The Identification of Therapeutic Targets and Virulence Factors of Clostridium difficile A thesis submitted in accordance with the conditions governing candidates for the degree of Master of Philosophiae in Cardiff University by Harsha Siani May 2014 School of Pharmacy and Pharmaceutical Sciences Cardiff University DECLARATION This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed (candidate) Date 19-May-2014 STATEMENT 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of MPhil. Signed (candidate) Date 19-May-2014 STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. The views expressed are my own. Signed (candidate) Date 19-May-2014 STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed (candidate) Date 19-May-2014 i ACKNOWLEDGEMENTS I would like to thank my supervisor Prof. Les Baillie for providing me with the opportunity to start my science career. His introduction into to the ‘wonderful’ world of C. difficile, Western blotting and 2DE has been nothing but eventful. I am grateful to Prof. Jean-Yves Maillard for his constant support and encouragement. I thank IQ Corporation for sponsoring this research. I extend my gratitude to Dr. Robin Howe and Dr. Lim Jones for assisting with the collection of serum samples. A special thank you to Ms Kerryn Money for keeping me sane during the ethics and RnD submissions. I would like to acknowledge the assistance of Dr. Anthony Hann and Guy Pitt (electron microscopy), Dr. Ian Brewis and Mrs Swee Nixon at CBS for helping me troubleshoot 2D gels and loaning of equipment and software. To the members (past and present) of the micro group, for all the amazing nights out (that we can remember), jokes/banter and most importantly friendship, Thank you. To Dr. Leon O’Malley, Dr. Ezra Linley, Dr. Baljinder Bains, Dr. Barbara Torrisi, Dr. Bettina Schelkle and Dr. Elzbieta Lis, thank you for all your advice, support, friendship and for always being there. I am grateful to Sue and Jeff who gave me a home away from home. To the most important people in my life, my family, whose love, support, patience and faith has been unwavering. I will forever be grateful. ii SUMMARY Clostridium difficile, a Gram-positive spore forming bacteria, is the leading cause of healthcare-associated diarrhoea in the UK and represents a major healthcare challenge. The vegetative form of the bacterium colonises the gut mucosa and produces two exotoxins, which are responsible for the pathology associated with the bacterium. We thus sort to characterise the host immune response directed against the surface of the bacterium and toxins A and B. Our studies revealed that the vegetative form of the pathogen is capable of altering its physiology in vitro to produce two distinct colony morphotypes. The differences observed in the cell surface, autolytic activity and bile salt sensitivity; suggest that the M2 morphotype may be better equipped to survive in the hostile conditions encountered in the gut. The mechanisms by which these changes are mediated are as yet unclear; however given the characteristics of the morphotypes it may involve one or more phase variable proteins. The identification of immunogenic proteins, including pyruvate-flavodoxin oxidoreductase, an anaerobic metabolism enzyme associated with oxidative stress warrants further investigation. To be effective, a future immuno-therapeutic should target the form of bacteria which is most often encountered during infection. Toxins A and B remain the primary virulence factors of C. difficile, with toxin neutralising antibodies targeting the receptor binding domains conferring protection and reducing recurrent infection. To characterise the antibody response directed against toxins A and B, the cell binding and translocation domains of each toxin were expressed in Escherichia coli. To identify immunogenic regions, the native, recombinant and toxoided proteins were subjected to enzymatic digestion with clostripain and probed with toxin neutralising animal sera and sera from C. difficile infected patients. The immune sera consistently identified two fragments of 40 and 60 kDa within both toxins A and toxin B, which may contain toxin neutralising epitopes and thus warrant further investigation. iii ABBREVIATIONS AAD Antibiotic-Associated Diarrhoea Ab Antibody Ag Antigen ANOVA Analysis of Variance BA Blood Agar BAPNA Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride BCA Bicinchoninic Acid BCR B Cell Receptor BHI Brain Heart Infusion BSA Bovine Serum Albumin bp Base Pair CCFA Cycloserine-Cefoxitin-Fructose Agar CDAD Clostridium difficile-Associated Diarrhoea CDI Clostridium difficile Infection C.D.M.N Clostridium difficile Moxalactam Norfloxacin CDT Clostridium difficile Binary Toxin CHAPS 3-Cholamidopropyl dimethylammonio-propanesulfonic Acid CROPs Combined Repetitive Oligopeptides CuSO4 Copper Sulfate CWP Cell Wall Protein diH2O De-ionised Water DMSO Dimethyl Sulfoxide DTT Dithiothreitol EDTA Ethylenediaminetetraacetic Acid EIA Enzyme Immunoassay ELISA Enzyme Linked Immunosorbent Assay Fc Fragment Crystalline FPLC Fast Performance Liquid Chromatography iv GI Gastrointestinal GMSC Genetic Modification Safety Committee GPS Global Proteome Server His Histidine HMW High Molecular Weight HPA Health Protection Agency HRP Horse Radish Peroxidase HSE Health and Safety Executive HuMAb Human Monoclonal Antibody IEF Isoelectric Focusing IFN Interferon Ig Immunoglobulin IL Interleukine IPTG Isopropyl -D-thiogalactopyranoside IVIg Intravenous Immunoglobulin kb Kilobase kDa Kilo Daltons LB Luria Bertani Broth LCT Large Clostridial Toxin LMW Low Molecular Weight mAb Monoclonal Antibody MCS Multiple Cloning Sites MHC Major Histocompatibility Complex MIC Minimum Inhibitory Concentration MLST Multilocus Sequence Typing MOPS Morpholinepropanesulfonic Acid Sodium Salt mRNA Messenger Ribonucleic Acid MW Molecular Weight NaOH Sodium Hydroxide v n Number of Replicates Ni Nickle OD Optical Density PAGE Polyacrylamide Gel Electrophoresis PAI Pathogenicity Island PaLoc Pathogenicity Locus PBS Phosphate Buffered Saline PBST Phosphate Buffered Saline+ Tween® 20 PCR Polymerase Chain Reaction PCR RT Polymerase Chain Reaction Ribotype PERK™ Protein Expression and Rescue Kit pI Isoelectric Point PMC Pseudomembranous Colitis PMF Peptide Mass Fingerprinting PPY Proteose Peptone Yeast PVP Poly(vinylpyridine) REC Research Ethics Committee R&D Research and Development IRAS Integrated Research Application System r.p.m. Revolutions per Minute SDS Sodium Dodecyl Sulfate S-Layer Surface Layer SLP Surface Layer Protein SSI Site-Specific Information σ Sigma Factor TBE Tris-Borate EDTA TCA Trichloroacetic Acid TC Cytotoxic T-cell TcdA Clostridium difficile Toxin A vi TcdB Clostridium difficile Toxin B TcnA Clostridium novyi Alpha Toxin TcsH Clostridium sordellii Haemorrhagic Toxin TcsL Clostridium sordellii Lethal Toxin TH T helper TEM Transmission Electron Microscopy TFB Transformation Buffer TpeL Clostridium perfringens Toxin tRNA Transfer Ribonucleic Acid SEM Scanning Electron Microscopy VH (L) Variable Heavy (Light) Chain VRE Vancomycin-Resistant Enterococcus v/v Volume per Volume w/v Weight per Volume 1-D One Dimensional 2-D Two Dimensional vii PUBLICATIONS During the course of this research the following abstracts were published: Conference Abstracts: Siani, H., Groen, H., Maillard, J. Y., Baillie, L. Characterisation of variant C. difficile morphotypes – microscopic examination and proteome analysis. 3rd International C. difficile Symposium, Bled, Slovenia (09/2010). Siani, H., Maillard, J. Y., Baillie, L. C. difficile – variant colony morphotypes and capsule production. 6th ClosPath International Conference, Rome, Italy (10/2009). viii ix TABLE OF CONTENTS DECLARATION ...................................................................................... Error! Bookmark not defined. ACKNOWLEDGEMENTS ........................................................................ Error! Bookmark not defined. SUMMARY ............................................................................................ Error! Bookmark not defined. ABBREVIATIONS................................................................................... Error! Bookmark not defined. PUBLICATIONS ..................................................................................... Error! Bookmark not defined. TABLE OF CONTENTS ...................................................................................................................... x LIST OF FIGURES ............................................................................................................................ xvii LIST OF TABLES ............................................................................................................................... xix CHAPTER ONE ........................................................................................................ 1 INTRODUCTION ...................................................................................................... 1 1.1 Introduction ..............................................................................................................................