Structural Characterization of Clostridium difficile TcdR, a Sigma Factor that Controls Bacterial Toxin Production
MetadataShow full item record
Clostridium difficile infections in the colon cause severe diarrhea and pseudomembranous colitis, with pathology stemming from two secreted bacterial toxins, TcdA and TcdB. With C. difficile infection rates on the rise in the United States, and with the growing threat of antibiotic resistant strains, hospitals are desperate for more effective treatments. Previous research has shown that the production of TcdA and TcdB depends on a regulatory sigma factor, TcdR, which directs RNA polymerase to the promoter regions of the toxins in order to initiate transcription. If TcdR function were to be knocked out by a small-molecule therapeutic, toxin production and subsequent C. difficile virulence could be reduced. Determining the protein structure of TcdR, as well as understanding its functionality in relation to RNA polymerase and toxin promoter regions, could accelerate inhibitor development. In this study, TcdR was expressed in Escherichia coli, and purified with co-eluting proteins by affinity and size-exclusion chromatography. Using mass spectrometry and electron microscopy, we were able to characterize a putative complex between TcdR and E. coli RNA polymerase. Electrophoretic mobility shift assays are currently being optimized to confirm the ability of the putative complex to bind toxin promoter DNA. The continuation of these studies will provide the basis for future functional screens for small-molecule inhibitors against the TcdR/RNA polymerase complex that could help combat C. difficile virulence and infection.