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Title: Sporulation initiation in Clostridium difficile
Author: Underwood, Sarah
ISNI:       0000 0004 2674 8316
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2009
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Clostridium difficile is a leading cause of hospital-acquired diarrhoea, responsible for over 30% of cases of antibiotic-associated colitis, nearly all cases of pseudomembranous colitis and costs the NHS over œ200 million per year. This bacterium is able to persist in the hospital environment to cause recurrent infection by the formation of stable spores, refractile to current decontamination procedures. A more comprehensive understanding of the sporulation signal transduction pathway is essential for the design of a decontamination regime effective in removing the spores from the nosocomial environment and the logical design of novel antimicrobial agents. This project aimed to elucidate the mechanism of sporulation initiation . regulation and the role of sporulation-associated proteins in other C. difficile virulence processes, such as toxin production and colonisation. Analysis of sporulation in response to various hospital cleaning agents showed that the combination of a neutral detergent (such as Hospec) with EDTA is a more effective cleaning agent than the chlorine-based agents currently used, as the combination product is uniquely able to both kill vegetative cells and inhibit spore formation. A variety of molecular approaches were used to elucidate information regarding the C. difficile sporulation initiation pathway and the relationship between sporulation and toxin production. Three putative C. difficile sporulation-associated sensor histidine kinases (CD1A, CD2A and CD3B) were identified and shown to be independently involved in sporulation initiation. Furthermore, CD3B has been shown to directly phosphorylate the master response regulator SpoOA, strongly suggesting that this pathway is a two-component system, as opposed to the extended phosphore lay pathway found in B. subtilis. Previous studies on bacteria capable of both toxin production and endospore formation have described links between the two processes. Data presented here indicates SpoOA has a role in indirectly regulating C. difficile toxin A and B production, as the protein is capable of specifically binding promoter regions of the toxin regulatory genes tcdC and tcdD. Inoculation of a triple-stage continuous-culture chemostat that modelled the human gut with C. difficile spoDA- mutant provided further evidence that SpoDA has a key role in both colonisation a!1d toxin production. Overall, this work adds to the growing body of evidence that SpaDA is a master global regulator and has a crucial role in the pathogenicity of C. difficile, making it an excellent target for future novel antimicrobial therapies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available