Repurposing auranofin to treat Clostridium difficile infection by targeting selenium. by Christine Roder B. Sci (Hons), M. Biotech Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Deakin University June, 2017 [Type here] Acknowledgements difficile [dif-i-seel] adjective 1. hard to deal with, satisfy or please. 2. hard to do; difficult “referring to the unusual difficulty that was encountered in its isolation and study” –Hall & O’Toole, 1935. Well they certainly weren’t wrong. A PhD is already a difficult mountain to climb and choosing to study an organism that was named for how difficult it is to grow only serves to make that mountain more challenging. As I stand here at the summit and reflect on the arduous uphill battle that was, I feel a sense of accomplishment and relief. I also feel overwhelming gratitude toward everyone who has helped me along the way. Unfortunately there are too many people to thank everyone by name, so I will take this opportunity to thank everyone who has contributed to my completing this thesis. Whether your contribution be small or large, it has meant a lot to me and I thank you for it. There were a number of people whose contribution was so significant they deserve special mention. Firstly I would like to thank my PhD supervisor Eugene Athan. You have provided ongoing support and encouragement throughout this endeavour and seem to have an unwavering confidence in my abilities which was especially important when I did not. It has been an absolute pleasure working with you and I look forward to our future projects together. Thank you to Deakin University for my scholarship, and also to Bellberry Limited for a top up scholarship and NHMRC for an equipment grant awarded to Deakin University, School of Medicine CMMR for the anaerobic cabinet “Annie”. I would also like to thank all the staff and students at CMMR and GCEID for all of their support and guidance. I would particularly like to thank the other members of my Lab group. I would like to acknowledge the contributions made by Dena Lyras and [Type here] Melanie Hutton from Monash University in both the donation of bacterial strains, and the assistance and use of facilities for the animal model. I would also like to thank John Stenos and Gemma Vincent from the Australian Rickettsial Reference Laboratory for donating the Vero cell line used in the tissue culture assays. On a personal note, I would like to thank my wonderful family and friends, who have given me a lot of love and support over the years. From coffee dates to wine nights, without these distractions to keep me sane I would not have made it through to the end. Many of you have also completed their PhDs, so to have a support network that understood the trials of PhD life was invaluable. To my parents, Margaret and Craig, the two of you raised me to be strong and independent, and have supported me emotionally and financially throughout my entire tertiary education. You have also read numerous drafts of papers, reports and finally my thesis. A task that was neither easy nor enjoyable, and for which I am eternally grateful. To my two beautiful dogs Charlie and Louie, for being the best fluffy study buddies a girl could ask for. Finally I thank my wonderful husband Kane. For every malteaser run, every time you picked me up in the middle of the night from Uni, for every high and low throughout this whole thing, you were there for me constantly. You are my rock and I would not be who I am without you. [Type here] List of publications 1. Roder, C. and Thomson, M.J. Auranofin: Repurposing an old drug for a golden new age. Drugs R & D. 15, 13-20 (2015). 2. Roder, C. et al. Population-based study of ribotypes and toxin-b production in clinical isolates of Clostridium difficile in Victoria, Australia. EC Microbiology. 1, 46-53 (2015). 3. Trubiano, J.A, et al. Australian Society of Infectious Diseases update guidelines for the management of Clostridium difficile infection in adults and children in Australia and New Zealand. Intern Med J. 46, 479-493 (2016). [Type here] Table of Contents Abstract ................................................................................................................... 1 Tables figures and abbreviations ............................................................................. 3 List of figures ...................................................................................................... 3 List of tables ........................................................................................................ 4 List of abbreviations ............................................................................................ 4 1. Introduction ..................................................................................................... 7 1.1. Background and epidemiology ................................................................. 7 1.2. C. difficile infection, pathology, the intestinal microbiota and the immune response ............................................................................................................... 9 1.2.1. The intestinal microbiota ................................................................... 9 1.2.2. Colonization of the intestinal epithelium ........................................ 10 1.2.3. Toxins and virulence ....................................................................... 12 1.2.4. The host immune response .............................................................. 18 1.3. Risk to public health ............................................................................... 19 1.3.1. Sources ............................................................................................ 19 1.3.2. Hypervirulent strains ....................................................................... 20 1.3.3. Recurrent disease ............................................................................ 20 1.3.4. Antibiotic resistance ........................................................................ 21 1.4. Current and emerging therapies .............................................................. 24 1.4.1. Diagnosis and treatment guidelines ................................................ 24 1.4.2. Antibiotics ....................................................................................... 24 1.4.3. Faecal microbiota transplant (FMT) ............................................... 26 1.4.4. Probiotics ......................................................................................... 28 1.4.5. Vaccines and antibodies .................................................................. 30 1.5. Auranofin ................................................................................................ 31 1.5.1. Background and overview of pharmacokinetics ............................. 31 1.5.2. Mechanisms of action ..................................................................... 33 1.5.3. Potential as a treatment for CDI ...................................................... 34 2. Methodology ................................................................................................. 36 [Type here] 2.1. Identifying selenoproteins in the C. difficile using in silico methods .... 36 2.2. Investigating auranofin as a treatment for CDI using in vitro methods . 38 2.2.1. Routine in vitro methodology ......................................................... 38 2.2.2. Growth Assays ................................................................................ 39 2.3. Preliminary testing of auranofin as a treatment for CDI using an in vivo animal model ..................................................................................................... 44 2.3.1. CDI prophylaxis animal model ....................................................... 44 3. Identifying selenoproteins in C. difficile using in silico methods. ................ 47 3.1. Background ............................................................................................. 47 3.2. Results .................................................................................................... 51 3.3. Discussion ............................................................................................... 58 4. Investigation of auranofin as a treatment for C. difficile infection ............... 63 4.1. Background ............................................................................................. 63 4.2. Results .................................................................................................... 68 4.2.1. Minimum inhibitory concentration (MIC) and subinhibitory concentration of auranofin ............................................................................ 68 4.2.2. Treatment concentrations of auranofin ........................................... 75 4.2.3. Comparison of the activity of auranofin with that of vancomycin and metronidazole ................................................................................................ 80 4.2.4. Preliminary testing of auranofin as a treatment for CDI using an in vivo animal model ......................................................................................... 87 4.3. Discussion ............................................................................................... 90 5. Discussion and conclusion ............................................................................ 97 6. References ..................................................................................................