Date of Award

2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

Abstract

Clostridium difficile is a leading cause of nosocomial infections, and recently emerged "hypervirulent" C. difficile strains have caused epidemics worldwide. We hypothesized that multiple factors were responsible for this phenotype, particularly the interaction of C. difficile with the epithelial cell, as well as toxin production and sporulation.

To test if host interaction varied in HV strains, we developed a quantitative host-cell adherence assay, and found that while C. difficile strains varied in adherence to human intestinal epithelial cells, hypervirulent strains were not significantly more adherent than other strains. The bacterial surface protein SlpA varied in both size and amount between strains. Both total C. difficile surface protein preparations and specific antibodies to SlpA significantly reduced adherence, indicating that SlpA specifically contributes to host epithelial cell adherence. SlpA contains two subunits, the high-molecular weight (HMW) and the low molecular weight (LMW) proteins. Sequencing of slpA from multiple Clostridium difficile strains indicated that the HMW subunit is more conserved in amino acid sequence, while the LMW subunit is more divergent. Recombinant protein experiments suggest that both subunits play a role, however, the LMW subunit may have a greater impact on adherence.

This study also examined the regulation of toxin production in hypervirulent C. difficile. Toxigenic C. difficile strains produce two toxins (TcdA and TcdB) during the stationary phase of growth. It has been suggested that hypervirulent C. difficile strains produce higher amounts of toxin, and that a mutation in a negative regulator (TcdC) allows for toxin production at all growth phases. Contrary to previous assumptions, toxins were undetectable during exponential growth, and toxin gene ( tcdA and tcdB), and toxin gene positive regulator ( tcdR) genes showed low, basal levels of transcription in both hypervirulent and non-hypervirulent strains. During stationary phase, hypervirulent strains produced robust but not significantly increased amounts of toxin. Interestingly, tcdC expression did not diminish in stationary phase, suggesting that TcdC may have a modulatory rather than a strictly repressive role. Hypervirulent isolates also produced significantly more spores. Increased sporulation, potentially in synergy with robust toxin production, may therefore contribute to the widespread disease now associated with hypervirulent C. difficile strains.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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