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. Testing the potential of mycobacteriophage endolysins fused to biodegradable nanobeads for controlling Mycobacteria A thesis presented in partial fulfilment of the requirements for the degree of Master of Natural Sciences at Massey University, Albany, New Zealand Courtney Grace Davies 2019 Abstract 10.4 million people are diagnosed with tuberculosis, worldwide, every year, according to the World Health Organisation. Mycobacterium tuberculosis is a Gram-positive bacterial pathogen that can easily be transmitted to health care professionals and people supplying aid in these nations. Fortunately, over the last 100 years, the bacteriophage has gained traction as a suitable therapeutic, antibiotic-alternative against bacterial pathogens, such as M. tuberculosis. Herein I describe my work utilising endolysins the lytic protein that bacteriophages usually employ to burst out of the cell, but instead using these proteins to lyse “from without”. In order to develop a proof-of-principle product, we used the expansive M. smegmatis bacteriophage collection and chose eight endolysin candidates for testing against M. smegmatis. These endolysins were bioinformatically analysed for active domains before being synthesised and inserted into an expression vector to produce fused biodegradable nanobeads made of polyhydroxyalkanoate. These nanobeads were tested for activity against M. smegmatis, a safe mycobacterium closely related to pathogenic tuberculosis. Four distinct tests were carried out to test the efficiency of these beads in causing cell death in different situations (45 minutes and 5 hours, across concentrations of 10mg/ml, 20mg/ml and 80mg/ml. Ultimately the nanobead fusions of endolysin Inca (lysin B) caused the most cell death at 80mg/ml after exposure to M. smegmatis for 5 hours in a standing culture, at 78.87% cell death ± 5.21. When the nanobeads were applied to filter paper to mimic application to a hospital mask as a proof- of-concept approach before spraying with a bacterial aerosol, we saw that endolysin nanobead Jaws (lysin B) caused the most cell death with 75.54% ± 3.15 at 80mg/ml. These results are promising and present a unique opportunity to take advantage of an existing natural mechanism to use as a prophylactic defense against pathogenic bacteria in hospital settings. i Acknowledgements First and foremost, I wish to acknowledge the wisdom and support of my supervisor and Chief Phage Hunter, Dr. Heather Hendrickson, whom opened my eyes to the small and incredibly versatile world of the humble bacteriophage. Her intellectual challenges and ongoing support throughout this research have enabled me to grow and succeed in many ways. Secondly, I would also like to acknowledge Dr. Eric Altermann, Kerri Reilly and the team at AgResearch, Palmerston North, for taking me under their wing and guiding me through the fascinating world of biodegradable nanobeads and their potential to save the world. I would also like to acknowledge my parents, Johanne and James, for the hours of devotion listening to my trials and tribulations, encouraging me to keep going and accompanying me to the laboratory in the evenings when a zombie movie has made me second guess my research path. Additionally, to my friends and colleagues who have been there through each practice talk, phage pun, mid-thesis crisis and procrastination lunch, I extend my sincere gratitude to you. My amazing friend Piper was my inspiration for beginning this field of research to find an antibiotic alternative to support her as she dealt with her unfair share of life-threatening bacterial infections. I dedicate this thesis to her and my best friend Chiff, who was and will always be, my shining light, who started this journey by my side and ended it in my heart. Last but not least my late family members Aunty Trish and Taid who would always encourage me to succeed and whose memories I cherish as I go forward into the future. ii Contents List of figures .......................................................................................................................................... v List of tables .......................................................................................................................................... vi List of abbreviations ............................................................................................................................ vii 1 Introduction ....................................................................................................................................... 1 1.1 Bacterial infection of Mycobacterium species ............................................................................ 1 1.1.1 Implications of untreated Mycobacterium in New Zealand ............................................... 2 1.1.2 Diagnosis and treatment of tuberculosis ............................................................................ 3 1.2 Antibiotic resistant tuberculosis.................................................................................................. 4 1.3 Transmission of tuberculosis ...................................................................................................... 4 1.3.1 Infection of health-care workers ........................................................................................ 5 1.4 Bacteriophages ............................................................................................................................ 7 1.4.1 Bacteriophage life cycle ..................................................................................................... 7 1.4.2 Bacterial resistance to bacteriophages................................................................................ 8 1.5 Gram-positive cell walls ........................................................................................................... 13 1.6 Endolysins ................................................................................................................................ 14 1.6.1 D29 mycobacteriophage lysin B demonstrates cell lysis from without ........................... 16 1.6.2 Endolysin diversity .......................................................................................................... 18 1.6.3 Advantages of endolysins ................................................................................................ 18 1.6.4 Complications associated with endolysins ....................................................................... 19 1.7 Mycobacterium smegmatis mc2155 as a model organism ......................................................... 20 1.8 PhagesDB.org ........................................................................................................................... 21 1.9 Mycobacteriophage clusters ..................................................................................................... 24 1.10 Biodegradable nanobeads ......................................................................................................... 24 1.10.1 PHA biodegradability ...................................................................................................... 25 1.10.2 PHA synthase ................................................................................................................... 26 1.11 Aims ......................................................................................................................................... 27 1.12 Summary for remainder of the thesis ........................................................................................ 29 2 Materials and Methods ................................................................................................................... 30 2.1 Bacteriophage endolysin selection through bioinformatics ...................................................... 30 2.1.1 Online databases and tools; PhagesDB and NCBI BLASTp annotations ........................ 30 2.1.2 Clustal Omega .................................................................................................................. 30 2.1.3 HHpred ............................................................................................................................. 31 2.1.4 Plasmid constructs ........................................................................................................... 32 2.2 Media ........................................................................................................................................ 33 2.2.1 Antibiotics, supplements, media, ..................................................................................... 33 2.2.2 Bacterial strains ................................................................................................................ 33 2.2.3 M. smegmatis growth ....................................................................................................... 33 2.2.4 Growing and maintaining bacteriophage ......................................................................... 34 iii 2.2.5 Transforming chemically-competent E. coli BL21 .......................................................... 34 2.3 Nanobead production ................................................................................................................ 34 2.3.1 Nile Red stain ..................................................................................................................