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The identification of therapeutic targets and virulence factors of Clostridium difficile

Siani, Harsha 2014. The identification of therapeutic targets and virulence factors of Clostridium difficile. MPhil Thesis, Cardiff University.
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Abstract

Clostridium difficile, a Gram-positive spore forming bacteria, is the leading cause of healthcare-associated diarrhoea in the UK and represents a major healthcare challenge. The vegetative form of the bacterium colonises the gut mucosa and produces two exotoxins, which are responsible for the pathology associated with the bacterium. We thus sort to characterise the host immune response directed against the surface of the bacterium and toxins A and B. Our studies revealed that the vegetative form of the pathogen is capable of altering its physiology in vitro to produce two distinct colony morphotypes. The differences observed in the cell surface, autolytic activity and bile salt sensitivity; suggest that the M2 morphotype may be better equipped to survive in the hostile conditions encountered in the gut. The mechanisms by which these changes are mediated are as yet unclear; however given the characteristics of the morphotypes it may involve one or more phase variable proteins. The identification of immunogenic proteins, including pyruvate-flavodoxin oxidoreductase, an anaerobic metabolism enzyme associated with oxidative stress warrants further investigation. To be effective, a future immuno-therapeutic should target the form of bacteria which is most often encountered during infection. Toxins A and B remain the primary virulence factors of C. difficile, with toxin neutralising antibodies targeting the receptor binding domains conferring protection and reducing recurrent infection. To characterise the antibody response directed against toxins A and B, the cell binding and translocation domains of each toxin were expressed in Escherichia coli. To identify immunogenic regions, the native, recombinant and toxoided proteins were subjected to enzymatic digestion with clostripain and probed with toxin neutralising animal sera and sera from C. difficile infected patients. The immune sera consistently identified two fragments of 40 and 60 kDa within both toxins A and toxin B, which may contain toxin neutralising epitopes and thus warrant further investigation.

Item Type: Thesis (MPhil)
Status: Unpublished
Schools: Pharmacy
Subjects: R Medicine > RS Pharmacy and materia medica
Uncontrolled Keywords: Clostridium difficile; Therapeutic targets; Virulent factors
Date of First Compliant Deposit: 30 March 2016
Last Modified: 19 Mar 2016 23:39
URI: http://orca.cf.ac.uk/id/eprint/59860

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