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Identification and Characterisation of Lysin Enzymes As Potential Therapeutics for the Treatment of Clostridium Difficile

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Identification and Characterisation of Lysin Enzymes As Potential Therapeutics for the Treatment of Clostridium Difficile Identification and characterisation of lysin enzymes as potential therapeutics for the treatment of Clostridium difficile ABDULLAH ALYOUSEF A thesis submitted to Cardiff University for the degree of Doctor of Philosophy March 2013 Cardiff University Cardiff School of Pharmacy & Pharmaceutical Sciences Redwood Building King Edward VII Avenue Cardiff CF10 3NB 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 22-06-2013 STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree of PhD. Signed (candidate) Date 22-06-2013 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 22-06-2013 STATEMENT 3 : PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loans after expiry of a bar on access previously approved by the Academic Standards & Quality Committee. Signed (candidate) Date 22-06-2013 Page | i Acknowledgements Firstly, I would like to thank my supervisors Professor L. W. J. Baillie and Dr. Jean- Yves Maillard for their help, support and guidance throughout my PhD studies. You encouraged me to do my best and your advice on article writing is unforgettable. Secondly, I would like to thank Dr. Jon Brazier and Dr. Val Hall for their kind donation of strains from the national anaerobic reference laboratory in Cardiff. I am thankful to Dr. Andrea Brancale for his help with protein modeling. Huge thanks to my friends and colleagues at Cardiff University, who helped me along my way and made it an excellent place to work. I would like to thank King Saud University (Riyadh) and Royal Embassy of Saudi Arabia (London) for their help and support during my study. On a personal note, I want to thank all my family, especially my father and mother, who have helped and encouraged me during my whole academic life, and I would also like to express great thanks to my wife ANFAL and my daughter LAYAL, whose patient love enabled me to complete this work . Abdullah Alyousef Page | ii Publications and Presentations relating to these studies Publication Joshi, L. T., Phillips, D.S., Williams, C.F., Alyousef, A., and Baillie, L. 2012. Contribution of spores to the ability of Clostridium difficile to adhere to surfaces. Applied and Environmental Microbiology 78(21), pp. 7671-7679. Patent Regarding characterization of bacteriophage endolysins as potential therapeutics for the treatment of Clostridium difficile, as a part of postgraduate study at Cardiff University. (2013) (pending). Presentations Isolation and characterization of lytic phage for treatment of Clostridium difficile. Welsh School of Pharmacy, Postgraduate Day, Cardiff, 2011. Poster presentation Evaluation of bacteriophages and their products as potential therapeutics for the treatment of Clostridium difficile. Welsh School of Pharmacy, Postgraduate Day, Cardiff, 2012. Oral presentation Page | iii Summary Clostridium difficile is the most common cause of hospital acquired infections. While the current treatments of choice, antibiotics, are generally effective in promoting recovery ,the increased incidence of C. difficile infections and treatment failure associated with antibiotic resistance combined with the emergence of hypervirulent strains highlights the need to develop therapeutic approaches that specifically target the pathogen without causing collateral damage to the protective microbiota. Several non-antibiotic approaches are currently being investigated, such as bacteriophage therapy. For this reason, we attempted to isolate C. difficile specific lytic bacteriophages which could form the basis of a treatment for C. difficile. While we were unable to isolate lytic phages, we were able to isolate twelve temperate bacteriophages from twenty-three clinical isolates of C. difficile using mitomycin C. Unfortunately we failed to identify a susceptible host strain capable of supporting the replication of these phages. This failure may in part be due to repeated episodes of phage infection over time, which have resulted in the emergence of “phage resistant” species mediated by systems such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). Employing a PCR-based approach using primers specific for the lysin genes of five previously isolated C. difficile phages, we found evidence to suggest that repeated bacteriophage infection is a common event for clinical isolates of C. difficile. Our inability to isolate a lytic bacteriophage prompted us to adopt an alternative approach in which we used endolysin enzyme of five previously identified C. difficile phages as recombinant protein. These lysins showed broad spectrum activity against the vegetative forms of a large collection of C. difficile ribotypes with little or no activity against other species, supporting their potential as therapeutic agents. We also identified a genome associated lysin (CD630, YP_001088405), which lysed vegetative C. difficile in a similar manner to the phage derived lysins. We also cloned and expressed a spore cortex lytic enzyme (SleC) which targeted the cortex of C. difficile spores. Unfortunately this enzyme was inactive against intact spores, suggesting that the outer layers of the spore act as a permeability barrier. The results of this study showed in vitro the applicability of endolysins against the vegetative form of C. difficile and the activity of spore cortex lytic enzyme against coatless spores, offering interesting perspectives for evaluation of the antibacterial activity of a mixture of endolysin and spore cortex lytic enzyme. Page | iv List of contents Declaration ........................................................................................................................... i Acknowledgements ............................................................................................................. ii Publications and Presentations relating to these studies ................................................... iii Summary ............................................................................................................................ iv List of contents .....................................................................................................................v List of tables ....................................................................................................................... xi List of figures .................................................................................................................. xiii Abbreviations ................................................................................................................. xvii Chapter One: Introduction ................................................................................................ 1 Chapter Two: General materials and methods .................................................................59 Chapter Three: Isolation of bacteriophages infecting Clostridium difficile ....................85 Chapter Four: The cloning and expression of the lysin genes of Clostridium difficile and its bacteriophages .....................................................................................................117 Chapter Five: The distribution of endolysins amongst clinical isolates of Clostridium difficile ........................................................................................................ 170 Chapter Six: Germination-specific cortex-lytic enzymes of Clostridium difficile ........203 Chapter Seven: General discussion ....................................................................... 248 References ....................................................................................................................... 264 Appendices .......................................................................................................................294 Appendix1 .........................................................................................................295 Appendix2 .........................................................................................................297 Appendix3 .........................................................................................................298 Appendix4 .........................................................................................................299 Page | v Chapter One: Introduction ...............................................................................................1 1.1 Historical Review ............................................................................................................... 2 1.2 Clostridium difficile Characterisation ................................................................................. 2 1.3 Spores of C. difficile ........................................................................................................... 4 1.4 Sporulation and germination of Spores .............................................................................. 6 1.5 Pathogenesis .................................................................................................................... .10 1.6 Incidence of C. difficile infection ...................................................................................
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