nisin Z and etoposide in melanoma cells. Synergism was calculated with the coefficient of drug interaction (CDI) , where CDI < 1 synergistic effect; CDI = 1 additive effect; CDI > 1 antagonistic effect. Nisin Z (150 µM) Etoposide concentration µM 25 50 100 200 400 CDI Etoposide 0.99 0.93 0.94 0.98 0.98 It is suggested that AMPs which also display anticancer activity be used in combination with conventional chemotherapeutic agents to enhance the effectiveness of these agents, prevent recurrence of cancer following treatment and possibly reduce instances of chemotherapy resistance (Gaspar et al., 2013, Swithenbank and Morgan, 2017). Previous studies have shown that AMPs hold the potential of enhancing the effectiveness of Chapter 5: Chemotherapeutic combinations 131 conventional chemotherapeutic agents. The cytotoxicity of etoposide and cisplatin could be enhanced through its combination with magainin A and magainin G, respectively (Ohsaki et al., 1992). More recently it was shown that the combination of melittin and 5-FU increased the cytotoxic effects against squamous skin cancer cells, while simultaneously reducing the toxicity to normal keratinocytes (Do et al., 2014). There are currently no AMPs that have entered into clinical trials or that are in preclinical development as cancer therapeutics. However, peptide-derived therapies are being recognised for the selectiveness and anticancer effectiveness; and have been investigated in clinical trials (Swithenbank and Morgan, 2017). For example the peptide asparagine (N)-glycine (G) arginine (R) tumour homing peptide (NGR-hTNF) has completed phase 1 clinical trials and is waiting to enter phase 2 clinical trials to test its effectiveness when used in combination with cisplatin for the treatment of several refractory solid tumours, including melanomas (Gregorc et al., 2011). The AMP nisin, which has GRAS status and is safe for human consumption, not only displays antibacterial but also anticancer activities. Although the use of nisin as adjuvant for conventional antibiotics has been extensively investigated, studies investigating nisin as adjuvant for conventional chemotherapeutic agents are few. Previously, as part of this study it was shown that nisin (nisin Z variant) not only induces selective toxicity to cancer cells, but also affects the bioenergetics (glycolysis and mitochondrial respiration), leads to the production of ROS and depolarisation of the mitochondrial membrane, and apoptosis in melanoma cells in vitro. These properties make nisin Z a promising anticancer agent to be used alone, or in combination with current chemotherapeutic agents to overcome chemotherapy resistance. In this study it was found that combinations of nisin Z with 5-FU, hydroxy urea and etoposide were able to enhance the cytotoxicity to melanoma cells, while no significant increase in toxicity towards non-malignant keratinocytes was observed. Especially of interest is the effect of nisin Z on the effectiveness of etoposide as etoposide resistance occurs in melanoma (Helmbach et al., 2002, Kalal et al., 2017). The combination of nisin Z with etoposide was able to significantly and selectively enhance the etoposide cytotoxic effect to melanoma cells. Synergism was furthermore observed when combining nisin Z and etoposide with regard to the toxic effect in melanoma cells. Based on these results and GRAS status of nisin Z it could therefore be considered to be used as an adjuvant for conventional chemotherapeutic agents. 5.4. Conclusion This study indicates that when used in combination with the conventional chemotherapeutic agents 5-FU, hydroxy urea and etoposide, nisin Z holds the potential of enhancing the effectiveness of these conventional chemotherapeutic agents. Synergism was observed Chapter 5: Chemotherapeutic combinations 132 between nisin Z and etoposide in melanoma cells. However, this study was only limited to the in vitro effect in melanoma cells with regards to cytotoxicity as measured with the MTT assay. For future in vitro studies it is suggested that more cancer cell lines be included. The mechanistic interaction between nisin Z and the chemotherapeutic agents should also be investigated. Furthermore, it is suggested that in vivo studies be conducted similar to that conducted by Preet et al. (2015) with the purpose of assessing whether the combination of nisin Z with these conventional chemotherapeutic agents are able to reduce melanoma tumorigenesis in vivo. The effective dosages also need to be determined with in vivo assays. 5.5. References The references used in this section are included in the final reference list at the end of this thesis. 133 Chapter 6: Summary, conclusion and future prospects 6.1. Exploring the multi-functionality of antimicrobial peptides as novel therapeutics An increase in antibiotic resistance in bacteria poses a threat to not only the successful treatment of bacterial infections but also cancer, due to antibiotic-resistant bacterial infections associated with cancer treatment (Gudiol and Carratala, 2014). Therefore, novel research is directed at finding alternatives to current antibiotics and/or strengthening our current arsenal of antibiotics. Likewise, the emergening resistance to conventional chemotherapy treatments associated with some cancers warrants research into novel anticancer agents and/or means of strengthening our current arsenal of chemotherapy treatments (Luqmani, 2005). Antimicrobial peptides (AMPs) are multifunctional as was highlighted in paper I in chapter 2 (Lewies et al., 2015), which describes potential use of AMPs for the treatment of neglected tropical diseases associated with bacterial infections as well as protozoa, parasites, helminths and viruses. Certain AMPs not only display activity towards Gram-positive and negative bacteria, but also have anticancer activities (Schweizer, 2009). This multi-functionality of AMPs makes them attractive candidates to be studied as alternatives to current antibiotics and/or cancer treatments, while the immune-modulatory properties, as well as the direct killing activity of cancer and bacterial cells, make them promising adjuvants for conventional antibiotics as well as chemotherapy agents (Do et al., 2014, Preet et al., 2015, Wright, 2016). The use of AMPs as adjuvants to current antibiotics with the aim of obtaining synergistic interactions and potentially reducing instances of antibiotic resistance is discussed in paper II in chapter 2. Nisin is one of the few AMPs that have received GRAS status and that is deemed safe for human consumption (Müller-Auffermann et al., 2015a). Currently, the antibacterial properties of nisin against Gram-positive bacteria are being utilised as a WHO and FDA approved food preservative (Cotter et al., 2005, Jones et al., 2005). Nisin is however not used/approved in medical therapies/drugs for humans. To date the in vitro efficacy profile of high-content nisin Z preparations against human skin cancer has not been investigated. There is also limited data on the in vitro efficacy of high-content nisin Z preparations against bacteria which are associated with skin infections. The aim of the current study was to investigate the antibacterial and anticancer properties of the antimicrobial peptide nisin Z in vitro. Chapter 6: Conclusion 134 6.2. The antibacterial activity of nisin Z (Chapter 3, paper III) The first hypothesis evaluated in this study was whether nisin Z could be used as an adjuvant with conventional antibiotics against Gram-positive and negative bacteria to achieve synergistic interactions and whether the antimicrobial activity of nisin Z towards Gram-negative bacteria could be enhanced using the chelating agent EDTA and/or through the entrapment in NLCs. This hypothesis was evaluated through the following set objectives:  Evaluating the potential of nisin Z to be used as an adjuvant for conventional antibiotics in Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and Gram-negative (Escherichia coli) bacteria.  Evaluating the potential of NLCs to enhance the entrapment efficiency of nisin Z compared to that of SLNs.  Evaluating the potential of EDTA and NLCs to enhance the antibacterial efficacy of nisin Z towards both Gram-positive and negative bacterial species. The potential of nisin Z to be used as an adjuvant for conventional antibiotics in Gram- positive (Staphylococcus aureus and S. epidermidis) as well as Gram-negative (Escherichia coli) bacteria was evaluated. Melittin (a broad spectrum potent AMP) was used as positive control. Compared to melittin, results indicated that nisin Z holds greater potential of being used as an adjuvant in combination studies with conventional antibiotics. Synergistic as well as additive interactions occurred between nisin Z and the conventional antibiotics. Melittin, on the other hand, displayed antagonistic interactions in addition to having additive interactions. Nisin Z furthermore displayed no cytotoxicity to non-malignant human keratinocytes (HaCat cells) even at concentrations above the respective minimum inhibitory concentrations (MICs) for each bacterial species tested, whereas melittin was cytotoxic at concentrations below the respective MICs. The most notable relations were the interaction between nisin Z and novobiocin against both Gram-positive bacterial species. Novobiocin is used for the treatment of mastitis in lactating cows (Brunton et al., 2012) and some nisin- based products have also been developed for the treatment of mastitis (Cotter et al., 2005, Pieterse and Todorov, 2010). The bacterial species used in this study are associated with mastitis. The synergistic interactions between nisin Z and novobiocin make this combination of special interest for developing novel formulations for the treatment of mastitis. Two major obstacles for the therapeutic use of nisin as an antibacterial agent are its limited activity against Gram-negative bacteria and its low solubility and low stability at physiological Chapter 6: Conclusion 135 pH. The activity of nisin was previously shown to be enhanced by the entrapment of nisin into SLNs (Prombutara et al., 2012). Also, the activity of nisin towards Gram-negative E.coli can be improved through the addition of EDTA at concentrations of 100 µM (Helander and Mattila-Sandholm, 2000). In the current study, the use of 200 µM disodium EDTA was shown to significantly enhance the antibacterial activity of nisin Z towards E.coli, while not leading to an increase in the cytotoxicity to non-malignant cells. The potential of NLCs (which are considered second generation SLNs) were investigated with regard to the entrapment efficiency compared to SLNs and the ability to enhance the activity towards Gram-positive and negative bacteria. The NLCs proved to be superior to SLNs for the entrapment of nisin Z, with a higher percentage entrapment. Furthermore, NLC formulations containing nisin Z showed promise for the use against both Gram-positive species at physiological pH. NLCs hold great potential for dermal application (Müller et al., 2016) and S. epidermidis as well as S. aureus are commonly found on the skin surface. Activity against Gram-negative bacteria was lacking. The combination of nisin Z-loaded NLCs with disodium EDTA displayed to further enhanced activity towards Gram-positive bacteria. Although the combination of nisin Z-loaded NLCs with disodium EDTA exhibited some activity against Gram-negative bacteria at the tested concentrations, it was not adequate and was lower than that of nisin Z in the presence (and even absence) of EDTA. Based on these findings, the first hypothesis could only be partially accepted. While it was found to be true that nisin Z could serve as adjuvant for conventional antibiotics to achieve synergistic interactions with these antibiotics against Gram-positive bacteria as was found for the nisin Z-novobiocin combination, more additive interactions were found in both Gram- positive and negative bacteria. Furthermore, although NLCs did display higher entrapment efficiency for nisin Z than SLNs; and it was possible to enhance the activity of nisin Z towards Gram-negative E. coli through nisin Z-EDTA combinations, NLCs and NLC-EDTA combinations were only effective against Gram-positive bacteria. This was the first study to show synergism between novobiocin and nisin Z in S. aureus and S. epidermidis. It was also the first study to evaluate the potential of NLCs to be used with nisin Z. 6.3. The anticancer activities of nisin Z (Chapter 4, paper IV and Chapter 5) The two hypotheses evaluated in this part of the study were that; i. Nisin Z holds the potential of not only inducing apoptosis and of preventing cell proliferation in cancer cells, but also of affecting the bioenergetics (glycolysis and mitochondrial respiration) and leading to an increase in ROS production which is associated with apoptosis. Chapter 6: Conclusion 136 ii. Nisin Z holds the potential to be used as adjuvant with conventional chemotherapy agents to achieve synergistic interactions. These hypotheses were evaluated through the following set objectives:  Evaluating the mechanism associated with the anticancer properties of nisin Z in melanoma regarding its effect on the mode of cell death (apoptosis vs necrosis), bioenergetics, ROS production, cell proliferation and its potential to prevent metastasis.  Evaluating the potential of nisin Z to enhance the cytotoxicity and selectivity of conventional chemotherapeutic agents, and to produce synergistic interactions with conventional chemotherapeutic agents. Nisin has been shown to display anticancer activities through induction of apoptosis and inhibition of cell proliferation (Joo et al., 2012, Kamarajan et al., 2015). Similarly, nisin has been shown to affect the expression of genes involved in energy and nutrient pathways in cancer cells (Joo et al., 2012). Both the altered energy metabolism and elevated ROS levels in cancer cells present targets for novel anticancer therapies which are selectively toxic to cancer cells (Trachootham et al., 2009, DeBerardinis and Chandel, 2016). In the current study, nisin Z was shown to be more selectively toxic to cultured melanoma (A375) cells, compared to non-malignant keratinocyte (HaCat) cells. It was found that nisin Z not only induces apoptosis and inhibits cell proliferation in cancer cells, but it is also able to increase the ROS production, depolarize the mitochondrial membrane potential, alter the bioenergetics (glycolysis and mitochondrial respiration) and inhibit the invasion (metastatic potential) of melanoma cells. The potential of nisin Z to enhance the cytotoxicity and selectivity of conventional chemotherapeutic agents and to produce synergistic interactions with conventional chemotherapeutic agents was also investigated. This study indicated that when used in combination with the conventional chemotherapeutic agents (5-Fluorouracil, hydroxy urea and etoposide), nisin Z holds the potential of enhancing the cytotoxicity of these conventional chemotherapeutic agents against cultured melanoma cells, without displaying toxicity to non-malignant cells. The combination of nisin Z with etoposide was able to significantly and selectively enhance the cytotoxic effect etoposide to melanoma cells. Synergism was observed when combining nisin Z and etoposide with regard to the toxic effect in melanoma cells. This interaction is of special interest and warrants further Chapter 6: Conclusion 137 investigation, as etoposide resistance has been on the rise in melanoma (Helmbach et al., 2002, Kalal et al., 2017). Based on these findings both the hypotheses could be accepted. All of the objectives were achieved. This was the first study to show that nisin Z, in addition to being able to induce apoptosis and prevent cell proliferation of cancer cells, also had the ability to affect the bioenergetics, increase ROS production and prevent metastasis of cancer cells. It was the first study to show that nisin Z is able to enhance cytotoxicity and selectivity of conventional chemotherapeutic agents, when used at concentrations that affected bioenergetics and increased ROS production and apoptosis. Synergism was achieved between nisin Z and etoposide, when combining etoposide with this concentration of nisin Z. 6.4. Important conclusions that were drawn from this study The fourth and final hypothesis for the current study was that nisin Z is a multi-functional peptide which does not display toxicity to non-malignant (“healthy”) cells that; can be considered an antibacterial peptide due to its activity against Gram-positive bacteria and as an anticancer peptide due to its activity towards cancer cells. This hypothesis could be evaluated by comparing the MICs of nisin Z for Gram-positive and negative bacteria with the IC50 values for A375 and HaCat cells, respectively (Chapter 3, paper III and Chapter 4, paper IV). The MIC and IC50 values were used as a measure of toxicity to bacterial and mammalian cells, respectively. It was proven in this study that nisin Z is selective to Gram-positive bacterial cells compared to non-malignant HaCat cells. The MICs for S. aureus and S. epidermidis were 10 µg/ml and 9.17 µg/ml respectively, whereas the IC50 value for HaCat cells was found to be 1536.50 µg/ml (439 µM). The IC50 value therefore is approximately 153x higher than the MIC values. Nisin Z is also more toxic to E. coli cells in the presence and absence of disodium EDTA. The MIC value of nisin Z for E. coli was 213.33 µg/ml in the absence of disodium EDTA, which is 7.2x lower than the IC50 value for HaCat cells. In the presence of 200 µM disodium EDTA the MIC value of nisin Z for E. coli was 16.67 µg/ml (92x lower than the IC50 value for HaCat cells). Furthermore, the addition of 200 µM disodium EDTA did not enhance the toxicity of nisin Z to the non-malignant cells. Nisin Z was found to be more selective towards cancer cells compared to the non-malignant cells. The IC50 value for melanoma cells was 659.75 µg/ml (188.5 µM), which is 2.3x lower than that of the non-malignant cells. Although this indicates a narrow therapeutic window, Chapter 6: Conclusion 138 nisin Z led to an increase in ROS production, depolarization of the mitochondrial membrane potential, apoptosis and lower bioenergetic capacity, even at the lowest concentration tested (175 µg/ml or 50 µM) without displaying toxicity to non-malignant cells. Furthermore, the IC50 value for melanoma is approximately 65x higher than the MIC values for Gram-positive bacteria and 3x higher than the MIC for Gram-negative bacteria in the absence of disodium EDTA. Therefore, it is possible that nisin Z would be able to simultaneously lead to the death of cancer cells and prevent bacterial infections when used at doses needed to induce cancer cell death. Based on these findings, it can be concluded that nisin Z can be considered an antibacterial peptide due to its activity towards Gram-positive bacteria, and an anticancer peptide due to its toxicity towards cancer cells. The final hypothesis could therefore bet accepted. The activity towards Gram-negative bacteria can be enhanced by the addition of disodium EDTA at concentrations that are not toxic to non-malignant cells. The current study indicated that nisin Z can be considered an adjuvant for conventional antibiotics and chemotherapeutic drugs by utilising the direct killing activity (toxic effect) of nisin Z towards bacterial as well as cancer cells. Synergistic interactions could be observed when combining nisin Z with the conventional antibiotic novobiocin against S. epidermidis and S. aureus respectively. Synergism could also be observed when combining nisin Z with the conventional chemotherapeutic agent etoposide. Some additive interactions occurred between ampicillin and nisin Z (S. aureus and S. epidermidis), gentamicin and nisin Z (S. aureus and E. coli), tetracycline and nisin Z (E. coli) as well as between 5-fluoruracil and nisin Z against melanoma cells (coefficient of drug interaction (CDI) = 1). It should be noted that although synergistic interactions are preferable, the significance of additive interactions should not be underestimated. Agents that are able to lower the effective therapeutic dose (of antibiotics or chemotherapy agents) can contribute to the fight against antibiotic and chemotherapy resistance respectively. The additive effect and lower effective therapeutic dose can assist in lowering the toxicity associated with conventional chemotherapeutic treatments. Based on the findings of this study, the fact that nisin Z has GRAS status, and is deemed safe for human consumption, nisin Z can be considered an attractive candidate to be developed as a novel therapeutic agent to be used alone or in combination with conventional treatment options for the treatment of bacterial infections and of cancer. The major limitation of the current study was that it was limited to evaluating the in vitro effects. Although in vitro experimentation is important for evaluating molecular mechanisms as well as molecular and Chapter 6: Conclusion 139 biomedical interactions of potential therapeutic agents, and this system offers some advantages such as that concentration and duration of the administered agents can be tightly controlled and the effects are easily measured under flexible culturing conditions, it possess some limitations. Due to the dynamic nature of in vivo models administered agents/drugs are adsorbed, distributed, metabolised and eliminated, leading to elevated plasma concentrations compared to the relatively constant concentrations observed in in vitro systems. However, in vivo experimentation are expensive and time consuming, therefore, in vivo experimentation is usually carried out on selected potential therapeutic agents, after in vitro evaluation has proven their potential therapeutic usefulness. This limitation is addressed in the following section. 6.5. Future prospects and recommendations The activity of nisin Z-loaded NLCs towards S. aureus and S. epidermidis could be enhanced by the addition of the non-antibiotic adjuvant, disodium EDTA. Disodium EDTA is commonly used in skin-care products and NLCs, as well as AMPs, are considered for topical application. It is advisable for future studies to focus on incorporating nisin Z and EDTA into NLCs simultaneously, to enhance the antibacterial activity towards Gram-positive bacteria associated with skin infections. Also, it is advised to test the effectiveness ex vivo and in vitro for topical application. NLC formulations were not able to enhance the activity of nisin Z towards Gram-negative bacteria, even in the presence of disodium EDTA, possibly due to the negative charge of the bacterial membrane as well as the particles which led to a repulsion of the NLCs. A layer-by-layer formulation approach (De Villiers et al., 2011) could be considered to reverse the net surface charge of the NLCs (i.e. make it positive) so as to enhance efficacy towards E.coli and other Gram-negative bacteria. Results indicate that apart from inducing apoptosis and reducing the proliferation of cancer cells, nisin Z exerts its anticancer activity by inducing cell membrane damage, ROS production and apoptosis associated with altered mitochondrial function and energy metabolism in cancer cells. However, although many of the identified mechanisms (especially energy mechanisms) are universally shared between most cancer cells (Armstrong, 2006, Constance and Lim, 2012), the exact molecular targets through which nisin Z exerts these activities still need to be elucidated. Although nisin Z was able to act as an adjuvant for conventional antibiotics and chemotherapeutic agents, this study was limited to the in vitro effect only. For future studies it is recommended that mechanistic interaction between nisin Z and the antibiotics as well as Chapter 6: Conclusion 140 chemotherapeutic agents be investigated as well. The effective dosages need to be determined with in vivo assays. Two types of adjuvants can be considered; type I adjuvants that act synergistically with conventional treatment options and type II adjuvants that are able to reinforce the host defence system through immunomodulation. In this current study the direct effect of nisin Z in combination with conventional antibiotics and chemotherapeutic agents on bacterial cells and cancer cells, respectively, was used to investigate the adjuvant potential of nisin Z to produce synergistic interactions. Future studies should investigate the immuno-modulatory effect of nisin Z. Finally, although the current study has indicated that nisin Z holds great potential of being developed as novel therapeutic for the treatment of both cancer and antibacterial infections when used alone and in combination with current therapeutics, future studies need to be undertaken that thoroughly examine the systemic peptide pharmacokinetics and pharmacodynamics. Through these studies the in vivo half-life and required dosing frequency can be determined rationally to minimise toxicity and optimise disease outcomes. 6.6. References The references used in this section are included in the final reference list at the end of this thesis. 141 Reference list AFANAS’EV, I. 2011. Reactive Oxygen Species Signaling in Cancer: Comparison with Aging. Aging and Disease, 2, 219-230. AHMAD, V., KHAN, M. S., JAMAL, Q. M., ALZOHAIRY, M. A., AL KARAAWI, M. A. & SIDDIQUI, M. U. 2017. Antimicrobial potential of bacteriocins: in therapy, agriculture and food preservation. Int J Antimicrob Agents, 49, 1-11. ARMSTRONG, J. S. 2006. Mitochondria: a target for cancer therapy. Br J Pharmacol, 147, 239-48. BARAN, Y., ZENCIR, S., CAKIR, Z., OZTURK, E. & TOPCU, Z. 2011. Imatinib-induced apoptosis: a possible link to topoisomerase enzyme inhibition. J Clin Pharm Ther, 36, 673-9. BARTOLONI, A., MANTELLA, A., GOLDSTEIN, B. P., DEI, R., BENEDETTI, M., SBARAGLI, S. & PARADISI, F. 2004. In-vitro activity of nisin against clinical isolates of Clostridium difficile. J Chemother, 16, 119-21. BEGDE, D., BUNDALE, S., MASHITHA, P., RUDRA, J., NASHIKKAR, N. & UPADHYAY, A. 2011. Immunomodulatory efficacy of nisin--a bacterial lantibiotic peptide. J Pept Sci, 17, 438-44. BEHRENS, H. M., SIX, A., WALKER, D. & KLEANTHOUS, C. 2017. The therapeutic potential of bacteriocins as protein antibiotics. Emerging Topics in Life Sciences, 1, 65–74. BERTINO, J. R., GOKER, E., GORLICK, R., LI, W. W. & BANERJEE, D. 1996. Resistance mechanisms to methotrexate in tumors. Stem Cells, 14, 5-9. BOLISETTY, S. & JAIMES, E. 2013. Mitochondria and Reactive Oxygen Species: Physiology and Pathophysiology. International Journal of Molecular Sciences, 14, 6306. BOSMA, T., KUIPERS, A., BULTEN, E., DE VRIES, L., RINK, R. & MOLL, G. N. 2011. Bacterial display and screening of posttranslationally thioether-stabilized peptides. Appl Environ Microbiol, 77, 6794-801. BRACCI, L., SCHIAVONI, G., SISTIGU, A. & BELARDELLI, F. 2014. Immune-based mechanisms of cytotoxic chemotherapy: implications for the design of novel and rationale-based combined treatments against cancer. Cell Death Differ, 21, 15-25. BRUMFITT, W., SALTON, M. R. & HAMILTON-MILLER, J. M. 2002. Nisin, alone and combined with peptidoglycan-modulating antibiotics: activity against methicillin resistant Staphylococcus aureus and vancomycin-resistant enterococci. J Antimicrob Chemother, 50, 731-4. - Reference list 142 BRUNTON, L. A., DUNCAN, D., COLDHAM, N. G., SNOW, L. C. & JONES, J. R. 2012. A survey of antimicrobial usage on dairy farms and waste milk feeding practices in England and Wales. Veterinary Record, 171, 296-296. CAO, L. T., WU, J. Q., XIE, F., HU, S. H. & MO, Y. 2007. Efficacy of nisin in treatment of clinical mastitis in lactating dairy cows. J Dairy Sci, 90, 3980-5. CARTMILL, C., LINGARD, L., REGEHR, G., ESPIN, S., BOHNEN, J., BAKER, R. & ROTSTEIN, L. 2009. Timing of surgical antibiotic prophylaxis administration: Complexities of analysis. BMC Medical Research Methodology, 9, 43. CHEN, L., YE, H. L., ZHANG, G., YAO, W. M., CHEN, X. Z., ZHANG, F. C. & LIANG, G. 2014. Autophagy inhibition contributes to the synergistic interaction between EGCG and doxorubicin to kill the hepatoma Hep3B cells. PLoS One, 9, e85771. CLEVELAND, J., MONTVILLE, T. J., NES, I. F. & CHIKINDAS, M. L. 2001. Bacteriocins: safe, natural antimicrobials for food preservation. Int J Food Microbiol, 71, 1-20. CONSTANCE, J. E. & LIM, C. S. 2012. Targeting malignant mitochondria with therapeutic peptides. Ther Deliv, 3, 961-79. COTTER, P. D., HILL, C. & ROSS, R. P. 2005. Bacteriocins: developing innate immunity for food. Nat Rev Microbiol, 3, 777-88. CREAGH, E. M. & COTTER, T. G. 1999. Selective protection by hsp 70 against cytotoxic drug-, but not Fas-induced T-cell apoptosis. Immunology, 97, 36-44. CRUZ-CHAMORRO, L., PUERTOLLANO, M. A., PUERTOLLANO, E., DE CIENFUEGOS, G. A. & DE PABLO, M. A. 2006. In vitro biological activities of magainin alone or in combination with nisin. Peptides, 27, 1201-9. DE VILLIERS, M. M., OTTO, D. P., STRYDOM, S. J. & LVOV, Y. M. 2011. Introduction to nanocoatings produced by layer-by-layer (LbL) self-assembly. Advanced Drug Delivery Reviews, 63, 701-715. DE VOS, W. M., MULDERS, J. W., SIEZEN, R. J., HUGENHOLTZ, J. & KUIPERS, O. P. 1993. Properties of nisin Z and distribution of its gene, nisZ, in Lactococcus lactis. Appl Environ Microbiol, 59, 213-8. DE, V. W. M., KUIPERS, O. P. & SIEZEN, R. J. 2003. Lantibiotics similar to nisin a, lactic acid bacteria which produce such lantibiotics, method for constructing such lactic acid bacteria and method for preserving foodstuffs with the aid of these lantibiotics and these lactic acid bacteria producing lantibiotics. Google Patents. DEBERARDINIS, R. J. & CHANDEL, N. S. 2016. Fundamentals of cancer metabolism. Sci Adv, 2, e1600200. DEEPA, P. R., VANDHANA, S., JAYANTHI, U. & KRISHNAKUMAR, S. 2012. Therapeutic and toxicologic evaluation of anti-lipogenic agents in cancer cells compared with non- neoplastic cells. Basic Clin Pharmacol Toxicol, 110, 494-503. Reference list 143 DEMPSEY, C. E. 1990. The actions of melittin on membranes. Biochim Biophys Acta, 1031, 143-61. DO, N., WEINDL, G., GROHMANN, L., SALWICZEK, M., KOKSCH, B., KORTING, H. C. & SCHAFER-KORTING, M. 2014. Cationic membrane-active peptides - anticancer and antifungal activity as well as penetration into human skin. Exp Dermatol, 23, 326-31. DOSLER, S. & GERCEKER, A. A. 2011. In vitro activities of nisin alone or in combination with vancomycin and ciprofloxacin against methicillin-resistant and methicillin- susceptible Staphylococcus aureus strains. Chemotherapy, 57, 511-6. DOSLER, S. & GERCEKER, A. A. 2012. In vitro activities of antimicrobial cationic peptides; melittin and nisin, alone or in combination with antibiotics against Gram-positive bacteria. J Chemother, 24, 137-43. FAO & WHO. 2010. JOINT FAO/WHO FOOD STANDARDS PROGRAMME CODEX COMMITTEE ON FOOD ADDITIVES. COMMENTS ON DRAFT AND PROPOSED DRAFT FOOD ADDITIVE PROVISIONS OF THE GSFA [Online]. Beijing, China. Available: ftp://ftp.fao.org/codex/Meetings/CCFA/ccfa42/fa42_05be.pdf. [Accessed 20 September 2017]. FDA. 2001. U.S. Food and Drug Administration. GRAS notices received in 2000 [Online]. Available: http://www.cfsan.fda.gov/ rdb/opa-gnoo.html.[Accessed 30 May 2017]. FERNANDEZ, L., DELGADO, S., HERRERO, H., MALDONADO, A. & RODRIGUEZ, J. M. 2008. The bacteriocin nisin, an effective agent for the treatment of staphylococcal mastitis during lactation. J Hum Lact, 24, 311-6. FIELD, D., COTTER, P. D., ROSS, R. P. & HILL, C. 2015. Bioengineering of the model lantibiotic nisin. Bioengineered, 6, 187-92. FOX, J. L. 2013. Antimicrobial peptides stage a comeback. Nat Biotechnol, 31, 379-82. FSANZ 2007. Food Standards Australia New Zealand (FSANZ) Final Assessment Report, Application A565 Use of Nisin in Processed Meat Products. GALLARATE, M., TROTTA, M., BATTAGLIA, L. & CHIRIO, D. 2009. Preparation of solid lipid nanoparticles from W/O/W emulsions: preliminary studies on insulin encapsulation. J Microencapsul, 26, 394-402. GARCIA-ORUE, I., GAINZA, G., GIRBAU, C., ALONSO, R., AGUIRRE, J. J., PEDRAZ, J. L., IGARTUA, M. & HERNANDEZ, R. M. 2016. LL37 loaded nanostructured lipid carriers (NLC): A new strategy for the topical treatment of chronic wounds. European Journal of Pharmaceutics and Biopharmaceutics, 108, 310-316. GASPAR, D., VEIGA, A. S. & CASTANHO, M. A. 2013. From antimicrobial to anticancer peptides. A review. Front Microbiol, 4, 294. Reference list 144 GIACOMETTI, A., CIRIONI, O., BARCHIESI, F. & SCALISE, G. 2000. In-vitro activity and killing effect of polycationic peptides on methicillin-resistant Staphylococcus aureus and interactions with clinically used antibiotics. Diagn Microbiol Infect Dis, 38, 115-8. GILL, S. R., FOUTS, D. E., ARCHER, G. L., MONGODIN, E. F., DEBOY, R. T., RAVEL, J., PAULSEN, I. T., KOLONAY, J. F., BRINKAC, L., BEANAN, M., DODSON, R. J., DAUGHERTY, S. C., MADUPU, R., ANGIUOLI, S. V., DURKIN, A. S., HAFT, D. H., VAMATHEVAN, J., KHOURI, H., UTTERBACK, T., LEE, C., DIMITROV, G., JIANG, L., QIN, H., WEIDMAN, J., TRAN, K., KANG, K., HANCE, I. R., NELSON, K. E. & FRASER, C. M. 2005. Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. J Bacteriol, 187, 2426-38. GOLDSTEIN, B. P., WEI, J., GREENBERG, K. & NOVICK, R. 1998. Activity of nisin against Streptococcus pneumoniae, in vitro, and in a mouse infection model. J Antimicrob Chemother, 42, 277-8. GRAVESEN, A., JYDEGAARD AXELSEN, A. M., MENDES DA SILVA, J., HANSEN, T. B. & KNOCHEL, S. 2002. Frequency of bacteriocin resistance development and associated fitness costs in Listeria monocytogenes. Appl Environ Microbiol, 68, 756- 64. GREGORC, V., DE BRAUD, F. G., DE PAS, T. M., SCALAMOGNA, R., CITTERIO, G., MILANI, A., BOSELLI, S., CATANIA, C., DONADONI, G., ROSSONI, G., GHIO, D., SPITALERI, G., AMMANNATI, C., COLOMBI, S., CALIGARIS-CAPPIO, F., LAMBIASE, A. & BORDIGNON, C. 2011. Phase I study of NGR-hTNF, a selective vascular targeting agent, in combination with cisplatin in refractory solid tumors. Clin Cancer Res, 17, 1964-72. GUDIOL, C. & CARRATALA, J. 2014. Antibiotic resistance in cancer patients. Expert Rev Anti Infect Ther, 12, 1003-16. GUT, I. M., BLANKE, S. R. & VAN DER DONK, W. A. 2011. Mechanism of inhibition of Bacillus anthracis spore outgrowth by the lantibiotic nisin. ACS Chem Biol, 6, 744-52. HAMPTON, M. B. & ORRENIUS, S. 1997. Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis. FEBS Lett, 414, 552-6. HANCOCK, R. E. 2015. Rethinking the Antibiotic Discovery Paradigm. EBioMedicine, 2, 629-30. HANCOCK, R. E. & DIAMOND, G. 2000. The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol, 8, 402-10. HANCOCK, R. E. & SAHL, H. G. 2006. Antimicrobial and host-defense peptides as new anti- infective therapeutic strategies. Nat Biotechnol, 24, 1551-7. Reference list 145 HE, J., STARR, C. G. & WIMLEY, W. C. 2015. A lack of synergy between membrane- permeabilizing cationic antimicrobial peptides and conventional antibiotics. Biochim Biophys Acta, 1848, 8-15. HELANDER, I. M. & MATTILA-SANDHOLM, T. 2000. Permeability barrier of the gram- negative bacterial outer membrane with special reference to nisin. Int J Food Microbiol, 60, 153-61. HELMBACH, H., KERN, M. A., ROSSMANN, E., RENZ, K., KISSEL, C., GSCHWENDT, B. & SCHADENDORF, D. 2002. Drug resistance towards etoposide and cisplatin in human melanoma cells is associated with drug-dependent apoptosis deficiency. J Invest Dermatol, 118, 923-32. HORINOUCHI, S., UEDA, K., NAKAYAMA, J. & IKEDA, T. 2010. 4.07 - Cell-to-Cell Communications among Microorganisms. Comprehensive Natural Products II. Oxford: Elsevier. HSU, S. T., BREUKINK, E., TISCHENKO, E., LUTTERS, M. A., DE KRUIJFF, B., KAPTEIN, R., BONVIN, A. M. & VAN NULAND, N. A. 2004. The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel antibiotics. Nat Struct Mol Biol, 11, 963-7. HWANG, B., HWANG, J. S., LEE, J. & LEE, D. G. 2011. The antimicrobial peptide, psacotheasin induces reactive oxygen species and triggers apoptosis in Candida albicans. Biochem Biophys Res Commun, 405, 267-71. INDRAN, I. R., TUFO, G., PERVAIZ, S. & BRENNER, C. 2011. Recent advances in apoptosis, mitochondria and drug resistance in cancer cells. Biochim Biophys Acta, 1807, 735-45. IQBAL, N. & IQBAL, N. 2014. Imatinib: a breakthrough of targeted therapy in cancer. Chemother Res Pract, 2014, 357027. JENSSEN, H., HAMILL, P. & HANCOCK, R. E. 2006. Peptide antimicrobial agents. Clin Microbiol Rev, 19, 491-511. JONES, E., SALIN, V. & WILLIAMS, G. W. 2005. Nisin and the Market for Commercial Bacteriocins. TAMRC Consumer and Product Research Report No. CP-01-05. [Online]. Available: http://wwww.ageconsearch.umn.edu/bitstream/90779/2/CP%2001%2005%20Nisin% 20Report.pdf [Accessed 18 August 2015. JOO, N. E., RITCHIE, K., KAMARAJAN, P., MIAO, D. & KAPILA, Y. L. 2012. Nisin, an apoptogenic bacteriocin and food preservative, attenuates HNSCC tumorigenesis via CHAC1. Cancer Med, 1, 295-305. JOZWIAK, P., FORMA, E., BRYS, M. & KRZESLAK, A. 2014. O-GlcNAcylation and Metabolic Reprograming in Cancer. Front Endocrinol (Lausanne), 5, 145. Reference list 146 KALAL, B. S., UPADHYA, D. & PAI, V. R. 2017. Chemotherapy Resistance Mechanisms in Advanced Skin Cancer. Oncol Rev, 11, 326. KAMARAJAN, P., HAYAMI, T., MATTE, B., LIU, Y., DANCIU, T., RAMAMOORTHY, A., WORDEN, F., KAPILA, S. & KAPILA, Y. 2015. Nisin ZP, a Bacteriocin and Food Preservative, Inhibits Head and Neck Cancer Tumorigenesis and Prolongs Survival. PLoS One, 10, e0131008. KAUR, S. & KAUR, S. 2015. Bacteriocins as Potential Anticancer Agents. Front Pharmacol, 6, 272. KAWAKITA, T. & LANDY, H. J. 2017. Surgical site infections after cesarean delivery: epidemiology, prevention and treatment. Maternal Health, Neonatology and Perinatology, 3, 12. KINDRACHUK, J., JENSSEN, H., ELLIOTT, M., NIJNIK, A., MAGRANGEAS-JANOT, L., PASUPULETI, M., THORSON, L., MA, S., EASTON, D. M., BAINS, M., FINLAY, B., BREUKINK, E. J., GEORG-SAHL, H. & HANCOCK, R. E. 2013. Manipulation of innate immunity by a bacterial secreted peptide: lantibiotic nisin Z is selectively immunomodulatory. Innate Immun, 19, 315-27. KLEANHAMMER, T. R., FREMAUX, C., AHN, C. & MILTON, K. 1993. Molecular Biology of Bacteriocins Produced by Lactobacillus. In: HOOVERAND, D. G. & STEENSON, L. R. (eds.) Bacteriocins of Lactic Acid Bacteria. New York: Academic press Inc. KOPERMSUB, P., MAYEN, V. & WARIN, C. 2011. Potential use of niosomes for encapsulation of nisin and EDTA and their antibacterial activity enhancement. Food Research International, 44, 605-612. KRYSTON, T. B., GEORGIEV, A. B., PISSIS, P. & GEORGAKILAS, A. G. 2011. Role of oxidative stress and DNA damage in human carcinogenesis. Mutat Res, 711, 193- 201. LE LAY, C., DRIDI, L., BERGERON, M. G., OUELLETTE, M. & FLISS, I. L. 2016. Nisin is an effective inhibitor of Clostridium difficile vegetative cells and spore germination. J Med Microbiol, 65, 169-75. LEWIES, A., WENTZEL, J. F., JACOBS, G. & DU PLESSIS, L. H. 2015. The Potential Use of Natural and Structural Analogues of Antimicrobial Peptides in the Fight against Neglected Tropical Diseases. Molecules, 20, 15392-433. LEWIES, A., WENTZEL, J. F., JORDAAN, A., BEZUIDENHOUT, C. & DU PLESSIS, L. H. 2017. Interactions of the antimicrobial peptide nisin Z with conventional antibiotics and the use of nanostructured lipid carriers to enhance antimicrobial activity. Int J Pharm, 526, 244-253. Reference list 147 LEWIES, A., WENTZEL, J. F., VAN DYK, H. & DU PLESSIS, L. H. 2018. The antimicrobial peptide nisin Z induces selective toxicity and apoptotic cell death in cultured melanoma cells. Biochimie. 144:28-40 LI, X., LIN, Z., ZHANG, B., GUO, L., LIU, S., LI, H., ZHANG, J. & YE, Q. 2016. beta-elemene sensitizes hepatocellular carcinoma cells to oxaliplatin by preventing oxaliplatin- induced degradation of copper transporter 1. Sci Rep, 6, 21010. LIOU, G. Y. & STORZ, P. 2010. Reactive oxygen species in cancer. Free Radic Res, 44, 479-96. LIU, C., BAYER, A., COSGROVE, S. E., DAUM, R. S., FRIDKIN, S. K., GORWITZ, R. J., KAPLAN, S. L., KARCHMER, A. W., LEVINE, D. P., MURRAY, B. E., M, J. R., TALAN, D. A., CHAMBERS, H. F. & INFECTIOUS DISEASES SOCIETY OF, A. 2011. Clinical practice guidelines by the infectious diseases society of america for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children. Clin Infect Dis, 52, e18-55. LONG, C. & PHILLIPS, C. A. 2003. The effect of sodium citrate, sodium lactate and nisin on the survival of Arcobacter butzleri NCTC 12481 on chicken. Food Microbiology, 20, 495-502. LONGLEY, D. B., HARKIN, D. P. & JOHNSTON, P. G. 2003. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer, 3, 330-8. LUBELSKI, J., RINK, R., KHUSAINOV, R., MOLL, G. N. & KUIPERS, O. P. 2008. Biosynthesis, immunity, regulation, mode of action and engineering of the model lantibiotic nisin. Cell Mol Life Sci, 65, 455-76. LUCKS, S. & MULLER, R. 1993. Medication Vehicles Made of Solid Lipid Particles (Solid Lipid Nanospheres-SLN). Google Patents. LUQMANI, Y. A. 2005. Mechanisms of drug resistance in cancer chemotherapy. Med Princ Pract, 14 Suppl 1, 35-48. LY, J. D., GRUBB, D. R. & LAWEN, A. 2003. The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update. Apoptosis, 8, 115-28. MARR, A. K., GOODERHAM, W. J. & HANCOCK, R. E. 2006a. Antibacterial peptides for therapeutic use: obstacles and realistic outlook. Curr Opin Pharmacol, 6, 468-72. MARR, A. K., GOODERHAM, W. J. & HANCOCK, R. E. W. 2006b. Antibacterial peptides for therapeutic use: obstacles and realistic outlook. Current Opinion in Pharmacology, 6, 468-472. MARTÍNEZ, B., RODRÍGUEZ, A. & SUÁREZ, E. 2016. Antimicrobial Peptides Produced by Bacteria: The Bacteriocins. In: VILLA, T. G. & VINAS, M. (eds.) New Weapons to Control Bacterial Growth. Cham: Springer International Publishing. Reference list 148 MARTINS, S., SARMENTO, B., FERREIRA, D. C. & SOUTO, E. B. 2007. Lipid-based colloidal carriers for peptide and protein delivery--liposomes versus lipid nanoparticles. Int J Nanomedicine, 2, 595-607. MATARACI, E. & DOSLER, S. 2012. In vitro activities of antibiotics and antimicrobial cationic peptides alone and in combination against methicillin-resistant Staphylococcus aureus biofilms. Antimicrob Agents Chemother, 56, 6366-71. MEHNERT, W. & MADER, K. 2001. Solid lipid nanoparticles: production, characterization and applications. Adv Drug Deliv Rev, 47, 165-96. MELCHIOR, M. B., VAARKAMP, H. & FINK-GREMMELS, J. 2006. Biofilms: a role in recurrent mastitis infections? Vet J, 171, 398-407. MULDERS, J. W., BOERRIGTER, I. J., ROLLEMA, H. S., SIEZEN, R. J. & DE VOS, W. M. 1991. Identification and characterization of the lantibiotic nisin Z, a natural nisin variant. Eur J Biochem, 201, 581-4. MÜLLER-AUFFERMANN, K., GRIJALVA, F., JACOB, F. & HUTZLER, M. 2015a. Nisin and its usage in breweries: a review and discussion. Journal of the Institute of Brewing, 121, 309-319. MÜLLER-AUFFERMANN, K., GRIJALVA, F., JACOB, F. & HUTZLER, M. 2015b. Nisin and its usage in breweries: a review and discussion. J. Inst. Brew, 121, 309-319. MÜLLER, R. H., ALEXIEV, U., SINAMBELA, P. & KECK, C. M. 2016. Nanostructured Lipid Carriers (NLC): The Second Generation of Solid Lipid Nanoparticles. In: DRAGICEVIC, N. & MAIBACH, H. I. (eds.) Percutaneous Penetration Enhancers Chemical Methods in Penetration Enhancement: Nanocarriers. Berlin, Heidelberg: Springer Berlin Heidelberg. MULLER, R. H., RADTKE, M. & WISSING, S. A. 2002. Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations. Adv Drug Deliv Rev, 54 Suppl 1, S131-55. NAGHMOUCHI, K., BAAH, J., HOBER, D., JOUY, E., RUBRECHT, C., SANE, F. & DRIDER, D. 2013. Synergistic effect between colistin and bacteriocins in controlling Gram-negative pathogens and their potential to reduce antibiotic toxicity in mammalian epithelial cells. Antimicrob Agents Chemother, 57, 2719-25. NAGHMOUCHI, K., LE LAY, C., BAAH, J. & DRIDER, D. 2012. Antibiotic and antimicrobial peptide combinations: synergistic inhibition of Pseudomonas fluorescens and antibiotic-resistant variants. Res Microbiol, 163, 101-8. NATRAJAN, N. & SHELDON, B. W. 2000. Efficacy of nisin-coated polymer films to inactivate Salmonella Typhimurium on fresh broiler skin. J Food Prot, 63, 1189-96. O’NEILL, J. 2016. The review on antimicrobial resistancs. Tackling drug-resistant infections globally: Final report and recommendations. [Online]. Available: https://amr- Reference list 149 review.org/sites/default/files/160518_Final%20paper_with%20cover.pdf [Accessed 16 January 2017]. OHSAKI, Y., GAZDAR, A. F., CHEN, H. C. & JOHNSON, B. E. 1992. Antitumor activity of magainin analogues against human lung cancer cell lines. Cancer Res, 52, 3534-8. OLIVEIRA, M., BEXIGA, R., NUNES, S. F., CARNEIRO, C., CAVACO, L. M., BERNARDO, F. & VILELA, C. L. 2006. Biofilm-forming ability profiling of Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates. Vet Microbiol, 118, 133-40. PAG, U. & SAHL, H. G. 2002. Multiple activities in lantibiotics--models for the design of novel antibiotics? Curr Pharm Des, 8, 815-33. PAIVA, A. D., BREUKINK, E. & MANTOVANI, H. C. 2011. Role of lipid II and membrane thickness in the mechanism of action of the lantibiotic bovicin HC5. Antimicrob Agents Chemother, 55, 5284-93. PARK, C. & LEE, D. G. 2010. Melittin induces apoptotic features in Candida albicans. Biochem Biophys Res Commun, 394, 170-2. PERICHON, B. & COURVALIN, P. 2009. VanA-type vancomycin-resistant Staphylococcus aureus. Antimicrob Agents Chemother, 53, 4580-7. PESCHEL, A. & SAHL, H.-G. 2006a. The co-evolution of host cationic antimicrobial peptides and microbial resistance. 4, 529. PESCHEL, A. & SAHL, H. G. 2006b. The co-evolution of host cationic antimicrobial peptides and microbial resistance. Nat Rev Microbiol, 4, 529-36. PIETERSE, R. & TODOROV, S. D. 2010. Bacteriocins - exploring alternatives to antibiotics in mastitis treatment. Braz J Microbiol, 41, 542-62. PREET, S., BHARATI, S., PANJETA, A., TEWARI, R. & RISHI, P. 2015. Effect of nisin and doxorubicin on DMBA-induced skin carcinogenesis--a possible adjunct therapy. Tumour Biol, 36, 8301-8. PROBIOTICSDB.COM. 2017. Lactococcus lactis - Probiotics Database [Online]. Available: http://probioticsdb.com/probiotic-strains/lactobacillus-lactis/ [Accessed 15 October 2017 2017]. PROMBUTARA, P., KULWATTHANASAL, Y., SUPAKA, N., SRAMALA, I. & CHAREONPORNWATTANA, S. 2012. Production of nisin-loaded solid lipid nanoparticles for sustained antimicrobial activity. Food Control, 24, 184-190. PRUDÊNCIO, C. V., DOS SANTOS, M. T. & VANETTI, M. C. D. 2015. Strategies for the use of bacteriocins in Gram-negative bacteria: relevance in food microbiology. Journal of Food Science and Technology, 52, 5408-5417. RISHI, P., PREET SINGH, A., GARG, N. & RISHI, M. 2014. Evaluation of nisin-beta-lactam antibiotics against clinical strains of Salmonella enterica serovar Typhi. J Antibiot (Tokyo), 67, 807-11. Reference list 150 RODRIGUEZ-ROJAS, A., RODRIGUEZ-BELTRAN, J., COUCE, A. & BLAZQUEZ, J. 2013. Antibiotics and antibiotic resistance: a bitter fight against evolution. Int J Med Microbiol, 303, 293-7. ROGERS, L. A. & WHITTIER, E. O. 1928. Limiting Factors in the Lactic Fermentation. J Bacteriol, 16, 211-29. SCHWEIZER, F. 2009. Cationic amphiphilic peptides with cancer-selective toxicity. Eur J Pharmacol, 625, 190-4. SHARMA, A. & SRIVASTAVA, S. 2014. Anti-Candida activity of two-peptide bacteriocins, plantaricins (Pln E/F and J/K) and their mode of action. Fungal Biol, 118, 264-75. SHIMANOVICH, U. & GEDANKEN, A. 2016. Nanotechnology solutions to restore antibiotic activity. Journal of Materials Chemistry B, 4, 824-833. SHIN, J. M., GWAK, J. W., KAMARAJAN, P., FENNO, J. C., RICKARD, A. H. & KAPILA, Y. L. 2016. Biomedical applications of nisin. J Appl Microbiol, 120, 1449-65. SINGH, A. P., PREET, S. & RISHI, P. 2014. Nisin/beta-lactam adjunct therapy against Salmonella enterica serovar Typhimurium: a mechanistic approach. J Antimicrob Chemother, 69, 1877-87. SOENGAS, M. S. & LOWE, S. W. 2003. Apoptosis and melanoma chemoresistance. Oncogene, 22, 3138-51. SWITHENBANK, L. & MORGAN, M. 2017. The Role of Antimicrobial Peptides in Lung Cancer Therapy. Journal of Antimicrobial Agents, 3:134. SYLVESTER, P. W., WALI, V. B., BACHAWAL, S. V., SHIRODE, A. B., AYOUB, N. M. & AKL, M. R. 2011. Tocotrienol combination therapy results in synergistic anticancer response. Front Biosci (Landmark Ed), 16, 3183-95. TARAI, B., DAS, P. & KUMAR, D. 2013. Recurrent Challenges for Clinicians: Emergence of Methicillin-Resistant Staphylococcus aureus, Vancomycin Resistance, and Current Treatment Options. J Lab Physicians, 5, 71-8. TIAN, H. & CRONSTEIN, B. N. 2007. Understanding the mechanisms of action of methotrexate: implications for the treatment of rheumatoid arthritis. Bull NYU Hosp Jt Dis, 65, 168-73. TIWARI, M. 2012. Antimetabolites: established cancer therapy. J Cancer Res Ther, 8, 510-9. TONG, Z., ZHANG, Y., LING, J., MA, J., HUANG, L. & ZHANG, L. 2014. An in vitro study on the effects of nisin on the antibacterial activities of 18 antibiotics against Enterococcus faecalis. PLoS One, 9, e89209. TRACHOOTHAM, D., ALEXANDRE, J. & HUANG, P. 2009. Targeting cancer cells by ROS- mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Discov, 8, 579-91. Reference list 151 VAN VUUREN, S. F., NKWANYANA, M. N. & DE WET, H. 2015. Antimicrobial evaluation of plants used for the treatment of diarrhoea in a rural community in northern Maputaland, KwaZulu-Natal, South Africa. BMC Complement Altern Med, 15, 53. VAN VUUREN, S. F., SULIMAN, S. & VILJOEN, A. M. 2009. The antimicrobial activity of four commercial essential oils in combination with conventional antimicrobials. Lett Appl Microbiol, 48, 440-6. WANG, G., LI, X. & WANG, Z. 2016. APD3: the antimicrobial peptide database as a tool for research and education. Nucleic Acids Res, 44, D1087-93. WARBURG, O. 1956a. On respiratory impairment in cancer cells. Science, 124, 269-70. WARBURG, O. 1956b. On the origin of cancer cells. Science, 123, 309-14. WARD, P. S. & THOMPSON, C. B. 2012. Metabolic reprogramming: a cancer hallmark even warburg did not anticipate. Cancer Cell, 21, 297-308. WEBB, A. L. B., FLAGG, R. L. & FINK, A. S. 2006. Reducing surgical site infections through a multidisciplinary computerized process for preoperative prophylactic antibiotic administration. Am J Surg, 192. WEI, X. Q., MA, H. Q., LIU, A. H. & ZHANG, Y. Z. 2013. Synergistic anticancer activity of 5- aminolevulinic acid photodynamic therapy in combination with low-dose cisplatin on Hela cells. Asian Pac J Cancer Prev, 14, 3023-8. WELLBROCK, C. 2014. MAPK pathway inhibition in melanoma: resistance three ways. Biochem Soc Trans, 42, 727-32. WEN, S., ZHU, D. & HUANG, P. 2013. Targeting cancer cell mitochondria as a therapeutic approach. Future Med Chem, 5, 53-67. WENTZEL, J. F., LOMBARD, M. J., DU PLESSIS, L. H. & ZANDBERG, L. 2017. Evaluation of the cytotoxic properties, gene expression profiles and secondary signalling responses of cultured cells exposed to fumonisin B1, deoxynivalenol and zearalenone mycotoxins. Arch Toxicol, 91, 2265-2282. WHO. 2016. Antimicrobial Resistance: Fact sheet N° 194 [Online]. Available: http://www.who.int/mediacentre/factsheets/fs194/en/ [Accessed 1 June 2017]. WILLEY, J. M. & VAN DER DONK, W. A. 2007. Lantibiotics: peptides of diverse structure and function. Annu Rev Microbiol, 61, 477-501. WISPLINGHOFF, H., SEIFERT, H., WENZEL, R. P. & EDMOND, M. B. 2003. Current trends in the epidemiology of nosocomial bloodstream infections in patients with hematological malignancies and solid neoplasms in hospitals in the United States. Clin Infect Dis, 36, 1103-10. WISSING, S. A., KAYSER, O. & MULLER, R. H. 2004. Solid lipid nanoparticles for parenteral drug delivery. Adv Drug Deliv Rev, 56, 1257-72. Reference list 152 WRIGHT, G. D. 2016. Antibiotic Adjuvants: Rescuing Antibiotics from Resistance. Trends in Microbiology, 24, 862-871. WU, J., HU, S. & CAO, L. 2007. Therapeutic effect of nisin Z on subclinical mastitis in lactating cows. Antimicrob Agents Chemother, 51, 3131-5. YANG, S. C., LIN, C. H., SUNG, C. T. & FANG, J. Y. 2014. Antibacterial activities of bacteriocins: application in foods and pharmaceuticals. Front Microbiol, 5, 241. YEAMAN, M. R. & YOUNT, N. Y. 2003. Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev, 55, 27-55. ZHEN, L., LI, X., ZHENG, L., XIAO-HONG, L., FEI, G. & LI, Y. 2010. Bovine Serum Albumin Loaded Solid Lipid Nanoparticles Prepared by Double Emulsion Method. Chemical Research in Chinese Universities, 26. ZIECH, D., FRANCO, R., PAPPA, A. & PANAYIOTIDIS, M. I. 2011. Reactive Oxygen Species (ROS)––Induced genetic and epigenetic alterations in human carcinogenesis. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 711, 167-173. 153 Appendix A: Validation of the modified BCA protein assay for the quantification of nisin Z Appendix A 154 Appendix A: Validation of the modified BCA protein assay for the quantification of nisin Z A1. Introduction The bicinchoninic acid (BCA) assay uses BCA for the colourimetric quantification of total protein in a sample (Smith et al., 1985). The principle of the method is based on the reduction of Cu2+ to Cu1+ by protein in an alkaline medium. A water-soluble purple chelate is then formed when Cu1+complexes with BCA, which is then measured at its absorption maximum at 562 nm (Figure A1). The range of the assay is typically 20-2000 µg/ml. A major advantage of this assay compared to other protein determination assays is that it is compatible with detergents (up to 5 % V/V). This method is, however, subject to lipid interference. To eliminate lipid interference, the BCA assay can be modified by the addition of the detergent sodium dodecyl sulphate (SDS) (Morton and Evans, 1992). Figure A1: Principal of the bicinchoninic acid (BCA) assay. Cu2+ is reduced to Cu1+ by protein (containing cysteine, cysteine, tyrosine and/or tryptophan amino acid residues) under alkaline conditions. Cu1+ then interacts with BCA to form a purple BCA-coper complex. Appendix A 155 A2. Aim The aims of this section are to discuss the validation of the modified BCA assay for the quantification of nisin Z and the accuracy of the chloroform extraction method used for the determination of entrapment efficiency of the lipid nanoparticles. A3. The modified BCA assay method The BCA assay kit from Sigma-Aldrich Chemie GmbH (Schelldorf, Germany) was used to perform the modified BCA assay as described by (Morton and Evans, 1992). A standard series of nisin Z was prepared and 20 µl of each sample was added to the wells of a 96 well plate. Then, 2% SDS (Bio-Rad, Hercules, California, United States) was added to each sample and the plates were shaken at 300 rpm at room temperature for 10 minutes. A BCA working solution was prepared by adding 50 parts of reagent A (BCA) to 1 part of reagent B (Copper-sulphate). After incubating the samples with SDS, 200 µl of the BCA working solution was added to each well. Plates were covered with adhesive seal and incubated at 60ºC for 30 minutes. Plates were then allowed to cool to room temperature for 15 minutes. Absorbance was measured at 562 nm using a microplate reader (SpectraMax ParadigmTM, Molecular Devices, USA). A4. Validation of the BCA assay The BCA assay was validated in terms of the limit of detection (LOD), lowest limit of quantitation (LLOQ), linearity, accuracy, precision and robustness. A4.1.Limit of detection The limit of detection (LOD) is defined as the lowest concentration of an analyte that can be detected but not necessarily quantified i.e. the lowest concentration of nisin Z that can be reliably differentiated from blank samples/background noise (Armbruster and Pry, 2008). The LOD was determined based on the standard deviation of the blank and the slope of a calibration curve (ICH, 2006). The LOD is expressed as: 𝐿𝑂𝐷 = 3.3𝜕 𝑆 Where; ∂ = the standard deviation of the blank S= the slope of the calibration curve Appendix A 156 A calibration curve was made using 0, 5, 25, 50, 125, 250 and 500 µg/ml nisin Z in 0.01 N HCl. The calibration curve with its correlation coefficient (R2), the slope of the regression line and the y-intercept is given in figure A2. Values represent the absorbance at 562 minus the absorbance of the blank samples at 562 nm. Excellent linearity was observed with an R2 value of 0.998 Figure A2: Calibration curve for nisin Z. n=3 Six determinations of the background were made and the standard deviation was calculated as given in table A1. Appendix A 157 Table A1: The absorbance of blank samples at 562 nm as determined by the BCA assay. Measurement Absorbance at 562 nm 1 0.1032 2 0.1042 3 0.1067 4 0.0994 5 0.0985 6 0.1009 Average 0.10215 Stdev 0.00311 %CV 3.048 The LOD is calculated as 7.902 ug/ml. A4.2.Lowest limit of quantitation The lowest limit of quantitation (LLOQ) is defined as the lowest concentration of an analyte that can be determined quantitatively with suitable accuracy (Armbruster and Pry, 2008). The LLOQ was also determined based on the standard deviation of the blank and slope of the calibration curve (fig A1) (ICH, 2006) The LLOQ is expressed as: 𝐿𝐿𝑂𝑄 = 10𝜕 𝑆 Where, ∂ = the standard deviation of the blank S= the slope of the calibration curve The calculated LLOQ is 23.9464 ug/ml. If during any of the entrapment efficiency analysis a concentration below the LLOQ was detected the results were considered unreliable and therefore rejected. A4.3.Linearity Linearity assesses the analytical methods ability (within a given range) to obtain results that are directly proportional to the concentration of the analyte in the sample (Tiwari and Tiwari, 2010). A minimum of five concentrations should be evaluated to assess linearity (ICH, 2006). A calibration curve was made using 0, 25, 50, 125, 250 and 500 µg/ml nisin Z in 1 mM HCl. The calibration curve with its correlation coefficient (R2), the slope of the regression line and Appendix A 158 the y-intercept is given in figure A3. Values represent the absorbance at 562 minus the absorbance of the blank samples at 562 nm. The R2 value is > 0.990 and is, therefore, represents good linearity within the range of 25 – 500 µg/ml nisin Z. Figure A3: Calibration curve for nisin Z. n=3 A4.4. Accuracy and precision To measure the accuracy and precision of the BCA assay for the quantification of nisin Z three concentrations of nisin Z were prepared (theoretical concentrations). The actual concentrations of the prepared solutions of nisin Z were calculated by interpolating the absorbance measurements into a calibration curve. The percentage recovery was then calculated and reflects the accuracy of the analytical method. This was done in triplicate per concentration and the average percentage recovery was calculated for each concentration as well as between the respective samples (table A2) (ICH, 2006). The (intraday) precision of the method was evaluated by measuring the coefficient of variation (%CV) for each concentration as well as between concentrations. Appendix A 159 Table A2: Accuracy and precision of the BCA assay for the quantification of nisin Z. Theoretical concentration (µg/ml)n n Actual concentration (µg/ml) % Recovery Average±Stdev %CV 25 1 25.341 101.364 102.715±1.351 1.316 2 25.679 102.715 3 26.017 104.067 125 1 128.691 102.95240 104.295±2.401 2.302 2 128.581 102.8644 3 133.834 107.06690 500 1 498.203 97.71110 99.163±1.434 1.446 2 495.989 99.19772 3 493.248 100.57920 Between samples 102.058±2.628 2.575 An analytical method is deemed accurate if the recovery is between 95 – 105 % (Walfish, 2006). The recovery for each sample was within the accepted range. The mean recovery was 102.1 % (table A2). The analytical method was therefore accurate. To achieve good precision, the %CV should be < 15 % (FDA, 2001). Results indicated good intraday precision with % CV < 5 (table A2). To evaluate the interday precision of the BCA assay fluctuations in the absorbance of a 500 µg/ml of nisin Z was evaluated for three days. Three measurements were made per day and the %CV calculated for each day as well as between days. The %CV should be < 15 % for interday precision (FDA, 2001). Results indicate good interday precision with % CV < 5 (table A3). Appendix A 160 Table A3: Interday precision of the BCA assay for the quantification of nisin Z. 500 ug/ml Nisin Z Absorbance at 562 nm Day 1 Day 2 Day 3 Between days 0.6498 0.6421 0.6240 0.6734 0.6783 0.6560 0.6355 0.6491 0.6922 0.6491 0.6509 0.6368 Average 0.6734 0.6491 0.6368 0.6531 Stdev 0.022 0.007 0.013 0.019 %CV 3.210 1.071 2.120 2.855 A4.5. Robustness It was necessary to validate the use of the modified BCA assay for the quantification of nisin Z in the presence of lipids. The absorbance of blank particles at 562 nm was 0.0198± 0.0036, which was calculated as a concentration of 10.775±2.760 µg/ml when interpolated into the calibration curve. This concentration was below the LLOQ (23.9464 ug/ml). The sample containing a 100 µg/ml Nisin Z in the presence of lipids gave an overestimation of the amount of nisin Z (114.5385±3.026157 µg/ml). This could be corrected to give a concentration of 103.7634±0.265451 µg/ml when the absorbance of the sample containing blank lipid nanoparticles was subtracted (figure A4). Therefore, the modified BCA assay proved to be suitable for the determination of the amount of nisin Z in the presence of lipids. Appendix A 161 Figure A4: Effect of lipid nanoparticles on the modified BCA assay. n=2 A5. Validation of the chloroform extraction method used to determine entrapment efficiency of lipid nanoparticles A methanol/chloroform precipitation method was initially tested to measure the entrapment of nisin Z in lipid nanoparticles, however; the yield of the nisin Z recovered with methanol precipitation was low. A chloroform extraction method, which gave acceptable yields of nisin Z, was used to determine the entrapment of nisin Z in lipid nanoparticles (Burianek and Yousef, 2000). The chloroform extraction method was validated in terms of linearity, accuracy, precision and robustness. Appendix A 162 A5.1. Linearity A calibration curve was made using 0, 25, 50, 125, 250 and 500 µg/ml nisin Z in 0.01 N HCl. This calibration curve was compared to a calibration curve subjected to the chloroform extraction method. The chloroform extraction was performed by adding 200 ul chloroform to each concentration respectively. To this 500 µl 0.01 N HCl was added and the samples were vigorously vortexed. Samples were then centrifuged (HERMLE Labortechnik GmbH, Germany) at 12,000 rpm and 22ºC for 1 hour. Nisin Z remained in the interface between the aqueous layer and the chloroform layer, the chloroform layer was carefully removed. The interface was then resuspended in the remaining 0.01 N HCl at 40 ºC and when dissolved completely the volume was adjusted to 500 µl. The BCA assay was then performed as described in section A1. The effect of the extraction method on linearity was evaluated. Good linearity was achieved with the chloroform extraction method > 0.990 (figure A4). When comparing the slopes of the two graphs in figure A5 the recovery of nisin Z following the chloroform extraction method was calculated as 96.313 %, indicating good accuracy (Walfish, 2006). Figure A5: Calibration curve for nisin Z and chloroform extracted nisin Z. n=3 Appendix A 163 A5.2.Accuracy and precision The accuracy and precision of the extraction method were evaluated by using 100 µg/ml nisin Z which was subjected to the chloroform extraction method. Three extractions were prepared and the measurements made in duplicate. The amount of nisin Z recovered was calculated by interpolating the absorbance measurements into a calibration curve. High yields of nisin Z could be recovered with the chloroform extraction method, without affecting the accuracy and precision of the analytical method (table A4). Average percentage recovery of 99.168 ± 5.205 (within the range of 95 -105 %) and %CV of 5.25 % (< 15 %). Table A4: Accuracy and precision of chloroform extraction method Extraction minus lipid n Amount recovered (µg/ml) Average±SD %CV (1) 94.68551 95.33559 95.01054±0.4596744 0.4838141 (2) 96.22945 98.7485 97.48897±1.781238 1.827117 (3) 107.1995 102.8115 105.0055±3.102785 2.954878 Between repeats (n1-3) 99.16833±5.204809 5.248458 A5.3. Robustness To evaluate the efficacy of the extraction method for the measurement of the entrapment efficiency of the lipid nanoparticles, 10 mg of blank lyophilised lipid nanoparticles were added to 100 µg/ml nisin Z which was then subjected to the chloroform extraction method. Blank lipid nanoparticles were also subjected to the chloroform extraction to compensate for lipid interference. The extraction method was found to be robust in the presence of lipids (table A5) with little interference from lipids (average percentage recovery 101.97 ± 2.125 and %CV 2.084). Table A5: Robustness of chloroform extraction method. Extraction plus lipid n Amount recovered (µg/ml) Average±SD %CV (1) 104.6805 98.7485 101.7145±4.194557 4.123854 (2) 93.7104 106.2583 99.98435±8.872704 8.874092 (3) 101.4663 106.9559 104.2111±3.881734 3.724876 Between repeats (n1 -3) 101.970±2.124923 2.083871 Appendix A 164 A high-resolution propionic acid/urea-PAGE (polyacrylamide gel electrophoresis) gel was made to confirm that the yield of nisin Z, when performing the chloroform extraction in the presence of lipids, was not caused by lipid interference and that it was a true representation of the amount of nisin Z recovered. The method as described by (Chettibi and Lawrence, 1989) was used with some adjustments. All reagents were purchased from Sigma-Aldrich Chemie GmbH (Schelldorf, Germany) unless stated otherwise. A 30 % Acrylamide/Bis-acrylamide solution from Sigma was used and diluted appropriately. The separating gel contained 22.5 % Acrylamide/Bis- acrylamide, 6 M urea, 2 % (V/V) propionic acid, 0.1% (V/V) APS (ammonium peroxodisulfate) and 0.008% (V/V) TEMED (tetramethylethylenediamine). The separating gel was poured into a Bio-Rad gel casting apparatus (Bio-Rad, Hercules, California, United States), overlaid with water-saturated isopropanol and left to set. After the gel was set the isopropanol was thoroughly removed by using filter paper. A stacking gel containing 8 % Acrylamide/Bis-acrylamide, 6 M urea, 2 % (V/V) propionic acid, 0.1% (V/V) APS and 0.008% (V/V) TEMED was then made and placed on top of the separating gel. A 10 well comb (Bio- Rad, Hercules, California, United States) was then placed into the gel and the gel was left to set at room temperature. Sample loading buffer contained 30 % (M/V) sucralose and 0.02 % (V/V) neutral red in 2 % (V/V) acetic acid. To load samples into the gel 10 µl samples containing 100 µg/ml nisin Z was mixed with 10 µl of the sample loading buffer. The samples were added to the respective wells. Electrophoresis buffer consisted of 2 % (V/V) Acetic acid. The current was conducted from the anode (positive) to the cathode (negative) at 150 V for 90 minutes. However, before sample was loaded the gel was pre-run for 3 hours at 140 V. After electrophoresis of the samples the gel was stained in Coomasie stain (45 % (V/V) methanol, 10 % (V/V) acetic acid, 0.4 % (M/V) coomasie brilliant blue R250) overnight by incubating it at room temperature and shaking it at 100 rpm. Following the overnight staining the gel was destained (45 % (V/V) methanol, 10 % (V/V) acetic acid) for 2 hours. The destain solution was exchanged three times within the 2 hours. Finally, the destained gel was rinsed and the gel was documented by scanning (HP digital document scanner). The Propionic acid/urea-PAGE gel (figure A6) indicates that the amount of nisin Z recovered when performing the chloroform extraction in the presence of lipids, compared to the same amount of nisin Z not subjected to the chloroform extraction in the absence of lipids. Appendix A 165 FigureA6: Propionic acid/Urea PAGE gel of 100 µg/ml Nisin Z (Lane A) and the recovery of 100 µg/ml Nisin Z following the chloroform extraction in the presence of lipid (Lane B). A4. Conclusion Based on the results it could be concluded that the modified BCA assay and the assay conditions were suitable for the quantification of nisin Z. The assay conditions provided excellent linearity, precision and accuracy for the quantification of nisin Z. Lipid interference could be eliminated with the modified BCA assay. The chloroform extraction method proved to be suitable for the extraction of nisin Z from the lipid nanoparticle formulations. References ARMBRUSTER, D. A. & PRY, T. 2008. Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev, 29 Suppl 1, S49-52. BURIANEK, L. L. & YOUSEF, A. E. 2000. Solvent extraction of bacteriocins from liquid cultures. Lett Appl Microbiol, 31, 193-7. CHETTIBI, S. & LAWRENCE, A. 1989. High resolution of honey bee (Apis mellifera) venom peptides by propionic acid/urea polyacrylamide gel electrophoresis after ethanol precipitation. Toxicon, 27, 781-7. FDA. 2001. Food and Drug Administration . Guidance for Industry: Bioanalytical Method Validation. US Department of Health and Human Services, FDA, Center for Drug Evaluation and Research; Rockville, MD: 2001 [Online]. Available: Appendix A 166 http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Gui dances/UCM070107.pdf. [Accessed 10 February 2015]. ICH. 2006. International Council for Harmonisation. Harmonized tripartite guideline, validation of analytical procedures, text and methodology. ICH Q2R1 [Online]. Available: http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guida nces/ucm073384.pdf. [Accessed 10 February 2015] MORTON, R. E. & EVANS, T. A. 1992. Modification of the bicinchoninic acid protein assay to eliminate lipid interference in determining lipoprotein protein content. Anal Biochem, 204, 332-4. SMITH, P. K., KROHN, R. I., HERMANSON, G. T., MALLIA, A. K., GARTNER, F. H., PROVENZANO, M. D., FUJIMOTO, E. K., GOEKE, N. M., OLSON, B. J. & KLENK, D. C. 1985. Measurement of protein using bicinchoninic acid. Anal Biochem, 150, 76- 85. TIWARI, G. & TIWARI, R. 2010. Bioanalytical method validation: An updated review. Pharmaceutical Methods, 1, 25-38. WALFISH, S. 2006. Analytical methods: a statistical perspective on the ICH Q2A and Q2B guidelines for validation of analytical methods. Biopharm. Int. 19. [Online]. Available: http://www.biopharminternational.com/analytical-methods-statistical-perspectiveich- q2a-and-q2b-guidelines-validation-analytical-methods. [Accessed 3 March 2015] . 167 Appendix B: Book Chapter The cytotoxic, antimicrobial and anticancer properties of the antimicrobial peptide nisin Z alone and in combination with conventional treatments. Angélique Lewies. Lissinda H. Du Plessis and Johannes F. Wentzel Book chapter accepted for publication InTech Open Cytotoxicity ISBN 978-953-51-5869-1. To be published February 2018. This section is written according to the guidelines as set by the publisher Appendix B 168 Appendix B 169 The cytotoxic, antimicrobial and anticancer properties of the antimicrobial peptide nisin Z alone and in combination with conventional treatments Angélique Lewies1, Lissinda H. Du Plessis1 and Johannes F. Wentzel1 1 Centre of Excellence for Pharmaceutical Sciences (PHARMACEN), North-West University, Potchefstroom Campus. Potchefstroom, 2520, South Africa. Abstract: Nisin is an antimicrobial peptide commonly used as a food preservative since 1969. This peptide has potent antimicrobial activity against several Gram- positive bacterial strains, including clinically important and resistant pathogens. The combination of nisin with conventional antibiotics has been shown to improve the antimicrobial activity of these antibiotic agents. Apart from the antimicrobial properties of nisin, this AMP also displays promising anticancer potential towards several types of malignancies. The nisin Z variant is able to induce selective cytotoxicity in melanoma cells compared to non-malignant cells. It was shown that nisin Z disrupts the cell membrane integrity of melanoma cells and that cytotoxicity is likely due to the activation of an apoptotic pathway. In addition, when used in combination with the conventional chemotherapeutic agents, nisin Z has the potential to enhance the cytotoxicity of these chemotherapeutic agents against cultured melanoma cells. Nisin Z has great potential for clinical application considering its low cytotoxicity to non-malignant cells and its effectiveness against Gram-positive bacterial strains and certain cancers. Keywords: Melanoma; Antimicrobial peptide nisin Z; Combination therapy; Selective cancer cytotoxicity; Chemotherapeutic agents; Antibiotic resistance Introduction Antimicrobial peptides (AMPs) are produced by all known living species and exhibit direct microbial killing activity while also playing an important role in the innate immune system [1]. This diverse group of peptides are found in all living species and may be promising alternatives or serve as additives to current antibiotics [2-4]. Many of the more than 2000 known AMPs have been demonstrated to exhibit broad- spectrum antibacterial activity [5] and bacteria are less likely to develop resistance to these peptides compared to conventional antibiotics [6,7]. The lantibiotic nisin, produced by Lactococcus lactis, has promising potential for clinical application with its Generally Regarded As Safe (GRAS) status. This AMP was approved by the World Health Organisation (WHO) in 1969 and the US Federal Food and Drug Administration (FDA) in 1988 for the use as a food preservative [8]. Despite being extensively utilized for food preservation for nearly 50 years, there is very little indication of resistant mutants arising in food products treated with this AMP [8,9]. Nisin is primarily used for its antibacterial activity. However, AMPs, and especially bacteriocins, display selectivity towards cancer cells [10]. Due to the toxicity associated with many conventional chemotherapeutic agents, as well as the development of chemotherapy resistance [11-13], there is a need for the Appendix B 170 development of novel anti-cancer therapies. Furthermore, to overcome chemotherapy resistance, the efficacy of chemotherapeutic agents can be enhanced by the co-administration of multi-functional agents to achieve synergistic interactions [14,15]. The ability of nisin to increase the activity of the chemotherapeutic drug doxorubicin was investigated in vivo by Preet and co-workers. Nisin, when used in combination with doxorubicin, enhanced the anti-cancer activities of doxorubicin. Apoptosis could be detected upon treatment of mice with induced skin carcinogenesis. However, the exact mechanism by which nisin exerts its anti-cancer activities was not known [16]. Antimicrobial properties of the antimicrobial peptide nisin A report published in 2016 projects that resistance to antibiotics could potentially lead to 10 million deaths per year by 2050 [17]. Moreover, the estimated economic impact of microbial resistance will be massive, costing nearly 100 trillion US dollars while leading to sharp decreases in the gross domestic product. Microbial resistance against conventional antibiotic agents is a serious hazard to the effective treatment of numerous diseases. This upsurge in antibiotic resistance has stimulated research into the development of alternative antimicrobial agents. Antimicrobial peptides are considered promising alternatives to current antibiotics and have the potential to replace certain antibiotics or to be used synergistically in combination with existing antimicrobial agents [2,18]. 2.1 Anti-bacterial effects of Nisin Nisin was discovered in the same year as penicillin but was quickly overshadowed by this antibiotic due to penicillin’s ease of mass production and low manufacturing costs [19]. Nisin is a 3.5 kDa polycyclic peptide consisting of 34 amino acids and is produced by the non-pathogenic bacteria Lactococcus lactis [20]. Two naturally occurring variants of this peptide are nisin A and nisin Z. These two variants are structurally identical with the exception a single amino acid at position 27 where histidine occurs in nisin A while asparagine is found in nisin Z [20]. Both variants display similar antimicrobial activity but nisin Z is more soluble at neutral pH [21,22]. In Gram-positive bacteria, nisin exhibits a dual mode of action by binding to lipid II on the bacterial membrane resulting in the inhibition of cell wall synthesis and the formation of pores in the bacterial cell membrane [23]. The antimicrobial effects of nisin Z against Gram-negative bacteria are largely inadequate. However, the activity towards Gram-negative bacteria can be improved by using ethylenediaminetetraacetic acid (EDTA) and the nonionic surfactant Tween®80 [24,25] (Figure 1). Appendix B 171 Figure 1. Minimum inhibitory concentrations (MIC) of nisin Z for Gram-positive and Gram-negative bacterial strains. The effect of EDTA (200 µM) on the MIC of E.coli is also demonstrated. The glycopeptide antibiotic, vancomycin, also binds to lipid II to inhibit cell wall synthesis, albeit at a different amino acid moiety. Vancomycin is one of the last line treatments against several Gram-positive antibiotic-resistant bacteria including methicillin-resistant Staphylococcus aureus (MRSA) [26,27]. Disturbingly, clinical variants of MRSA have been isolated of which the lipid II pentapeptide have mutated to acquire resistant towards vancomycin. These strains contain the vanA-type gene cluster where the terminal D-Ala has been changed to D-Lactate in the lipid II pentapeptide [28]. Due to its different binding motif, nisin remains active against the vanA-type resistant strains [29]. This shows the potential of nisin to bolster the antimicrobial defences against antibiotic-resistant bacterial strains. Nisin has promising potential for clinical application with its GRAS status and approval by both the FDA and WHO, considering its low cytotoxicity and the fact that it considered safe for human consumption. Currently is employed as a food preservative in nearly 50 countries to guard food against spoilage resulting from pathogens such as Staphylococcus aureus, Listeria monocytogenes, Clostridium botulinum [30]. In addition, nisin has also been demonstrated to possess antibacterial activity against several clinically relevant pathogens including vancomycin-resistant Enterococci, Streptococcus pneumonia and methicillin-resistant Staphylococcus aureus [31.32]. Mastitis-causing Staphylococcus strains have a tendency to develop resistance to antibiotics [33,34]. Nisin has been successfully applied as a sanitizer against mastitis Appendix B 172 causing Staphylococcus and Streptococcus species in lactating cows even when these species are antibiotic resistant [35.36]. Three nisin based products were developed for the treatment of bovine mastitis, namely Ambicin N® (Applied Microbiology, Inc., New York) and Mast Out® as well as Wipe Out® Dairy wipes (ImmuCel Corporation, Maine, USA) [30]. In vivo nisin has also been shown to be an effective and safe alternative to antibiotics in the treatment of staphylococcal mastitis during lactation in pregnant women [37]. Antibacterial agents possessing various modes of action are particularly of interest in the fight against antimicrobial resistance as it is considered to be more challenging for bacteria to develop resistance against multiple mechanisms concurrently. This has proven true in the case of nisin, as there is very little evidence of transmissible and stable resistance occurring after nearly 50 years of treating food products with this AMP [37-39]. 2.2 AMPs as antibiotic adjuvant therapy The discovery and subsequent development of a wide range of antibiotics have revolutionized modern health care. Over the last century, the introduction of antibiotics drastically reduced morbidity and mortality. Today, antibiotics are readily available to the global population and effective antibiotic agents have been developed against the majority of illness-causing bacteria. Ironically, the success of antibiotics has resulted in these drugs being misused, leading to the accelerated development of antimicrobial resistance amongst many bacterial species. Antibiotic resistance is making the effective treatment of numerous infections no longer achievable and there is a pressing need for alternative therapeutic approaches. Antibiotic adjuvant therapy (to achieve synergistic interactions, although additive interactions are also favoured) can be considered a promising strategy to combat antibiotic resistance. Combination of antibiotics and AMPs that possess different modes of action are valuable in the fight against antimicrobial resistance as it is unlikely for bacteria to develop resistance against multiple mechanisms simultaneously. Several studies have demonstrated synergism between nisin and conventional antibiotics. Nisin displayed synergism with the antibiotics, colistin and clarithromycin, against the common Gram-negative bacteria, Pseudomonas aeruginosa [40]. Synergistic effects were also observed with streptomycin, penicillin, rifampicin and lincomycin against P. fluorescens as well as the antibiotic-resistant variants of this strain [41]. Daptomycin, teicoplanin and ciprofloxacin displayed synergism against MRSA biofilms [4]. In a study by Dosler and Gerceker, nisin- antibiotic combinations were shown to have synergistic interactions against clinical isolates of methicillin-susceptible S. aureus (MSSA), MRSA and Enterococcus faecalis. A major finding from their study was that a high incidence of synergistic interactions occurred with a nisin-ampicillin combination against MSSA and nisin- daptomycin combination against E. faecalis strains [42]. When nisin is combined with penicillin, chloramphenicol or ciprofloxacin, biofilm formation of E. faecalis was significantly reduced [43]. In a previous study, we also evaluated the interaction of the nisin Z variant with conventional antibiotics [24]. Antibiotic-nisin Z combinations (1:1) were evaluated on Staphylococcus epidermidis (ATCC 12228) and Staphylococcus aureus (ATCC 12600) seen as nisin is principally effective against Gram-positive bacterial species. Appendix B 173 Several conventional antibiotics with different mechanisms of action against Gram- positive bacteria were selected and included methicillin; vancomycin; ampicillin; tetracycline; gentamicin and novobiocin. The minimum inhibitory concentration (MIC) was used as a reflection of the bacterial cytotoxicity following exposure to the antibiotic-nisin Z combinations. The MIC was determined using a modified broth microdilution method [44], where the p-iodonitrophenyltetrazolium violet (INT) was replaced with the yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT). The interactions between the antibiotics and nisin were determined using the fractional inhibitory concentrations (FIC) [45] and values were interpreted as ƩFIC ≤ 0.5 synergistic, ƩFIC > 0.5 to 1.0 additive, ƩFIC > 1.0 and < 4.0 indifferent and ƩFIC ≥ 4.0 antagonistic. Bacterial treatment with nisin Z-antibiotic combinations resulted in the identification of three additive and two synergistic combinations. Nisin Z displayed an additive effect (ƩFIC > 0.5 to 1.0) when combined with ampicillin and gentamicin in S. aureus (Table 1). Table 1. MIC values and ƩFIC values for antibiotic-nisin Z combinations Bacterial Strain MIC of Nisin Z (µg/ml) MIC of Antibiotic (µg/ml) Nisin Z:Antibiotic (1:1) ƩFIC S. epidermidis Nisin Z (9.17) Methicillin (1.88) 2.68 Vancomycin (2.50) 2.55 Ampicillin (16.67) 0.71 Tetracycline (80.00) 3.65 Gentamicin (1.04) 2.23 Novobiocin (1.46) 0.50 S. aureus Nisin Z (10.00) Methicillin (1.88) 1.06 Vancomycin (2.50) 2.50 Ampicillin (1.04) 0.66 Tetracycline (0.47) 1.40 Gentamicin (6.67) 0.94 Novobiocin (2.29) 0.17 Highlighted values represent ƩFIC values which indicate positive interactions between nisin Z and antibiotics where; ≤0.5 synergistic, >0.5-1.0 additive, 1.1-3.9 indifferent and ≥ 4.0 antagonistic Appendix B 174 Furthermore, S. epidermidis treated with ampicillin-nisin Z combination also showed an additive interaction. Novobiocin-nisin Z combinations showed synergistic interactions when used against S. epidermidis and S. aureus. Novobiocin, as part of the aminocoumarins antibiotic group, is able to indirectly block DNA replication by effectively inhibiting bacterial DNA gyrase. Novobiocin-nisin Z combination was particularly effective in the treatment of S. aureus as a dramatic reduction in the ƩFIC was witnessed. This may be due to the different, but complementary, mechanisms of actions of nisin Z and novobiocin. As the lipid II-nisin Z complex forms pores in the bacterial membrane, hydrophobic novobiocin can pass through the cell membrane to interact with the DNA gyrase of S. aureus. This is only speculation and the exact synergistic mechanism of should be examined further. This in vitro study shows the potential of nisin Z for the use as an adjuvant with conventional antibiotics. AMP-antibiotic combination therapy may aid in reinforcing the defences against resistant organisms by making it more challenging for a bacterial strain to adapt to multiple antimicrobial mechanisms. Furthermore, novobiocin is used for the treatment of mastitis in lactating cows [46] and as previously mentioned some nisin based products have been developed for the treatment of mastitis. The synergistic interactions between nisin and novobiocin make this combination especially of interest for developing novel formulations for the treatment of mastitis. 3 Cytotoxic effects of nisin on non-malignant mammalian cells It is clear that nisin is an effective antimicrobial agent which can inhibit the growth of/kill several Gram-positive bacterial species, including food-borne pathogens such as Staphylococcus aureus, Listeria monocytogenes and Clostridium botulinum as well as exhibiting activity against many clinical important pathogens such as vancomycin-resistant Enterococci (VRE), Streptococcus pneumonia and MRSA [32,47,48]. Despite having exceptional antimicrobial activity, many AMPs also exhibit high toxicity to mammalian cells. An example of a cytotoxic AMP is melittin, the main active component of apitoxin (bee venom). Melittin has excellent antibacterial activity and the antimicrobial mechanism of this AMP is most likely the permeabilisation of cell membranes by pore formation resulting in cell lysis and death [49]. Although melittin has effective broad-spectrum antimicrobial activity, this AMP is extremely toxic to mammalian cells even at very low concentrations. As mentioned before, nisin has a Generally Regarded as Safe (GRAS) status and is considered safe for human consumption. The Accepted Daily Intake (ADI) of nisin was determined by the FDA as 2.94 mg/per day (0.049 mg/kg body weight/day) in 1988, prior to receiving GRAS status [50]. In a study by Joo and co-workers, mice were exposed to a concentration of nisin more than x1000 (150 mg/kg body weight/day) the recommended ADI over a period of 3 weeks with no signs of cytotoxicity [51]. In another study, mice were treated with doses of 800 mg/kg body weight/day (more than 10 000 times higher than the recommended ADI) ultra-pure nisin Z for 3 weeks without any evidence of toxicity [52]. In both these studies, long- term (> 3 weeks) treatment with high concentrations of nisin did not result in any observable toxicity. We also investigated cytotoxicity of nisin Z towards mammalian cells using the MTT assay to measure metabolic activity and the lactate dehydrogenase (LDH) assay to indicate membrane integrity. The non-malignant human immortalised keratinocyte Appendix B 175 (HaCaT) cells were employed for cytotoxicity testing and cultured under normal conditions [24]. Briefly, HaCat cells were seeded in a 96-well plate and incubated until ~90% confluent. Synthetic melittin was used (≥ 97% HPLC from Sigma-Aldrich) as a positive AMP control for cytotoxicity. After 24 hours exposure to nisin Z or melittin (2.5-40 µg/ml), the MTT assay was performed as described previously [24]. The ability of NAD(P)H-dependent cellular oxidoreductase enzymes to reduce MTT to formazan is considered a reflection of the number of viable cells present. Cell viability is expressed as a percentage relative to the untreated control, which was set as being 100 % viable. For an assay positive control, cells were exposed to 0.01% Triton-X 100 (Sigma-Aldrich, St Louis, MO, USA). To investigate the effect of the two AMPs on cell membrane integrity, the CytoTox- ONETM Homogeneous Membrane Integrity Assay (Promega, Madison, WO, USA) was employed. This assay determines the release of lactate dehydrogenase (LDH) into the culture media from cells with impaired cell membranes. HaCat cells were exposed to melittin and nisin Z as described earlier. A lysis solution (Promega) was used as a maximum LDH release positive control. The LDH release assay was performed as described previously [24]. Results are conveyed relative to the untreated control (set to 0% LDH release) and the maximum release sample (set to 100% LDH release). Cytotoxicity data (Figure 2) shows that nisin Z did not negatively affect the cell viability of HaCat cells. Figure 2. Cytotoxicity assay of HaCat cells exposed to the AMPs melittin and nisin Z. (A) MTT assay. (B) LDH release assay. Vehicle control groups are represented by 0 mg/ml. Values represent mean stdev n = 3. ***p < 0.001 compared to the vehicle control group. The MTT assay indicates that the ability of NAD(P)H-dependent cellular oxidoreductase enzymes to reduce MTT to formazan was not affected by the exposure to the tested nisin Z concentrations. Indicating that nisin Z did not Appendix B 176 negatively affect the cell viability of HaCat cells. The LDH assay also showed that there was no significant increase in the release of LDH, indicating that nisin Z did not cause any measurable membrane damage. On the other hand, both the MTT and LDH assays showed that relatively low concentrations of melittin led to a considerable increase in cytotoxicity in HaCat cells. These in vitro results eco many of the recent in vivo findings, showing that nisin exposure to non-malignant cells has very little to no cytotoxic effects. Even at concentrations of nisin Z that exceeds the MICs for S. aurues and S. epidermids (figure 1) no toxicity was observed. Keeping in mind that nisin is an effective antimicrobial agent against several Gram-positive bacterial species including clinical important and resistant pathogens, this AMP shows promising potential for clinical application. 4 Cytotoxic effect of nisin on malignant cells Over the last few decades, great strides have been made in cancer treatment and therapies, leading to the steady decline of cancer death rates [53]. Despite these developments, many current cancer therapies are still associated with high cytotoxicity and lack specificity. There is consequently still a need for the development of novel anti-cancer therapies. AMPs, especially bacteriocins, display selectivity towards cancer cells [10]. These AMPs are therefore potential alternative candidates to current chemotherapeutic agents. AMPs can also be applied as adjuvants to chemotherapeutic agents to lower the therapeutic doses needed with the intention of quelling the toxicity of these treatments. Studies have previously investigated the anti-tumour potential of nisin in vitro and in vivo for head and neck squamous cell carcinoma (HNSCC) [52]. The study by Joo and co-workers indicated that nisin has the ability to selectively induce apoptosis, cell cycle arrest and reduce cell proliferation in HNSCC cells, compared to primary keratinocytes in vitro [51]. In vivo, nisin treatment reduced the overall tumour burden compared to non-nisin treated groups, in a floor-of-mouth oral cancer xenograft mouse model. Also, to examine the mechanism by which nisin facilitates its anti- proliferative and pro-apoptotic effects on HNSCC cells, the effect of nisin-treatment on the expression of 39 000 genes was examined by using Affymetrix gene arrays. The expression of multiple genes was altered, including those in the apoptotic and cell cycle pathways, membrane physiology, energy and nutrient pathways, ion transport, and signal transduction and protein binding pathways. The CHAC1 gene, a cation transport regulator and apoptosis mediator were dramatically up-regulated. This study was the first to show that the antibacterial food preservative nisin could effectively reduce and prevent tumorigenesis both in vitro and in vivo. 4.1 Cytotoxic effects of nisin Z on melanoma cells We also evaluated the potential of nisin Z to induce selective cytotoxicity to human melanoma cells in vitro. Melanoma is the leading cause of skin cancer-related deaths [54,55]. Contrary to most types of cancer, the frequency of melanoma has been increasing over the last three decades [55]. In addition to a high mortality rate, Melanoma cells also have a sinister tendency to rapidly develop resistance to mainstream chemotherapeutic agents [12,13]. In vitro cytotoxicity of the nisin Z was determined by employing the MTT assay, LDH assay and flow cytometric apoptosis and necrosis analyses. The non-malignant human keratinocyte (HaCat) cell line was Appendix B 177 used as a control. The MTT and LDH assays were performed as described previously [56]. The flow cytometric FITV Annexin V apoptosis assay (BD PharmigenTM, BD Biosciences, San Jose, CA, USA) was employed for the detection of apoptotic cytotoxicity. FITC Annexin emits green fluorescence and its presentation indicates early apoptotic events while propidium iodide (PI) emanates red fluorescence and is associated with late apoptotic or necrotic cells. The quantitative colourimetric MTT assay was used to investigate the cytotoxic effect of nisin Z on cultured melanoma cells as well as non-malignant keratinocytes. There is a clear concentration-dependent decline in cell viability observed in melanoma cells exposed to nisin Z (Figure 3 A). Figure 3. Cytotoxic effects of nisin Z on melanoma (A375) cells. (A): Cell viability was determined using the MTT assay. (B) LDH release from cells following treatment with nisin Z. Keratinocytes (HaCat) were used as a non- malignant control. p < 0.001 compared to the control groups. Appendix B 178 A significant increase in cytotoxicity is observed in melanoma cells after exposure to relatively low concentrations of nisin Z. The IC50 value of melanoma cells exposed to nisin Z is approximately 180 µM. Conversely, the non-malignant keratinocytes exposed to nisin Z presented with considerably higher cell viability, with an IC50 value more than double that of its malignant counterpart. To examine whether the observed cytotoxicity of melanoma cells exposed to nisin Z is the result of membrane damage, the LDH assay was performed. This assay measures the release of lactate dehydrogenase, the cytosolic enzyme, as a result of cellular plasma membrane damage. Results suggest that the exposure of melanoma cells to nisin Z concentrations of 150 µM and higher (Figure 3 B) lead to in a significant increase in LDH release. No significant LDH release was detected in the non- malignant keratinocytes after nisin Z exposure, indicating very little membrane damage. Both the basic cytotoxicity assays (MTT and LDH assay) suggest that nisin Z is selectively more toxic towards cultured melanoma cells compared to non- malignant cells. Flow cytometry was used to investigate whether the cytotoxicity observed in melanoma cells was of apoptotic or necrotic origin. For the non-malignant keratinocyte cells, the flow cytometric analysis indicated that >98% of the cells exposed to 50 µM nisin Z could be considered viable and is comparable to the untreated control (Figure 4). Figure 4. Pie graphs representing the cell population sizes of viable, apoptotic and necrotic non-malignant keratinocyte (HaCat) and melanoma (A375) cells after exposure to 50-200µM of nisin Z for 24 hours. Appendix B 179 A small increase in cytotoxicity is observed at higher concentration. Melanoma cells exposed to 50 µM nisin Z showed a much larger early apoptosis (>17%) population than their non-malignant counterparts. A significant increase in cytotoxicity is observed in melanoma cells exposed to higher concentrations of nisin Z, resulting in approximately half of the cancer cells undergoing apoptosis/necrosis after being exposed to nisin Z concentrations of 100 µM or higher. These results confirm the basic viability data that nisin Z is more selectively cytotoxic towards melanoma cells and give an indication that the cell death observed in these cells is probably due to the activation of an apoptotic pathway. 4.2 The potential of nisin Z to increase the cytotoxicity and selectivity of conventional chemotherapeutic agents Due to the toxicity associated with some conventional chemotherapeutic agents, as well as the constant threat of malignancies evolving chemotherapy resistance [11- 13], there is a necessity for the development of novel anti-cancer therapies. To combat chemotherapy resistance, the efficacy of chemotherapeutic agents can be enhanced by the co-administration of multi-functional agents to achieve synergistic interactions [14,15]. As stated earlier, there is an abundance of studies which investigated the use of nisin as an adjuvant to conventional antibiotics [4, 40-42, 57]. It has been shown that nisin displays anticancer properties; however, inadequate focus has been given to applying nisin as an adjuvant for chemotherapeutic agents. The ability of nisin to increase the activity of the chemotherapeutic drug, doxorubicin, was investigated in vivo by Preet and co-workers [16]. Doxorubicin (Adriamycin) is traditionally employed to treat breast cancer, bladder cancer, lymphoma, and acute lymphocytic leukaemia, to name a few. When combining nisin with doxorubicin, enhanced anti-cancer activities were observed and apoptosis could be detected upon treatment of mice with induced skin carcinogenesis as well as a slight increase in oxidative stress. However, the exact mechanism by which nisin exerts its anti-cancer activities was not determined [16]. It is suggested that AMPs which display anticancer activity should be used in combination with conventional chemotherapeutic agents to enhance the effectiveness of these treatments, prevent recurrence of cancer following treatment and possibly reduce instances of chemotherapy resistance [58,59]. Other studies have also shown that AMPs have the potential to enhance the effectiveness of conventional chemotherapeutic agents. The cytotoxicity of etoposide and cisplatin could be enhanced through the combination with magainin A and magainin G respectively [60]. More recently it was shown that the combination of melittin and 5-fluorouracil enhanced cytotoxic effects against squamous skin cancer cells, while simultaneously reducing the toxicity to normal keratinocytes [61]. There are currently no AMPs that have entered into clinical trials or that are in preclinical development as cancer therapeutics. However, peptide-derived therapies are being recognized for the selectivity and anticancer effectiveness and have been investigated in clinical trials [59]. For example, the peptide asparagine-glycine arginine tumour homing peptide (NGR-hTNF) has completed phase 1 clinical trials and is waiting to enter phase 2 clinical trials to test its effectiveness when used in combination with cisplatin for the treatment of several refractory solid tumours including melanomas [62]. Based on the findings that nisin Z is more selectively cytotoxic to melanoma cells, the cytotoxic effect of the combination of nisin Z with conventional chemotherapeutic Appendix B 180 agents was investigated in cultured melanoma cells. The effect of combinations of nisin Z with conventional chemotherapeutic agents (5-fluorouracil, etoposide, hydroxyurea) on A375 (melanoma) and HaCat (non-malignant keratinocyte) cells was determined by the MTT assay. Cells were exposed to different concentrations of the respective chemotherapeutic agents independently and in combination with 150 µM of nisin Z for 24 hours. Following exposure, the MTT assay was performed as described earlier. Blank and background measurements were subtracted and cell viability is expressed as a percentage relative to the untreated control, which was set as 100 % viable. Possible synergistic interactions were evaluated by comparing the cytotoxicity of combination treatment with mono-treatment on melanoma cells. The chemotherapeutic agent 5-Fluorouracil can inhibit RNA and DNA synthesis leading to cell death. The combination of nisin Z with 5-Fluorouracil increased the cytotoxicity to melanoma cells over the entire concentration range tested compared to the mono-treatment of 5-Fluorouracil (p < 0.05) (Figure 5A), with no significant increase in toxicity to non-malignant keratinocytes (Figure 5B). Appendix B 181 Figure 5. Cytotoxicity of chemotherapeutic agents in combination with nisin Z on melanoma (A375) cells and non-malignant keratinocytes (HaCat) as determined by the MTT assay. (A) Melanoma cells exposed to 5-Fluorouracil (FU) combinations. (B) HaCat exposed to 5-FU combinations. (C) Melanoma cells exposed to etoposide combinations. (D) HaCat exposed to etoposide combinations. (E) Melanoma cells exposed to hydroxyurea combinations. (F) HaCat exposed to hydroxyurea combinations. Vehicle control groups were included and are represented by 0 µM. Results are expressed relative to the untreated controls which were set as being 100 percent viable. Bars represent the standard deviation, n= 4. *p< 0.05, **p<0.01 and *** p< 0.001 for combination compared to chemotherapeutic agent alone. Appendix B 182 The 5-Fluorouracil treatment is initially cytotoxic at 50 µM (p< 0.01 compared to the control) whereas the combination of 5-Fluorouracil and nisin Z only begins to induce toxicity at 200 µM (p < 0.001 compared to the control) in the non-malignant keratinocytes. Results indicate that the 5-Fluorouracil-nisin Z combination is more cytotoxic to melanoma cells compared to the mono-treatment. The anti-cancer activity of 5-Fluorouracil may, therefore, be enhanced by combination treatment with nisin Z. Etoposide is a chemotherapeutic agent that is able to induce DNA strand breaks in cancer cells by interfering with type II topoisomerase, ultimately inducing apoptosis. When combining etoposide with nisin Z it was found that the activity towards melanoma cells was enhanced compared to mono-treatment across the entire concentration range (p < 0.001) (Figure 5 C), with no increase in cytotoxicity to non-malignant keratinocytes (Figure 5 D). In melanoma cells, the combination of nisin Z with etoposide had a higher level of activity at the lowest concentration tested compared to the highest concentration for mono-treatment (p< 0.001). The anti- cancer activity of etoposide can, therefore, be significantly enhanced through the combination of nisin Z. Hydroxyurea is able to induce DNA damage and inhibit DNA synthesis. The combination of nisin Z with hydroxyurea was able to increase the cytotoxicity to melanoma cells at concentrations of between 25 -400 µM compared to the mono-treatment of hydroxyurea (p < 0.01) (Figure 5 E), with no significant increase in toxicity to non-malignant keratinocytes (Figure 5 F). To evaluate if possible synergistic interactions occurred between the chemotherapeutic agents and nisin Z, the cytotoxicity of melanoma cells following the mono-treatment of the respective chemotherapeutic agents (50 µM) was compared to that of the mono-treatment of nisin Z (150 µM), followed by that of the combination (50 µM chemotherapeutic agent + 150 µM nisin Z). Synergism occurs when the combined effects of the different components are greater than their individual effects. The cell viability of melanoma cells was significantly lower for all combinations compared to mono-treatment with the chemotherapeutic agent alone (p< 0.05) (Figure 6). However, the only combination that displayed synergism was the combination of nisin Z with etoposide. Figure 6. Cytotoxicity results for mono-treatment and combinations of chemotherapeutic agents (50 µM) and nisin Z (150 µM) as determined by the MTT assay. Nisin Z was combined with (A) etoposide, (B) 5-Fluorouracil and (C) hydroxyurea. Bars represent the average and error bars the standard deviation, n= 4. **p< 0.01 and ***p< 0.001 for combination compared to chemotherapeutic agent alone. #p<0.05 and ##p<0.01 for combination compared to nisin Z alone Appendix B 183 The AMP nisin, which is considered safe for human consumption, not only displays antibacterial properties but also anti-cancer activities. Although the use of nisin as an adjuvant for conventional antibiotics has been investigated extensively, there are few studies investigating nisin as an adjuvant for conventional chemotherapeutic agents. Nisin also exhibits immune-modulatory properties. We have shown that nisin Z induces selective cytotoxicity to melanoma cells through an apoptotic pathway. These properties make nisin Z an attractive anti-cancer agent to be used alone or in combination with current chemotherapeutic agents to enhance anti-cancer properties of these agents while also potentially combatting chemotherapy resistance. Here it was shown that combinations of nisin Z with 5-Fluorouracil, hydroxyurea and etoposide was able to enhance the cytotoxicity to melanoma cells, while no significant increase in toxicity toward non-malignant keratinocytes were observed. Especially of interest is the consequence of nisin Z on the effectiveness of etoposide, seeing as etoposide resistance is known in melanoma [63,64]. The combination of nisin Z with etoposide was able to significantly and selectively enhance the cytotoxic effect etoposide to melanoma cells. Synergism was also observed when combining nisin Z and etoposide with regards to the cytotoxic effect in melanoma cells. Based on all the properties of nisin Z and its GRAS status it could, therefore, be considered an adjuvant for conventional chemotherapeutic agents. Conclusion The majority of AMPs exhibit direct microbial killing activity and occur in all living species as an important part of their innate immune system. Due to the co-evolution of AMPs and bacteria, bacterial species are less likely to develop resistance to these peptides compared to conventional antibiotics. The lantibiotic, nisin, has promising potential for clinical application as it exhibits very low cytotoxicity to mammalian cells while displaying potent antimicrobial activity against several common foods borne and clinically important Gram-positive bacteria. The use of nisin against Gram- negative bacteria still remains limited. Nisin can be considered a promising adjuvant for antibiotics in the treatment of Gram-positive bacteria. Antibiotic-nisin combinations can potentially be used to lower the therapeutic dose of antibiotic treatments while also enhancing the antimicrobial activity by employing multiple modes of action. With multiple antimicrobial mechanisms concurrently in play, these combinations can hinder the development of antibiotic resistance. We have demonstrated that nisin Z displays synergism when combined with novobiocin against S. aureus. This bacterial species is associated with mastitis. Both nisin based products and novobiocin are used for the treatment of mastitis. The synergistic interactions between nisin and novobiocin make this combination especially of interest for developing novel formulations for the treatment of mastitis (Figure 7 A). Appendix B 184 Figure 7. Summary of the antimicrobial and anticancer properties of nisin Z alone and in combination with conventional therapies. (A) The antimicrobial effects and mechanisms of action of nisin Z and selected antibiotics alone and in combination on gram-positive bacteria. (B) The cytotoxic effect of nisin Z on cultured melanoma cells and combinations of this AMP with conventional chemotherapeutic agents. Apart from the antimicrobial properties of nisin, this AMP also displays promising anticancer potential towards several types of malignancies. This chapter discussed the anti-cancer potential of nisin Z towards cultured melanoma cells. Results showed that this AMP is more cytotoxic to melanoma cells compared to non-malignant keratinocytes. It was shown that nisin Z disrupts the cell membrane integrity of melanoma cells while also inducing apoptosis in the majority of exposed malignant cells (Figure 7 B). Taking into account these anticancer properties of nisin Z, the cytotoxicity of nisin Z-chemotherapeutic agent combinations to melanoma cells was compared to the mono-treatment with selected conventional chemotherapeutic agents. This study indicated that when used in combination with the conventional chemotherapeutic agents (5-Fluorouracil, hydroxyurea and etoposide), nisin Z has the potential to enhance the cytotoxicity of these conventional chemotherapeutic agents against cultured melanoma cells. Synergism was observed between the nisin Z and etoposide combination. However, this study was only limited to the in vitro effect in melanoma cells with regards to cytotoxicity as measured by the MTT assay. For future in vitro studies, it is suggested that more cancer cell lines be included. The mechanistic interaction between nisin Z and the chemotherapeutic agents should also be investigated. It is also suggested that in vivo studies be conducted similarly to that by Preet and co-workers To assess whether the combination of nisin Z with these conventional chemotherapeutic agents are able to reduce melanoma tumorigenesis in vivo [16]. The effective dosages also need to be determined with in Appendix B 185 vivo assays. Nisin Z has great potential for clinical application considering its low cytotoxicity to non-malignant cells and the effectiveness of this AMP against Gram- positive bacterial strains and certain cancers. However, detailed antimicrobial and anticancer mechanistic interaction studies analysis are lacking and many in vitro results must still be confirmed within in vivo systems. Acknowledgements AL is grateful for financial assistance from the National Research Foundation (NRF) of South Africa (Grant number 94942). Opinions expressed and conclusions arrived at are those of the authors and are not to be attributed to the NRF. Appendix B 186 References [1] Hancock RE, Diamond G. The role of cationic antimicrobial peptides in innate host defences. Trends in microbiology. 2000;8:402-10 [2] Fox JL. Antimicrobial peptides stage a comeback. Nature biotechnology. 2013;31:379-82:10.1038/nbt.2572 [3] Lewies A, Wentzel JF, Jacobs G, Du Plessis LH. The Potential Use of Natural and Structural Analogues of Antimicrobial Peptides in the Fight against Neglected Tropical Diseases. Molecules. 2015;20:15392- 433:10.3390/molecules200815392 [4] Mataraci E, Dosler S. In vitro activities of antibiotics and antimicrobial cationic peptides alone and in combination against methicillin-resistant Staphylococcus aureus biofilms. Antimicrobial agents and chemotherapy. 2012;56:6366-71:10.1128/AAC.01180-12 [5] Wang G, Li X, Wang Z. APD3: the antimicrobial peptide database as a tool for research and education. Nucleic acids research. 2016;44:D1087-93:10.1093/nar/gkv1278 [6] Marr AK, Gooderham WJ, Hancock REW. Antibacterial peptides for therapeutic use: obstacles and realistic outlook. Current Opinion in Pharmacology. 2006;6:468-72:http://dx.doi.org/10.1016/j.coph.2006.04.006 [7] Peschel A, Sahl HG. The co-evolution of host cationic antimicrobial peptides and microbial resistance. Nature reviews Microbiology. 2006;4:529-36:10.1038/nrmicro1441 [8] Shin JM, Gwak JW, Kamarajan P, Fenno JC, Rickard AH, Kapila YL. Biomedical applications of nisin. Journal of applied microbiology. 2016;120:1449-65:10.1111/jam.13033 [9] Cleveland J, Montville TJ, Nes IF, Chikindas ML. Bacteriocins: safe, natural antimicrobials for food preservation. International journal of food microbiology. 2001;71:1-20 [10] Kaur S, Kaur S. Bacteriocins as Potential Anticancer Agents. Frontiers in pharmacology. 2015;6:272:10.3389/fphar.2015.00272 [11] Luqmani YA. Mechanisms of drug resistance in cancer chemotherapy. Medical principles and practice : international journal of the Kuwait University, Health Science Centre. 2005;14 Suppl 1:35-48:10.1159/000086183 [12] Soengas MS, Lowe SW. Apoptosis and melanoma chemoresistance. Oncogene. 2003;22:3138- 51:10.1038/sj.onc.1206454 [13] Wellbrock C. MAPK pathway inhibition in melanoma: resistance three ways. Biochemical Society transactions. 2014;42:727-32:10.1042/BST20140020 [14] Sylvester PW, Wali VB, Bachawal SV, Shirode AB, Ayoub NM, Akl MR. Tocotrienol combination therapy results in synergistic anticancer response. Frontiers in bioscience. 2011;16:3183-95 [15] Wei XQ, Ma HQ, Liu AH, Zhang YZ. Synergistic anticancer activity of 5-aminolevulinic acid photodynamic therapy in combination with low-dose cisplatin on Hela cells. Asian Pacific journal of cancer prevention : APJCP. 2013;14:3023-8 [16] Preet S, Bharati S, Panjeta A, Tewari R, Rishi P. Effect of nisin and doxorubicin on DMBA-induced skin carcinogenesis--a possible adjunct therapy. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 2015;36:8301-8:10.1007/s13277-015-3571-3 [17] O’Neill J. The review on antimicrobial resistancs. Tackling drug-resistant infections globally: Final report and recommendations. [Online]. 2016. Available: https://amr- review.org/sites/default/files/160518_Final%20paper_with%20cover.pdf [Accessed 16 January 2017]. [18] Hancock RE, Sahl HG. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nature biotechnology. 2006;24:1551-7:10.1038/nbt1267 [19] Rogers LA, Whittier EO. Limiting Factors in the Lactic Fermentation. Journal of bacteriology. 1928;16:211-29 [20] Mulders JW, Boerrigter IJ, Rollema HS, Siezen RJ, de Vos WM. Identification and characterization of the lantibiotic nisin Z, a natural nisin variant. European journal of biochemistry. 1991;201:581-4 [21] De Vos WM, Mulders JW, Siezen RJ, Hugenholtz J, Kuipers OP. Properties of nisin Z and distribution of its gene, nisZ, in Lactococcus lactis. Applied and environmental microbiology. 1993;59:213-8 [22] De VWM, Kuipers OP, Siezen RJ. Lantibiotics similar to nisin a, lactic acid bacteria which produce such lantibiotics, method for constructing such lactic acid bacteria and method for preserving foodstuffs with the aid of these lantibiotics and these lactic acid bacteria producing lantibiotics. Google Patents; 2003. [23] Pag U, Sahl HG. Multiple activities in lantibiotics--models for the design of novel antibiotics? Current pharmaceutical design. 2002;8:815-33 Appendix B 187 [24] Lewies A, Wentzel JF, Jordaan A, Bezuidenhout C, Du Plessis LH. Interactions of the antimicrobial peptide nisin Z with conventional antibiotics and the use of nanostructured lipid carriers to enhance antimicrobial activity. International journal of pharmaceutics. 2017;526:244-53:10.1016/j.ijpharm.2017.04.071 [25] Natrajan N, Sheldon BW. Efficacy of nisin-coated polymer films to inactivate Salmonella Typhimurium on fresh broiler skin. Journal of food protection. 2000;63:1189-96 [26] Liu C, Bayer A, Cosgrove SE, Daum RS, Fridkin SK, Gorwitz RJ, et al. Clinical practice guidelines by the infectious diseases society of america for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 2011;52:e18-55:10.1093/cid/ciq146 [27] Tarai B, Das P, Kumar D. Recurrent Challenges for Clinicians: Emergence of Methicillin-Resistant Staphylococcus aureus, Vancomycin Resistance, and Current Treatment Options. Journal of laboratory physicians. 2013;5:71-8:10.4103/0974-2727.119843 [28] Perichon B, Courvalin P. VanA-type vancomycin-resistant Staphylococcus aureus. Antimicrobial agents and chemotherapy. 2009;53:4580-7:10.1128/AAC.00346-09 [29] Hsu ST, Breukink E, Tischenko E, Lutters MA, de Kruijff B, Kaptein R, et al. The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel antibiotics. Nature structural & molecular biology. 2004;11:963-7:10.1038/nsmb830 [30] Cotter PD, Hill C, Ross RP. Bacteriocins: developing innate immunity for food. Nature reviews Microbiology. 2005;3:777-88:10.1038/nrmicro1273 [31] Bartoloni A, Mantella A, Goldstein BP, Dei R, Benedetti M, Sbaragli S, et al. In-vitro activity of nisin against clinical isolates of Clostridium difficile. Journal of chemotherapy. 2004;16:119-21:10.1179/joc.2004.16.2.119 [32] Dosler S, Gerceker AA. In vitro activities of nisin alone or in combination with vancomycin and ciprofloxacin against methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains. Chemotherapy. 2011;57:511-6:10.1159/000335598 [33] Gill SR, Fouts DE, Archer GL, Mongodin EF, Deboy RT, Ravel J, et al. Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. Journal of bacteriology. 2005;187:2426-38:10.1128/JB.187.7.2426-2438.2005 [34] Melchior MB, Vaarkamp H, Fink-Gremmels J. Biofilms: a role in recurrent mastitis infections? Veterinary journal. 2006;171:398-407:10.1016/j.tvjl.2005.01.006 [35] Cao LT, Wu JQ, Xie F, Hu SH, Mo Y. Efficacy of nisin in treatment of clinical mastitis in lactating dairy cows. Journal of dairy science. 2007;90:3980-5:10.3168/jds.2007-0153 [36] Wu J, Hu S, Cao L. Therapeutic effect of nisin Z on subclinical mastitis in lactating cows. Antimicrobial agents and chemotherapy. 2007;51:3131-5:10.1128/AAC.00629-07 [37] Fernandez L, Delgado S, Herrero H, Maldonado A, Rodriguez JM. The bacteriocin nisin, an effective agent for the treatment of staphylococcal mastitis during lactation. Journal of human lactation : official journal of International Lactation Consultant Association. 2008;24:311-6:10.1177/0890334408317435 [38] Gravesen A, Jydegaard Axelsen AM, Mendes da Silva J, Hansen TB, Knochel S. Frequency of bacteriocin resistance development and associated fitness costs in Listeria monocytogenes. Applied and environmental microbiology. 2002;68:756-64 [39] Willey JM, van der Donk WA. Lantibiotics: peptides of diverse structure and function. Annual review of microbiology. 2007;61:477-501:10.1146/annurev.micro.61.080706.093501 [40] Giacometti A, Cirioni O, Barchiesi F, Scalise G. In-vitro activity and killing effect of polycationic peptides on methicillin-resistant Staphylococcus aureus and interactions with clinically used antibiotics. Diagnostic microbiology and infectious disease. 2000;38:115-8 [41] Naghmouchi K, Le Lay C, Baah J, Drider D. Antibiotic and antimicrobial peptide combinations: synergistic inhibition of Pseudomonas fluorescens and antibiotic-resistant variants. Research in microbiology. 2012;163:101- 8:10.1016/j.resmic.2011.11.002 [42] Dosler S, Gerceker AA. In vitro activities of antimicrobial cationic peptides; melittin and nisin, alone or in combination with antibiotics against Gram-positive bacteria. Journal of chemotherapy. 2012;24:137- 43:10.1179/1973947812Y.0000000007 [43] Tong Z, Zhang Y, Ling J, Ma J, Huang L, Zhang L. An in vitro study on the effects of nisin on the antibacterial activities of 18 antibiotics against Enterococcus faecalis. PloS one. 2014;9:e89209:10.1371/journal.pone.0089209 Appendix B 188 [44] Van Vuuren SF, Nkwanyana MN, de Wet H. Antimicrobial evaluation of plants used for the treatment of diarrhoea in a rural community in northern Maputaland, KwaZulu-Natal, South Africa. BMC complementary and alternative medicine. 2015;15:53:10.1186/s12906-015-0570-2 [45] Van Vuuren SF, Suliman S, Viljoen AM. The antimicrobial activity of four commercial essential oils in combination with conventional antimicrobials. Letters in applied microbiology. 2009;48:440-6:10.1111/j.1472- 765X.2008.02548.x [46] Brunton LA, Duncan D, Coldham NG, Snow LC, Jones JR. A survey of antimicrobial usage on dairy farms and waste milk feeding practices in England and Wales. Veterinary Record. 2012;171:296-:10.1136/vr.100924 [47] Brumfitt W, Salton MR, Hamilton-Miller JM. Nisin, alone and combined with peptidoglycan-modulating antibiotics: activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. The Journal of antimicrobial chemotherapy. 2002;50:731-4 [48] Goldstein BP, Wei J, Greenberg K, Novick R. Activity of nisin against Streptococcus pneumoniae, in vitro, and in a mouse infection model. The Journal of antimicrobial chemotherapy. 1998;42:277-8 [49] Dempsey CE. The actions of melittin on membranes. Biochimica et biophysica acta. 1990;1031:143-61 [50] Müller-Auffermann K, Grijalva F, Jacob F, Hutzler M. Nisin and its usage in breweries: a review and discussion. J Inst Brew. 2015;121:309-19 [51] Joo NE, Ritchie K, Kamarajan P, Miao D, Kapila YL. Nisin, an apoptogenic bacteriocin and food preservative, attenuates HNSCC tumorigenesis via CHAC1. Cancer medicine. 2012;1:295-305:10.1002/cam4.35 [52] Kamarajan P, Hayami T, Matte B, Liu Y, Danciu T, Ramamoorthy A, et al. Nisin ZP, a Bacteriocin and Food Preservative, Inhibits Head and Neck Cancer Tumorigenesis and Prolongs Survival. PloS one. 2015;10:e0131008:10.1371/journal.pone.0131008 [53] Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA: a cancer journal for clinicians. 2017;67:7- 30:10.3322/caac.21387 [54] NIH. SEER (Surveillance, Epidemiology and End Results) [Online]. 2017. Available: https://seer.cancer.gov/statfacts/html/melan.html [Accessed 18 August 2017]. [55] ACS. American Cancer Society - Key Statistics for Melanoma Skin Cancer [Online]. 2017. Available: https://www.cancer.org/cancer/melanoma-skin-cancer/about/key-statistics.html [Accessed 13 April 2017]. [56] Wentzel JF, Lombard MJ, Du Plessis LH, Zandberg L. Evaluation of the cytotoxic properties, gene expression profiles and secondary signalling responses of cultured cells exposed to fumonisin B1, deoxynivalenol and zearalenone mycotoxins. Archives of toxicology. 2017;91:2265-82:10.1007/s00204-016-1872-y [57] Rishi P, Preet Singh A, Garg N, Rishi M. Evaluation of nisin-beta-lactam antibiotics against clinical strains of Salmonella enterica serovar Typhi. The Journal of antibiotics. 2014;67:807-11:10.1038/ja.2014.75 [58] Gaspar D, Veiga AS, Castanho MA. From antimicrobial to anticancer peptides. A review. Frontiers in microbiology. 2013;4:294:10.3389/fmicb.2013.00294 [59] Swithenbank L, Morgan M. The Role of Antimicrobial Peptides in Lung Cancer Therapy. Journal of Antimicrobial Agents. 2017;3:134:10.4172/2472-1212.1000134 [60] Ohsaki Y, Gazdar AF, Chen HC, Johnson BE. Antitumor activity of magainin analogues against human lung cancer cell lines. Cancer research. 1992;52:3534-8 [61] Do N, Weindl G, Grohmann L, Salwiczek M, Koksch B, Korting HC, et al. Cationic membrane-active peptides - anticancer and antifungal activity as well as penetration into human skin. Experimental dermatology. 2014;23:326-31:10.1111/exd.12384 [62] Gregorc V, De Braud FG, De Pas TM, Scalamogna R, Citterio G, Milani A, et al. Phase I study of NGR- hTNF, a selective vascular targeting agent, in combination with cisplatin in refractory solid tumors. Clinical cancer research : an official journal of the American Association for Cancer Research. 2011;17:1964-72:10.1158/1078- 0432.CCR-10-1376 [63] Helmbach H, Kern MA, Rossmann E, Renz K, Kissel C, Gschwendt B, et al. Drug resistance towards etoposide and cisplatin in human melanoma cells is associated with drug-dependent apoptosis deficiency. The Journal of investigative dermatology. 2002;118:923-32:10.1046/j.1523-1747.2002.01786.x [64] Kalal BS, Upadhya D, Pai VR. Chemotherapy Resistance Mechanisms in Advanced Skin Cancer. Oncology reviews. 2017;11:326:10.4081/oncol.2017.326 189 Appendix C: Conference poster presentation Synergistic interactions of the antimicrobial peptide nisin Z with conventional antibiotic and the use of nanostructured lipid carriers to enhance antibacterial actiivty. Angélique Lewies, Johannes F. Wentzel and Lissinda H. Du Plessis This poster was presented at the 7th European Molelcular Biology Organisation (EMBO) meeting. which was held in Mannheim, Germany 10 - 13 September 2017. Appendix C 190 Appendix C 191 192 Appendix D: Certificate of analysis for ultra-pure nisin Z Appendix D 193 194 Appendix E: Additional publications E1. Lewies, A., Van Dyk, E., Wentzel, J.F. and Pretorius P.J. 2014. Using a medium- throughput comet assay to evaluate the global DNA methylation status of single cells. Frontiers in genetics. 5:215, 1-6. E2. Wentzel, J.F., Lewies, A., Bronkhorst A. J., Van Dyk, E., Du Plessis, L.H. and Pretorius P.J. 2017. Exposure to high levels of fumarate and succinate leads to apoptotic cytotoxicity and altered global DNA methylation profiles in vitro. Biochimie;135:18-34. doi:10.1016/j.biochi.2017.01.004 A detailed description of author contributions is given at the end of each article. Appendix E 195 Appendix E 196 Appendix E 197 Appendix E 198 Appendix E 199 Appendix E 200 Appendix E 201 Appendix E 202 Appendix E 203 Appendix E 204 Appendix E 205 Appendix E 206 Appendix E 207 208 Appendix F: Proof of ethical training Appendix F 209 210 Appendix G: Proof of language editing Appendix G 211 212 Appendix H: Permission for use of copyright material Appendix H 213 Article I is published in Molecules, the book chapter (Appendix B) is accepted for publication in an InTech Open Book and the addition article (Appendix E1) is publised in Frontiers in Genetics. All of these are open acces articles/publications and are distrubuted under the terms and conditions of the Creative Common Attribution (CC-BY) License*. The current version is CC-BY, version 4.0 (http://creativecommons.org/licenses/by/4.0/) In accordance there is no copyright transfer to the publishers and authors retain copyright to their work. *According to these terms and conditions you are free to: Share- Copy and redistribute the material in any medium or format Adapt – remix, transform, and build upon the material for any purpose, even commercially And the licensor cannot provoke these freedoms as long as the license terms are followed Terms:  Attribution- You must give appropriate credit, provide a link to the license, and indicated if changes were made. You may also do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use The terms and conditions of each of the publishers can be found at: Molecules: http://www.mdpi.com/about/terms-and-conditions InTech Open: https://www.intechopen.com/open-access-policy.html Frontiers in Genetics: https://www.frontiersin.org/Copyright.aspx Appendix H 214 Article III and IV as well as the additional article (Appendix E2) was published in Elsevier journals. Elsevier grants the author the right to include the article(s) in a thesis. The rights of an author are given below. It can also be found at: http://support.elsevier.com/app/answers/detail/a_id/565/ WHAT RIGHTS DO I RETAIN AS AN AUTHOR? As an author, you retain rights for a large number of author uses, including use by your employing institute or company. These rights are retained and permitted without the need to obtain specific permission from Elsevier. These include:  The right to make copies of the article for your own personal use, including for your own classroom teaching use.  The right to make copies and distribute copies (including through e-mail) of the article to research colleagues, for the personal use by such colleagues (but not commercially or systematically, e.g. via an e-mail list or list serve).  The right to post a pre-print version of the article on Internet web sites including electronic pre-print servers, and to retain indefinitely such version on such servers or sites (see also our information on electronic preprints for a more detailed discussion on these points.).  The right to post a revised personal version of the text of the final article (to reflect changes made in the peer review process) on the author's personal or institutional web site or server, with a link to the journal home page (on elsevier.com).  The right to present the article at a meeting or conference and to distribute copies of such paper or article to the delegates attending the meeting.  For the author’s employer, if the article is a ‘work for hire’, made within the scope of the author’s employment, the right to use all or part of the information in (any version of) the article for other intra-company use (e.g. training).  Patent and trademark rights and rights to any process or procedure described in the article.  The right to include the article in full or in part in a thesis or dissertation (provided that this is not to be published commercially).  The right to use the article or any part thereof in a printed compilation of works of the author, such as collected writings or lecture notes (subsequent to publication of the article in the journal). 215