Access Type

Open Access Dissertation

Date of Award

January 2013

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Andrew L. Feig

Abstract

Clostridium difficile infections cause one of the most common and vital hospitalacquired

diseases often associated with broad-spectrum antibiotic usage. TcdA and TcdB

are the key virulence factors involved in major patho-physiology. While standard

antibiotics provide some respite, due to the high relapse rates and the emergence of more

severe disease presentations, antibiotics alone have often proven to be suboptimal.

Therefore there is a desperate need to develop an effective non-antimicrobial

therapeutics. Part of this work focuses on identification and further characterization of

peptide therapeutic that target the major virulence factor TcdA/TcdB. Towards

development of mechanistic-based anti-toxin agent, phage display was used to identify

peptides that bind to the catalytic domain of C. difficile Toxin A. Characterization of the

binding and inhibitory activity revealed that the lack of parent peptide ability to inhibit

the cells in vivo. Further derivatization of above parent peptides in to irreversible binders

lead to protects cells in vivo. Mass spectroscopy approaches revealed the peptide

inhibition was mainly due to cross-linking of modified peptide in to key catalytic residues

in active site. While there are still several steps required to further explore in terms of the

stability of these compounds, agents like these could be potentially used prophylactically

163

to avoid extensive cellular damage during treatment with broad spectrum antibiotics or in

populations prone to CDI.

The other area, focused on this thesis, is identification of the functional role of a

negative regulator (TcdC) involved in toxin gene expression. In this work, we used a

variety of biochemical and genetic approaches and characterized TcdC is not repressor

instead acts as an Extra Cytoplasmic Class (ECF) anti-sigma factor and was able to

propose a new mechanistic model regarding the regulatory role of TcdC. As well as here

we have successfully developed GFP-based reporter system which has a potential to be

an adaptable tool for investigating fine details on toxin genes tuning. Being able to adopt

in host environment is vital for survival and propagation of a pathogenic bacteria. Thus,

exploring the regulatory nodes on PaLoc gene expression can be lead to exploit potential

therapeutic opportunities hidden within such systems.

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