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 hospitalacquireddiseases often associated with broad-spectrum antibiotic usage. TcdA and TcdBare the key virulence factors involved in major patho-physiology. While standardantibiotics provide some respite, due to the high relapse rates and the emergence of moresevere disease presentations, antibiotics alone have often proven to be suboptimal.Therefore there is a desperate need to develop an effective non-antimicrobialtherapeutics. Part of this work focuses on identification and further characterization ofpeptide therapeutic that target the major virulence factor TcdA/TcdB. Towardsdevelopment of mechanistic-based anti-toxin agent, phage display was used to identifypeptides that bind to the catalytic domain of C. difficile Toxin A. Characterization of thebinding and inhibitory activity revealed that the lack of parent peptide ability to inhibitthe cells in vivo. Further derivatization of above parent peptides in to irreversible binderslead to protects cells in vivo. Mass spectroscopy approaches revealed the peptideinhibition was mainly due to cross-linking of modified peptide in to key catalytic residuesin active site. While there are still several steps required to further explore in terms of thestability of these compounds, agents like these could be potentially used prophylactically163to avoid extensive cellular damage during treatment with broad spectrum antibiotics or inpopulations prone to CDI.The other area, focused on this thesis, is identification of the functional role of anegative regulator (TcdC) involved in toxin gene expression. In this work, we used avariety of biochemical and genetic approaches and characterized TcdC is not repressorinstead acts as an Extra Cytoplasmic Class (ECF) anti-sigma factor and was able topropose a new mechanistic model regarding the regulatory role of TcdC. As well as herewe have successfully developed GFP-based reporter system which has a potential to bean adaptable tool for investigating fine details on toxin genes tuning. Being able to adoptin 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 potentialtherapeutic opportunities hidden within such systems.