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Title: Assembling the surface of Clostridium difficile
Author: Willing, Stephanie
ISNI:       0000 0004 6348 0427
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2015
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Clostridium difficile is a Gram-positive, spore forming, obligate anaerobe and pathogen of humans. C. difficile infection (CDI) is a potentially fatal gastrointestinal disease, typically acquired following antibiotic treatment. As a serious nosocomial pathogen, C. difficile also inflicts a large economic cost on healthcare systems. The symptoms of CDI are primarily the result of two toxins, and these have been the focus of much research. However, less well understood is how C. difficile manages to colonise the gastrointestinal tract. The surface of the vegetative cell is likely to prove very important in the colonisation process. The vegetative cell is covered by a paracrystalline array known as a surface layer. There are 28 paralogues of the surface layer protein, SlpA, found on the C. difficile 630 genome; these comprise the cell wall protein family. Each protein within this family has in common a 100 amino acid motif (CWB 2; PF04122) repeated in triplicate at either the N or C-terminus. This motif is found in many species of Gram-positive bacteria, but despite being widespread nothing is known about its anchoring mechanism. This study demonstrates that the CWB 2 repeats have likely evolved into a discrete pseudo-trimer domain such that each repeat is necessary for cell wall protein anchoring. Amino acids that are conserved in the CWB 2 sequence are investigated and residues important for cell wall anchoring identified. A cell wall associated polysaccharide is identified as a ligand of the CWB 2 repeats. A genetic locus that putatively encodes for the biosynthesis of this cell wall associated polysaccharide is investigated, and aberrations in the surface layer demonstrated when these genes are knocked-down. The potential of the CWB 2 repeats to mediate lateral interactions between the surface layer protein SlpA is analysed in experiments that suggest a role for Ca2+ in surface layer assembly. Additionally, a mutant with an unusual colony morphology obtained whilst attempting to inactivate a putative cell wall biosynthesis gene is investigated, revealing a link between a transcription coupled repair factor and toxin expression.
Supervisor: Fairweather, Neil Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral