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Chemical Probes of Surface Layer Biogenesis in Clostridium Difficile

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Chemical Probes of Surface Layer Biogenesis in Clostridium Difficile Chemical probes of surface layer biogenesis in Clostridium difficile By Thi Hoai Tam Dang A thesis submitted to Imperial College London in candidature for the degree of Doctor of Philosophy of Imperial College. Department of Chemistry Imperial College London Exhibition Road London SW7 2AZ Februar 2011 1 Abstract The bacterium Clostridium difficile is responsible for recent epidemics of gastroenteritis and currently causes over twice as many deaths per year as the other major hospital ‘superbug’, MRSA. Since the bacterium is resistant to conventional antibiotics there is an urgent need to develop novel therapies. C. difficile secretes a family of proteins that are held in place on the cell wall by non-covalent forces, producing a proteinaceous coat (the surface layer or S- layer) that surrounds the entire cell. These S-layer proteins (SLPs) are immunogenic in humans and play a role in binding to host cells. Synthesis of the C. difficile S-layer involves site-specific proteolytic cleavage of the SlpA precursor by an as yet unidentified C. difficile protease. Identification of the protease that processes SlpA has proven challenging, due in part to a lack of established genetic tools in C. difficle. Here the development of novel chemical probes that can disrupt S-layer formation by inhibiting the protease are described, and found to be powerful chemical proteomic tools for both protease identification and exploring the process of S-layer formation. Screening and inhibition experiments were first performed to identify novel synthetic irreversible protease inhibitors combining an electrophilic warhead with a specific sequence element matching the SlpA cleavage. These compound series were shown to possess structure- dependent activity, and inhibited cultures were also more sensitive to lysozyme-induced cell lysis, suggesting that correct processing and assembly of the S-layer is important for cell envelope integrity. Optimised inhibitors were further developed into ‘activity-based probes’ (ABPs) carrying an affinity tag, and were successfully used to isolate and identify de novo the key protease involved in cleavage of SlpA, Cwp84, using a combination of different labelling and proteomics approaches. These probes also permitted identification of Cwp84 activity across a wide range of clinical strains. In later work, the scope of the warhead element was explored in more detail, and the potential antibacterial effects of the inhibitors were investigated in both wild type and genetically engineered strains. Finally, the contributions of this work in terms of both chemical technology and C. difficile biology are critically assessed. 2 Acknowledgements I would like to thank everyone who has made this thesis possible as well as having made my time at Imperial College and in London an unforgettable experience. First and foremost, I would like to thank my supervisors: Dr. Ed Tate not just for offering me a PhD position in his lab, but also for his endless patience to guide, support and motivate me throughout the three years. Thanks for your countless and always valued advice as well as your help and listening when I was in trouble; Prof. Neil Fairweather for providing me a microbiology working corner in Flowers and teaching me about C. difficile. For assistance in technical aspects, I must thank Dr. Robert Fagan for teaching me all required microbiological techniques to handle bacteria and turning me from an organic chemist into a “C. difficile killer” and half-microbiologist, Dr. Lucia de la Riva Perez for the wonderful collaboration and constantly encouraging me as a good friend, and Dr. Paul Hitchin for practical training in proteomics. I am indebted for the help of Dr. William Heal with helpful discussion, valuable advice in the chemistry lab and sometimes beyond the research-related matters. You seem always to have a right answer for any of my concern. Many thanks to the vital proof-reading team: Will, Neki, Megan, Alex, Lucy, Chris, Helen and Lucia, your time and effort are much appreciated. A general thank you as well goes to all the members of the Tate, Fairweather and Leatherbarrow research groups for making my time in London in general and at Imperial specifically, very productive and enjoyable. I will miss a lot how much fun we had during the conference stays, the together cooking and the special meals not just Vietnamese food but also European cheese and wine. For financial support, I am very grateful to the Chemistry Department at Imperial College which, courtesy of EPSRC, provided the funding for my PhD and the RSC, GdCH, SGM, Marie Curie and Biochemical Society for funding the travel and stay of my numerous conferences and providing me the chance to present my work national and international-wide. Finally, and most importantly, my biggest thank you goes to my family in Germany, my husband in Vietnam and my friends for all their warmly love and unconditional support. 3 Publications and Presentations Arising from this Thesis Publications W. P. Heal, T. H. T. Dang and E. W. Tate " Activity-based probes: discovering new biology and new drug targets ", Chem. Soc. Reviews. 2011, 40, 246-257. T. H. T. Dang, L. de la Riva, R. P. Fagan, E. M. Storck, W. P. Heal, C. Janoir, N. F. Fairweather and E. W. Tate "Chemical probes of surface layer formation in Clostridium difficile", ACS Chem. Biol. 2010, 5 (3), 279–285. Oral Presentations T. H. T. Dang et al. Chemical probes of surface layer biogenesis in Clostridium difficile. Biological and Medicinal Chemistry Symposium for Postgraduates, Cambridge, UK (2009) † T. H. T. Dang et al. Activity-based probes reveal a putative S-layer processing complex in C. difficile. Activity-based probes meeting, Leiden, Holland (2009) Poster Presentations T. H. T. Dang et al. Activity-based probes reveal an S-layer processing complex in Clostridium difficile. Chemical biology ISACS, San Francisco, USA (2010) T. H. T. Dang et al. Activity-based probes reveal an S-layer processing complex in Clostridium difficile. SET for Britain, London, UK (2010) T. H. T. Dang et al. Activity-based probes reveal a putative S-layer processing complex in C. difficile. Pfizer poster competition, London, UK (2009) T. H. T. Dang et al. Novel chemical probes of Surface-Layer formation in Clostridium difficile. Clostpath 2009, Rome, Italy (2009) T. H. T. Dang et al. Novel chemical probes of Surface-Layer formation in Clostridium difficile. 42nd IUPAC congress, Glasgow, UK (2009) 4 T. H. T. Dang et al. Novel chemical probes of Surface-Layer formation in Clostridium difficile. RSC South East Regional Organic Meeting, UCL, UK (2009) T. H. T. Dang et al. Novel chemical probes of Surface-Layer formation in Clostridium difficile Biological and Medicinal Chemistry Symposium for Postgraduates, Cambridge, UK (2008) T. H. T. Dang et al. Novel chemical probes of Surface-Layer formation in Clostridium difficile. BioCamp Novartis, UK (2008) T. H. T. Dang et al. Secrection of cell surface proteins in Clostridium difficile. 2nd EuCheMS Congress in Torino, Italy (2008) †Presentation awarded a prize due to content and delivery 5 Contents Abstract .............................................................................................................................................. 2 Acknowledgements ........................................................................................................................ 3 Publications and Presentations Arising from this Thesis ................................................ 4 Publications ........................................................................................................................ 4 Oral Presentations ............................................................................................................. 4 Poster Presentations .......................................................................................................... 4 Contents .............................................................................................................................................. 6 Abbreviations ................................................................................................................................. 12 Chapter 1 Introduction ............................................................................................................ 17 1.1 Chemical proteomics ................................................................................................ 18 1.1.1 Strengths and limitations of chemical proteomics ............................................ 20 1.2 Activity-based protein (or proteome/proteomic) profiling (ABPP) ......................... 21 1.3 Activity-Based Probes (ABPs) ................................................................................... 22 1.3.1 Designing ABPs ................................................................................................... 22 1.3.2 Click chemistry ................................................................................................... 26 1.3.3 Affinity enrichment of labelled proteins............................................................ 29 1.4 Application of ABPs to different protease families .................................................. 31 1.4.1 Proteases ............................................................................................................ 31 1.4.2 Serine and Threonine proteases ........................................................................ 33 1.4.3 Cysteine proteases ............................................................................................
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