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Title: Chemical proteomic profiling to investigate lipoprotein biogenesis in Clostridium difficile
Author: Charlton, Thomas
ISNI:       0000 0004 5922 7605
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2015
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Bacterial lipoproteins feature an N-terminal Type II signal peptide, containing a lipobox motif which targets these proteins for post-translational lipidation. Following secretion, pre-prolipoprotein diacylglyceryl transferase (Lgt) catalyses the addition of an S-diacylglyceryl moiety to the lipobox cysteine thiol. The signal peptide sequence is then cleaved by a lipoprotein signal peptidase (Lsp), leaving the modified cysteine as the N-terminal residue of the mature Gram-positive lipoprotein. Lipoproteins are surface exposed and play an important role at the host-pathogen interface, as well as being implicated in nutrient uptake, sporulation and antibiotic resistance. Clostridium difficile is a Gram-positive, spore forming, obligate anaerobe which causes severe gastrointestinal disease in humans. Spores are the transmissible agent of C. difficile, with infection typically occurring via the faecal-oral route. Lipoproteins of C. difficile are known to function in nutrient uptake and adhesion and the lipoproteome is likely to be important in transmission and colonisation. Bacterial lipidation is difficult to study by traditional methods, however, metabolic tagging with bioothogonally-tagged lipid analogues has recently emerged as a powerful method to study lipidated proteins. This thesis describes the development and optimisation of metabolic tagging and quantitative chemical proteomics to investigate lipidation in C. difficile. The application of this approach to profile the lipoproteomes of 630 Δerm and the clinically relevant 'hypervirulent' strain R20291 is described. This work includes the use of these probes, in combination with genetic and chemical inactivation of lgt, lspA and lspA2, to investigate lipoprotein biogenesis in C. difficile and to demonstrate the presence of two active Lsps. A combination of quantitative proteomics and phenotypic analysis has identified new functions for the C. difficile lipoproteome, including a role in the regulation of flagella and toxin production and in the initiation of sporulation.
Supervisor: Tate, Edward ; Fairweather, Neil Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral