Structure-function studies on Clostridium difficile anti-sigma factor TcdC
Incidences of C. difficile infections have
increased dramatically in the past decade due to the spread of a
As the causative agent of pseudomembranous colitis and antibiotic associated diarrhea, Clostridium difficile is an important health care- associated pathogen. Two large toxin proteins, encoded by the tcdA and tcdB genes are the primary virulence factors that determine the severity of the disease. The toxin genes are positively regulated by TcdR, an alternate sigma factor and are negatively regulated by TcdC, an anti sigma factor. During the current decade there has been a dramatic increase in the incidence and severity of C. difficile infections due to the emergence of an unique C. difficile isolate. This hyper toxigenic strains carry mutations in tcdC gene that results in truncated TcdC proteins. This defective toxin gene negative regulator was speculated to be responsible for the high toxin production in these epidemic strains. Despite TcdC's importance in C. difficile pathogenesis, very little is known about its structure and function. TcdC is a small acidic protein with an amino terminal transmembrane domain and a central coiled coil domain. We have created different tcdC constructs with defined deletions in specific domains.
In Specific Aim 1, we will test the effect of TcdC variants on toxin gene transcription. We will purify TcdC protein variants and will test their ability to interfere with the TcdR mediated in vitro transcriptions of tcdA and tcdB promoters. In this aim we propose to map the TcdC domain that is critical in its function as a negative regulator.
In Specific Aim 2, we will purify TcdC variants in large scale for crystallization studies. Significance: Determining crystal structure of TcdC and defining its active domain for toxin repression will give more information on its structure and function, which will advance our knowledge in C. difficile toxin gene regulation. It will further help us to find new ways to control and prevent the C. difficile associated disease.
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This project is supported by grants from the National Center for Research Resources (5 P20 RR017708) and the National Institute of General Medical Sciences (8 P20 GM 103420).