Development of peptide inhibitors targeting Clostridium difficile toxins A/B and characterizing the regulatory role of a putative negative regulator TcdC in Clostridium difficile toxin gene expression.

摘要

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.