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Title: Chemical probes of surface layer biogenesis in Clostridium difficile
Author: Dang, Thi Hoai Tam
ISNI:       0000 0004 2702 8185
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2011
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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.
Supervisor: Tate, Ed ; Fairweather, Neil Sponsor: EPSRC ; RSC ; GdCH ; SGM ; Marie Curie ; Biochemical Society
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral