Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. ANTIMICROBIAL PEPTIDES ISOLATED FROM OVINE BLOOD NEUTROPHILS A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, Palmerston North, New Zealand. Rachel C Anderson 2005 Abstract ABSTRACT The aim of the research presented in this thesis was to investigate the properties of the antimicrobial peptides found in ovine blood, in order to assess their potential as a high-value product. Due to the large number of lambs and sheep that are slaughtered New Zealand (approximately 25 million lamb and 5 million sheep per year), there are considerable volumes of ovine blood available for processing (approximately 40 million litres per year). Currently this blood is dried and sold as a low value product. The first objective of this research was to purify and characterise the antimicrobial peptides isolated from ovine neutrophils. A number of proline/arginine-rich peptides, as well as two small fragments of larger proteins, that displayed antimicrobial activity were identified. The second objective of this research was to investigate the mechanism of action of ovine antimicrobial peptides. For this investigation, three ovine peptides, a-helical SMAP29 and proline/arginine-rich OaBac5mini and OaBac7.5mini, were synthesised. Of these, SMAP29 was the most potent. The three peptides all bound Gram-negative bacterial LPS and caused the outer membrane to be permeabilised. SMAP29 caused significant depolarisation of the cytoplasmic membrane that led to cell lysis. However, the other two peptides only caused slight depolarisation of the cytoplasmic membrane, which indicates that they probably passed through the membrane to interact with the inner cellular contents. The third objective of this research was to investigate the morphological changes to bacterial cells induced by the ovine antimicrobial peptides. Transmission electron microscopy and atomic force microscopy confirmed that SMAP29 caused significant damage to the membranes of bacterial cells and induced cell lysis; whereas, OaBac5mini caused minor alterations to the bacterial membranes but did not induce cell lysis. The fourth objective of this research was to determine the effect of the environmental conditions on the activity of the peptides. The peptides were very stable over a range of pH values and when heated to temperatures up to 80°C. The activity of the peptides decreased slightly in the presence of monovalent cations and was inhibited by the presence of divalent cations. The peptides were significantly more active in combination than individually, and they were strongly synergistic with polymyxin B, a peptide antibiotic. The final objective of this research was to develop a pilot-scale extraction process for the isolation of antimicrobial peptides from ovine blood. The laboratory-scale process was simplified and adapted to design a process that could be used industrially. The crude pilot-plant extract was active against a broad-range of food pathogens and disease causing organisms. The antimicrobial peptides found in ovine blood have the potential to be used as biopreservatives for chilled lamb products, or in a topical cream for cuts and grazes; therefore it is recommended that further research is carried out to investigate the above applications and. if successful, the feasibility of commercialising the technology. iii Acknowledgements ACKNOWLEDGEMENTS First and foremost I would like to thank my supervisors. Or Pak-Lam Yu, thank you for taking a keen interest in my project and for teaching me all I need to know to be a successful researcher. Or Brian Wilkinson, thank you for being around when I needed that extra bit of help or advice. Professor [an Maddox, thank you for joining the team to help me with the preparation of this manuscript. I would also like to thank Professor Robert Hancock and his team for allowing me to visit their laboratory at the Department of Microbiology and Immunology, University of British Columbia, for three months, and for supervising and assisting with my bacterial membrane interaction experiments. This work was made possible by the financial support I received from Meat and Wool New Zealand (formerly MeatNZ), in the form of both a doctoral scholarship and project funding. The project was also partially funded by the Massey University Research Fund (MURF), and my research trip to UBC was funded by the C. Alma Baker Trust. This work was made easier by help [ received from numerous people including the ITE technical staff, especially Anne-Marie Jackson and Mike Sahayam, and the staff of Feilding Lamb Packers, who collected the sheep blood for my experiments. [ also received valuable help from the undergraduate and foreign-intern students that assisted on various parts of this project, including David Houlding (laboratory extraction process), Adi Sugiarto (RP-HPLC), Marie Bourin (crude extract MICs) and Andrew Lister (pilot-scale extractions). I received assistance from Aaron Hicks (Institute of Veterinary, Animal and Biomedical Sciences) to prepare the TEM samples, HortResearch to image the TEM samples, and Associate Professor Richard Haverkamp to image the AFM samples. Finally, [ would like to thank family and friends who helped keep me sane throughout this whole process. Other postgrads, especially Craig, Stephen, Roland and Anna, it always helped to know that there were others who shared the same, or worse, difficulties - Good luck to you all. Regan, thank you for caring enough to wade through this thesis to find the spelling and grammatical mistakes - a best friend who doubles as a proof-reader, what more could I ask for? Dad, I would never have made it this far without the support of you and "The Anderson Trust" - I think I was the best fed undergraduate student in town. And finally, Peter, there are not words to describe how much I appreciate you - I look forward to the future we will spend together. I dedicate this thesis to my mother, who [ know would have been proud. Her encouragement, support and love will be with me always. v Table of Contents TABLE OF CONTENTS Abstract ................................................................................................................................ iii Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Table of contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi List of figures . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii List of tables .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi List of abbreviations . . . . . . . . . . . . . . . .. . . . . . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi i i List of publications . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . xx CHAPTER 1 PROJECT INTRODUCTION 1.1 Reason for the research ................................................................................................ 1 1.2 Project objectives .......................................................... . . ........................ . ................ .... 2 CHAPTER 2 ANTIMICROBIAL PEPTIDES LITERATURE REVIEW 2.1 Introduction . .. ............................ ....... . . ......................................................................... 4 2.2 Antimicrobial peptides ................................................................... . ............................. 5 2.2.1 Animal antimicrobial peptides ................. ............................................................. 5 2.2.2 Plant antimicrobial peptides ........................................................ . . ...................... 11 2.2.3 Microbial antimicrobial peptides ........................................................... ........... ... 15 2.3 The animal immune system ........................................................................................ 18 2.3.1 Innate immunity ................. .. . .......... .................................................................... 19 2.3.2 Adaptive immunity ... . .......... . .......... . ............ . . .. . . . . .. ..... ........................................ 24 2.3.3 Role of antimicrobial peptides in animal immune systems . ................... .............. 26 2.4 Potential applications of antimicrobial peptides .......................................................... 28 2.4.1 Applications for antimicrobial peptides ............................... ......... ....................... 29 2.4.2 Possible applications for ovine blood antimicrobial peptides ............................... 31 2.5 Purification and characterisation of animal antimicrobial peptides .............................. 35 2.5.1 Techniques to purify and characterise antimicrobial peptides .............................. 36 2.5.2 Livestock blood antimicrobial peptides . . ......... .................................................... 39 2.5.3 Ovine antimicrobial peptides .............................................................................. 45 2.6 Mechanism of action of animal antimicrobial peptides ............................................... 46 2.6.1 Techniques to determine mechanisms of action of antimicrobial peptides ........... 47 vi Table of Contents 2 .6 .2 Mechanisms of action .... . .... . ................................ ... .... .. . . . . .. ................................ 48 2 .6 .3 Mechanism of action of ovine antimicrobial peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1 2 .7 Morphological changes to microbial cells induced by animal antimicrobial peptides .. 5 1 2 .7 . 1 Techniques to investigate morphological changes ..................... ......... . . .. . . . ...... . . . . 52 2 .7 .2 Morphological changes . . ... ............... . ........ . . . ......... . . . .. . . . .. . . . ........................... . ..... 53 2 .7 .3 Morphological changes induced by ovine antimicrobial peptides . . . . . . . . . . . . . . . . . . . . . . . . 5 3 2 . 8 Effect of environmental condit ions on activity of animal antimicrobial peptides ... ..... 54 2 .8 . 1 Techniques to determine effect of environmental conditions ............... .... . . . ......... 54 2 .8 .2 Effects of environmental condit ions ........... . . . . ...... .. ......... . .......... . . . ...................... 55 2 .8 . 3 Effects of environmental conditions on ovine antimicrobial peptides . . ................ 56 2 .9 Pilot-scale extraction of animal antimicrobial peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 2 . 1 0 Conclusions ...... ........ ... . . . ........... . ... .... . ......... .... ....................... . . . ..... .. ............. .. . ..... . . .. 57 CHAPTER 3 MATERIALS AND METHODS 3 . 1 Materials and methods used for peptide purification . .... . ......... .. . .... . . .. ......................... 59 3 . 1 . 1 Crude extraction ... . . ... . .... . ... . .......................... . . ...... ..... . . ..... . ......... . . . . . . . . . .. . .... . . .. . . . 59 3 . 1 .2 Gel electrophoresis ................ . .... . . . . .. . .. ... . . ... . . ............................. ................... . .. . . 60 3 . 1 . 3 Gel filtration .................. . ................. . . . . ......... . . ................. . .................................. 61 3 . 1 .4 Cationic-exchange chromatography .... . . . . . . . . . . . .... ..... . . . . . . . . . . . ......... ... .. ...... . . .. ......... 6 1 3 . 1 . 5 Peptide purification using HPLC ............................ . .... . . . . ... . . . . ... ......................... 62 3 . 1 .6 Radial diffusion plate assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 3 . 1 . 7 Radial diffusion plate assay MIC method . ...................................... ............ . .. . . . ... 63 3 . 1 . 8 Mass spectroscopy ......... . .. ... . . . . . . . . ..... . . ....................... . . . . . . ...... . . .. ...... . . . . . . . . . .. . . . . . ... 64 3 . 1 .9 N -terminal sequencing ... . ..... . . . .... . .............................. .. . . . . . . ........ . .... . . . .. . ..... . . . . ..... 65 3 . 1 . 1 0 Peptide characterisat ion ...... ... . . . . . . . . . ....................................... .... . . . ............. ... . . . .. . 65 3 . 1 . 1 1 Analysis of proline/arginine-rich sequences ... . .......... . . . . .. . . . .. .. .. . . . ..... . . . .. . . ... . ..... ... 66 3 .2 Materials and methods used for mechanism of action tests . . .. .. ..... ....... . . ........ .. . . . . . . . . . . . 66 3 .2 . 1 Peptide synthesis ................................... . ........ .......... ............ .............................. 66 3 .2 .2 Micro-broth dilution MIC method ...... ... . . . ......................................................... .. 67 3 .2 . 3 Circular dichroism spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 3 .2 .4 LPS binding assay . ........ .... . . . ............... . . ........ . . ........... .... ...... .. . . . ........... . .... . ... . .... 69 3 .2 . 5 Outer membrane permeabil isation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 3 .2 .6 Cytoplasmic membrane depolarisation ..... . ........ .. . ............................................... 70 vii Table of Contents 3 .2 .7 Optical density and viable cell counts over time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1 3 .2 .8 Peptide-DNA binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1 3 .3 Materials and methods used to investigate bacterial cell morphological changes . . . . . . . . 72 3 . 3 . 1 Transmission electron rrUcroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 3 . 3 . 2 AtorrUc force rrUcroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 3 .4 Materials and methods used to assess the effect of condit ions on peptide activity . . . . . . . 74 3 .4. 1 Salt effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 3 .4 .2 Cation effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 3 .4 .3 pH effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 3.4.4 Temperature effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 3 .4 .5 Synergistic effects between test peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 3 .4 .6 Synergistic effects between test peptides and com on antibiotics . . . . . . . . . . . . . . . . . . . . . . . 76 3 . 5 Materials and methods used for the pilot-scale extraction of antimicrobial peptides from ovine blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 3 . 5 . 1 Crude extraction process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 3 . 5 .2 Minimum inhibitory concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 3 . 5 . 3 Transmission electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 3 . 5 .4 Yield calculat ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 CHAPTER 4 ISOLATION AND CHARACTERISATION OF ANTIMICROBIAL PEPTIDES FROM OVINE NEUTROPHILS 4. 1 Introduction . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 4.2 Extraction of crude antimicrobial solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1 4 .3 Purification of antimicrobial peptides using gel filtration and RP-HPLC . . . . . . . . . . . . . . . . . . . . 83 4.4 Characterisation of OaBac5 and variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 4.5 Characterisation of truncated OaBac7.5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 4.6 Characterisation ofOaBac l l and truncates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 4 .7 Minimum inh ibitory concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 4 .8 Sequence analysis of proline/arginine-rich peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 4.9 Purification of antimicrobial peptides using cationic exchange chromatography and RP- HPLC ............................................................ ............................................................. 98 4. 1 0 Other predicted cathelicidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 03 4.11 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 vii i Table of Contents CHAPTER 5 SPECTRUM OF ACTIVITY AND BACTERIAL MEMBRANE INTERACTIONS O F SYNTHETIC OVINE CATHELlCIDINS 5.1 I ntroduction .................................... ........................... . ... ............. ............................. 106 5.2 Minimum inh ibitory concentrations .... .... ... . . ........................................................... . 108 5.3 Circular d ichro ism spectroscopy ................................. ............................................. 111 5.4 LPS binding assay ................................ ................................................................ .... 112 5.5 Outer membrane permeabil isation ........ .......................... . .................. ....................... 116 5.6 Cytoplasmic membrane depolarisation ......................... .............................. .. .. . ... ...... 119 5.7 Kill curves .. ............................................................. ................................................ 123 5.8 DNA binding ................. . ............... . ............................ . ............................... ............. 124 5.9 Conc lusions ............................. .... .... .. . ..... .... ............................................................ 126 CHAPTERS MORPHOLOGY OF BACTERIAL CELLS TREATED WITH SYNTHETIC OVINE CATHELlCIDINS 6.1 Introduction ................... . ...... .......... . . ..... .................................................................. 128 6.2 E. coli TEM results ...... ..................................................................... .............. ......... 128 6.3 S. aureus TEM results ............................................... .. ... . ........... . .......... ................... 129 6.4 S. aureus AFM method development ..................... ............. . .................................... 132 6.5 S. aureus AFM results .............................................................................................. 135 6.6 E. coli AFM method development problems .. . ......................................................... 139 6.7 Conclusions ................... ................. ............ .. ................. .......................................... 141 CHAPTER 7 FACTORS AFFECTING THE ANTIMICROBIAL ACTIVITY OF SYNTHETIC OVINE CATHELlC IDINS AGAINST E. COLl0157:H7 7. 1 Introduction .................. .............. ........... . . ............................................................ . . . . 143 7.2 Effect of salt .......... ........... ... ........... .. . ...................................................................... 144 7.3 Effect of metal ions ................................................ ............................. ..................... 145 7.4 Effect of pH ................ ............................. ................................................................ 148 7.5 Effect of temperature ...... ........................ . . . .............................................................. 150 7.6 S ynergy between peptides ............................................ .......... ..................... ............. 151 7.7 Synergy between peptides and known antibiotics .. . . . . . .. . . ... . ..... . . . ... .. . . . .... ..... ...... .... . . . 153 ix Table of Contents 7 .8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 54 CHAPTER 8 PILOT -SCALE EXTRACTION OF ANTIMICROBIAL PEPTIDES F ROM OVINE BLOOD 8 . 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 57 8 . 2 Crude extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 58 8 . 3 Minimum inhibitory concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 1 6 1 8 . 4 Transmission electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 63 8 . 5 Y ield calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 65 8 .6 Industrial-scale process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 66 8 . 7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 69 CHAPTER 9 CONCLUSIONS AND RECOMMENDATIONS 9 . 1 Summary of research conclusions . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 1 7 1 9 .2 Recommendations for future research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 1 74 9 . 3 Final conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 76 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 77 APPENDIX A1 RAW DATA AND CALCULATIONS FROM C HARACTERISATION STUDIES A 1 . 1 Mass Spectra of the purified HPLC peaks . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 97 A 1 .2 Example calculation of confidence intervals from plate assay raw data . . . . . . . . . . . . . . . . . . . . 203 A1 . 3 Raw data, calculated MICs and 95% confidence intervals for the MICs of the purified peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 APPENDIX A2 RAW DATA AND CALCULATIONS FROM MECHANISM OF ACTION STUDIES A2. 1 Raw data from the micro-broth dilution MIC method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 A2 .2 Example calculation of the mean MIC and confidence intervals for the mean from the raw data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 1 A2 .3 Raw Data from the LPS binding assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 2 A2 .4 Calculation of Imax and I so from LPS binding assay raw data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 5 A2.5 Raw data from the outer membrane permeabil isat ion assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 7 x Table of Contents A2.6 Analysis of variance ofNPN uptake data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 9 A2 .7 Raw data from the outer inner membrane depolarisation assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 A2. 8 Analysis of variance ofDiSC35 release data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 APPENDIX A3 RAW DATA AND CALCULATIONS FROM EFFECT OF CONDITIONS STUDIES A3 . 1 Raw data of MICs at different salt concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 223 A3 .2 Raw data ofMICs at different cation concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 A3 . 3 Raw data of MICs at different pH values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 A3 .4 Raw data of MICs after heating to different temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 A3 . 5 Example calculation of the mean MIC and confidence intervals for the mean from the raw data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 A3 .6 Analysis of variance of MIC data from different conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 APPENDIX A4 RAW DATA AND CALCULATIONS FROM PILOT -SCALE EXTRACTION STUDIES A4. 1 Raw data from the micro-broth dilution MIC method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 1 A4.2 Example calculation of the mean MIC and confidence intervals for the mean from the raw data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 A4.3 Calculation of the settling velocit ies of different types of blood cells . . . . . . . . . . . . . . . . . . . . . . . 233 APPENDIX A5 PEER-REVIEWED PUBLICATIONS A5 . 1 Ovine antimicrobial peptides: new products from an age-old industry . . . . . . . . . . . . . . . . . . . . . . 236 A5 .2 Iso lation and characterisation of proline/arginine-rich cathelicidin peptides from ovine neutrophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244 A5 .3 Antimicrobial activity and bacterial membrane interaction of ovine-derived cathelicidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 A5.4 Investigation of morphological changes to S. aureus induced by ovine-derived antimicrobial peptides using TEM and AFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 A5 .5 Factors affecting the antimicrobial activity of ovine-derived cathelicidins against E. coli 0 1 57 :H7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266 xi List of Figures LIST OF FIGURES Figure 2 . 1 - Examples of the four structural classes of cationic ant imicrobial peptides . . . . . . . . . 6 Figure 2 .2 - Examples of the three groups of defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Figure 2 .3 - Schematic diagram of a cathelicidin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Figure 2 .4 - Schematic diagram of the gene for the human cathelicidin LL-37 . . . . . . . . . . . . . . . . . . . . 1 1 Figure 2 .5 - Structure of thionins. 'A' shows the secondary structures of thionins with six and eight cysteine residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 Figure 2 .6 - Structure of plant defensins. 'A' shows the secondary structures of plant defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 Figure 2 . 7 - Structure of lipid transfer proteins. 'A' shows the secondary structures of lipid transfer proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 Figure 2 . 8 - Structure of hevein- and knottin-type pept ides. 'A' shows the secondary structures of hevein- and knottin-type peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4 Figure 2 .9 - Structures of four Class I bacterioc ins. Nisin A, epidermin and lacticin 48 1 are class la bacterioc ins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6 Figure 2 . 1 0 - Schematic diagram showing the principle mechanisms of innate and adaptive immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 8 Figure 2. 1 1 - Functions of the epithelia in innate immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9 Figure 2 . 1 2 - Mechanism of phagocytosis and intracellular kill ing of microbes. NO is nitric oxide and ROI is reactive oxygen intermediate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Figure 2 . 1 3 - Functions of the natural kil ler (NK) cells. A) NK cells kill infected host cells and B) NK cells activate macrophages to kil l phagocytosed microbes. I L- 1 2 in interleukin- 1 2 and IFN-y is interferon-y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Figure 2 . 1 4 - Pathways of activation of the complement system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Figure 2 . 1 5 - Types and mechanisms of adaptive immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Figure 2 . 1 6 - Specificity and memory in adaptive immunity illustrated by primary and secondary immune response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 5 Figure 2 . 1 7 - Proposed roles of antimicrobial peptides within the innate immune system . . . . . 27 Figure 2 . 1 8 - Schematic diagram of the proposed mechanisms of permeability change o f cytoplasmic membranes caused by antimicrobial peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Figure 3 . 1 - Graph of Ln peptide concentration versus clearing size showing the relationsh ip between the line-of-best-fit and the bounds of the 95% confidence intervals for the line . ..... ... ... . . . ......... . . .. . . . . ....... . ... . . . . . ...... . . . . . . . ... . .. . . ....... . . .. . . . . . .... .. . .. .. . . ..... . .. . . 64 xii List of Figures Figure 3 .2 - Graph of Ln peptide concentration versus c learing size showing the relationship between the l ine-of-best-fit and the bounds of the 95% confidence intervals for the line when the bounds do not cross the x-axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Figure 4 . 1 - Flowchart showing the process used to extract the crude antimicrobial solution from ovine blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1 Figure 4 .2 - Images of typical stained blood samples during the extraction process . . . . . . . . . . . . 82 Figure 4 .3 - Images of typical plate assay results of neutrophil crude extract against three test organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 F igure 4.4 - Typical gel filtration chromatograph resulting from the addition of an ovine neutrophil crude extract into a P 1 0 gel filtrat ion column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Figure 4 .5 - I mage of a typical SDS-PAGE gel of ovine neutrophil extract gel filtration fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Figure 4 .6 - RP-HPLC chromatograph of the second gel filtration fraction (F2) of the ovine neutrophil crude extract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Figure 4.7 - Hydrophobicity plots of the prol ine/arginine-rich cathelic idin peptides . . . . . . . . . . . 96 Figure 4 .8 - Polarity plots of the proline/arginine-rich cathel ic idin peptides . . . . . . . . . . . . . . . . . . . . . . . . 97 Figure 4.9 - Ion-exchange chromatograph for the addit ion of the ovine neutrophil crude extract to a weak cationic exchange co lumn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Figure 4. 1 0 - RP-HLPC chromatograph of the cationic fraction ( F3 and F4) of the ovine crude extract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 00 Figure 5 . 1 - Schematic diagram showing the proposed mechanism of action of antimicrobial peptides against Gram-negative bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 08 Figure 5 .2 - C ircular dichro ism spectra of25mM synthetic ovine antimicrobial peptides . . I I I Figure 5 . 3 - Schematic diagram showing the mechanism invo lved in the lipopolysaccharide binding assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 2 F igure 5 .4 - A typical run showing the changes in the fluorescence of dansyl po lymyxin B due to the addition of SMAP29 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 1 1 3 Figure 5 . 5 - Lineweaver-Burke plot for a typical run of the SMAP29-LPS binding assay. 1 1 4 F igure 5 .6 - Schematic diagram showing the mechanism invo lved in the I -N-phenylnapthyl- amine (NPN) uptake assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 6 F igure 5 . 7 - Uptake of 1 -N-phenylnapthylamine (NPN) by E. coli UB 1 005 cells caused by synthetic ovine peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 8 F igure 5 . 8 - Schematic diagram showing the mechanism involved in the 3 ,3- dipropylthiacarbo-cyanine (DiSC35) assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 20 xiii List of Figures Figure 5.9 - Release of 3,3-dipropylthiacarbocyanine (DiSC35) dye fro m the cytoplasmic membrane of E. coli DC2 cells caused by synthetic ovine peptides . . . . . . . . . . . . . . . . 1 22 Figure 5 . 1 0 - Optical density and viable cell count over time for E. coli 0 I I I treated with synthetic ovine peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 24 Figure 5 . 1 1 - DNA gel showing the running pattern of different ratios of DNA and synthet ic ovine peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 25 Figure 6. 1 - Transmission electron microscope images taken of E. coli 0 I I I cells treated with SMAP29 and OaBac5 mini for one hour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 0 Figure 6.2 - Transmission electron microscope images taken of S. aureus 4 1 63 NCTC cel ls treated with SMAP29 and OaBac5 mini for one hour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 1 Figure 6.3 - AFM images of S. aureus NCTC 4 1 63 cells trapped on a polycarbonate membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 3 Figure 6.4 - AFM images of S. aureus NCTC 41 63 cells grouped together on a polycarbonate membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 33 Figure 6 .5 - AFM 3D representation of S. aureus NCTC 4 1 63 cells grouped together on a po lycarbonate membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 3 Figure 6.6 - AFM images of S. aureus NCTC 4 1 63 treated with 25).!g/mL nisin . . . . . . . . . . . . . . . 1 3 5 Figure 6 .7 - Far away AFM images o f S. aureus NCTC 4 1 63 cells on a g lass slide treated with SMAP29 and OaBac5mini for 30 minutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 36 Figure 6.8 - Close up AFM images of S. aureus NCTC 4 1 63 cells on a g lass sl ide treated with SMAP29 and OaBac5 mini for 30 minutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 7 Figure 6 .9 - AFM images of E. coli 0 1 1 1 debris after being suspended in dist i l led water. 1 39 Figure 6. 1 0 - AFM images of E. coli 0 1 1 1 cells covered with dried Muel ler-H inton broth. 1 40 Figure 6. 1 1 - AFM images of crystals that formed when E. coli 01 1 1 was suspended in phosphate buffer and dried on a g lass sl ide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 40 Figure 7. 1 - The effect of salt concentrat ion on the minimum inhibitory concentration (MIC) of synthetic ovine peptides against E. coli 01 5 7 : H7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 44 Figure 7.2 - The effect of metal ion concentrations on the minimum inhibitory concentration (MIC) of synthetic ovine peptides against E. coli 0 1 5 7 : H 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 46 Figure 7.3 - The effect of media pH on the minimum inhibitory concentration (MIC) of synthetic ovine peptides against E. coli 0 1 57:H7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 49 Figure 7.4 - The effect of heating on the minimum inhibitory concentration (MIC) of synthetic ovine peptides against E. coli 0 1 57:H7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 50 F igure 7.5 - Diagram of a microtitre plate for a typical synergy test for OaBac5 mini and OaBac7.5mini . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 52 xiv List of Figures Figure 8 . 1 - Flow diagram showing the pilot-scale process used to extract antimicrobial peptides from ovine blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 59 Figure 8 .2 - Photograph of the pi lot-scale d isk-stack centrifuge used to separate white blood cells from plasma and red blood cells . . . . . . .. . ......... . . .. . ........... .... .. . . . . . . . . . . . . . . . . . . . . 1 60 Figure 8 . 3 - Transmission e lectron rnicroscopy images of control cells treated with 0 .0 1 % acetic acid ( left) and cells treated with ovine neutrophil crude extract (right). 1 64 Figure 8 .4 - Steps in an industrial process to produce a crude antimicrobial extract from ovine blood . .. . . . . . . . . . . . . . . . . ..... . ..... . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 67 xv list of Tables LIST OF TABLES Table 2. 1 - Amino acid sequences of the p-defensins found in l ivestock blood . . . . . . . . . . . . . . . . . . 4 1 Table 2 . 2 - Amino acid sequences of cathelicidins rich in one or more amino ac ids found in l ivestock blood. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Table 2 .3 - Amino ac id sequences of a-helical cathe licidins found in l ivestock blood . . . . . . . 44 Table 2 .4 - Amino ac id sequences of cathelic idins containing disulphide bonds found in l ivestock blood . . . . . . . . . . . .. . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Table 3 . 1 - Sequences of ovine ant imicrobial pept ides used for this research . . . . . . . . . . . . . . . . . . . . . 66 Table 3 .2 - Microorganisms used for micro-broth di lution lTllmmUm inhibitory concentration tests and their sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Table 3 .3 - Microorganisms used for crude extract minimum inhibitory concentration tests and their sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . 78 Table 4. 1 - Antimicrobial activity of typical ovine neutrophil extract gel fi ltration fractions against test organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Table 4.2 - Antimicrobial act ivity of RP-HPLC peaks from the second gel filtration fraction of the ovine neutrophil crude extract against test organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Table 4.3 - Comparison of masses and N-terminal sequences of Pa and Pc purified from the ovine neutrophil crude extract to known Bac5 peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Table 4.4 - Comparison of mass and N-terminal sequence of Pb purified from the ovine neutrophil crude extract to OaBac7.5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1 Table 4.5 - Comparison of masses and N-terminal sequences ofPd and Pf purified from the ovine neutrophil crude extract to Bac l l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1 Table 4.6 - Minimum inhibitory concentrations of peptides purified from ovine neutrophil extract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Table 4.7 - I dent ification of repeats in the sequences of the pro line/arg inine-rich cathelic idin peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Table 4.8 - Antimicrobial act ivity of typical ovine neutrophil extract cationic-exchange chromatography fractions against test organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Table 4.9 - P late assay results and molecu lar weights of antimicrobial peptides iso lated from the cationic fraction of ovine neutrophil extract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 1 Table 4. 1 0 - Comparison of the N-terminus of cationic Peak 1 8 to the cathel in- l ike precursor of SMAP29 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 02 Table 4. 1 1 - Comparison of the N-terminus of cationic Peak 24 to the signal peptide of T-cell surface g lycoprotein CD4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 02 xvi List of Tables Table 5 . 1 - Sequences of synthetic ovine antimicrobial peptides used for this research . . . . 1 06 Table 5 .2 - Minimum inhibitory concentrations (MIC) of synthetic ovine antimicrobial peptides against various microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 09 Table 5 . 3 - Data collected and calculated for the change in dansyl polymyxin B fluorescence due to the addition of SMAP29 in a typical run . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 4 Table 5 .4 - The abil ity of synthetic ovine peptides to bind to E. coli lipopolysaccharide (LPS) using the dansyl po lymyxin B (DPX) displacement assay . . . . . . . . . . . . . . . . . . . 1 1 5 Table 5 . 5 - Data collected and calculated for change in I -N-phenylnapthylamine (NPN) fluorescence due to the addition of SMAP29 for a typical run . . . . . . . . . . . . . . . . . . . . . . . 1 1 7 Table 5 .6 - Data collected and calculated for change in 3 ,3 -dipropylthiacarbocyanine (DiSC35) fluorescence due to the addit ion of SMAP29 for a typical run . . . . . . . . 1 20 Table 7. 1 - Concentrations of metal ions in lean trimmed, raw lamb meat. . . . . . . . . . . . . . . . . . . . . . . 1 48 Table 7 .2 - Antibiotics used in the synergy tests and their mechanisms of actions . . . . . . . . . . . 1 53 Table 7 .3 - Fractional inhibitory concentrations of synthet ic ovine peptides in combination with common ant ibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 54 Table 8 . 1 - Minimum inhibitory concentrations of ovine neutrophil crude extract from the pilot-scale extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 62 Table 8 .2 - Yields for pilot-scale extractions of antimicrobial peptides from ovine blood . 1 66 xvii List of Abbreviations AFM Bac BMAP BSA CD cDNA CFU ChBac DiSC35 DNA DPX EDTA FIC HLPC I so I FN-y IL- 1 2 Imax LPS LTPs MHB MIC MRSA MAP NF-KB NK cells NMR NO NPN NCLSS xviii LIST OF ABBREVIATIONS atomic force microscopy bactenicin bovine myeloid antimicrobial peptide bovine serum albumin circular dichro ism complementary DN A colony forming units Capra hircus bactenecin 3,3-dipropylthiacarbocyanine deoxyribonucleic acid dansyl po lymyxin B ethy lened iaminetetraacetic acid fractional inhibitory concentration high performance l iquid chromatography concentration of peptide required to displace half the of the maXimum displacement amount of DPX from LPS interferon-y interleukin- 1 2 maximum percentage of DPX that could be displaced from LPS by the peptides lipopolysaccharide l ipid transfer proteins Mueller-Hinton broth minimum inhibitory concentration methic il lin resistant Staphylococcus aureus myeloid antimicrobial peptides nuclear factor KB natural killer cells nuclear magnetic resonance nitric oxide I -N-phenyl-napthylamine National Committee of Laboratory Safety Standards NCPF NCTC OaBac OaDode OD PBSX PMAP PMN RP-HPLC SBD SDS SDS-PAGE SEM SMAP TEM TEMED TFA TFE TLRs TSB National Collection of Pathogenic Fungi National Collection of Type Cultures Ovine aries bactenicin Ovine aries dodecapeptide optical density phosphate buffered saline plus magnesium chloride porcine myeloid ant imicrobial peptide polymorphonuclear leukocytes reverse-phase high performance liquid chromatography sheep �-defensin sodium dodecyl sulphate sodium dodecyl sulfate - polyacrylamide gel electrophoresis scanning electron microscopy sheep myeloid ant imicrobial peptide transmission electron microscopy N,N,N',N'-tetramethylethylenediamine trifluoroacetic acid 2,2,2-trifluroethanol Toll-like receptors tryptic-soy broth List of Abbreviations xix List of Publications LIST OF PUBLICATIONS Most of the research presented in this thesis has been peer-reviewed and published in journals and/or presented at conferences. These publ icat ions are listed below. The full text of the journal art ic les are given in Appendix AS. Journal Articles Anderson RC, and Yu PL. (2003) I so lat ion and characterization of prol ine/arginine-rich cathe licidin peptides from ovine neutrophils. Biochemical and Biophysical Research Communications 3 1 2(4), 1 1 39- 1 1 46. Anderson RC, Wilkinson B, and Yu PL. (2004) Ovine ant imicrobial peptides: new products from an age-old industry. Austra lian Journal of Agricultu ral Research, SS( I ), 69-7S . Anderson RC, Hancock REW, and Yu PL. (2004) Antimicrobial activity and bacterial membrane interaction of ovine-derived cathel icidins. Antimicrobial Agents and Chemotherapy, 48(2), 673-676. Anderson RC, Haverkamp R and Yu PL. (2004) Invest igat ion of morpho logical changes to S. aureus induced by ovine-derived antimicrobial peptides using TEM and AFM. FEMS Microbiology Letters, 240( 1 ), 1 OS - 1 1 O . Anderson RC and Yu PL.(200S) Factors affect ing the antimicrobial activity of ovine-derived cathe l icidins against E. coli 0 I S7 :H7. International Journal of Antimicrobial Agents, 2S(3 ), 20S-2 1 O. Anderson RC and Yu PL. Purification and characterisation o f two protein fragments with ant imicrobial activity from ovine blood, inc luding part of the cathel icidin precursor. (wait ing for Meat and Wool NZ approval to submit) Anderson RC and Yu PL. P i lot-scale extraction and antimicrobial activity of crude extract from ovine neutrophils. (wait ing for Meat and Wool NZ approval to submit) Conference Proceedings Anderson RC, Hancock REW and Yu PL (2003) Mechanism of action of ovine-derived ant imicrobial peptides. New Zealand Institute of Chemistry Conference, 30th Nov- 4th Dec 2003, Nelson, New Zealand. Anderson RC, Wilkinson B and Yu PL (2002) Separation and activity of antimicrobial peptides from ovine blood. American Society of M icrobiology General Meeting, 1 9- 24th May, Salt Lake C ity, Utah, USA. Anderson RC, Wilk inson B and Yu PL (200 1 ) Purificat ion of antimicrobial peptides from sheep' s blood. Proceedings of the Molecules for Life Conference, 6-9th November 200 1 , Napier, New Zealand. Yu PL and Anderson RC (2004) Ovine Antimicrobial peptides: How much do we know? New Zealand M icrobiological Society Annual Conference. 1 7th - 1 9th November 2004, Palmerston North, New Zealand xx