Clostridium difficile is the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-infections (CDI) because it disrupts normal protective gut flora and enables C. difficile to colonize the colon. The current treatment for CDI, administration of additional antibiotics, is increasingly ineffective and often results in relapse of the disease. New strategies to treat this important pathogen are urgently needed and one such approach is to target its virulence. Toxigenic C. difficile strains produce two toxins, toxin A and toxin B that are considered to be the major virulence factors. The toxins encoding genes, tcdA and tcdB are part of a pathogenicity locus, which also carry the gene encodes for the toxin genes positive regulator tcdR. TcdR is an alternate sigma factor that is specifically required for expression of tcdA and tcdB. In a preliminary study we found that tcdR in C. difficile to affect both toxin production and sporulation. It is hypothesized that a small molecule that inhibits TcdR would block toxin production along with sporulation and will serve as an anti-pathogenic agent against C. difficile. Different TcdR activated promoter-reporter fusions were developed and have been shown in E. coli to be appropriate for high-throughput screening. In the first specific aim, we propose to develop series of recombinanat TcdR activated promoter reporter fusions in E. coli and in B. subtilis for high throughput screening. Employing these reporter fusions, we will conduct a pilot screening of small molecule libraries from the KU-HTS core for compounds that inhibit TcdR activity. Second aim of the grant will focus on understanding the influence of TcdR on sporulation. We propose to study the influence on TcdR on sin (sporulation inhibition) locus transcription in C. difficile.
Alternate sigma factors are known to regulate virulence and virulence associated genes in many pathogenic bacteria. Including toxin genes, TcdR may regulate other virulence-associated genes in C. difficile. We have created and characterized, tcdR mutant in two different C. difficile strains. Mutation in tcdR affected both toxin production and sporulation in C. difficile. Microarray analysis revealed many differentially expressed sporulation-associated genes in tcdR mutant. In this project in our first aim, we propose to test the role of TcdR in C. difficile sporulation. In our second aim, we are proposing to monitor TcdR dependent promoter expression at cellular level, using a novel reporter system. During the current decade there has been a dramatic increase in the incidence and severity of C. difficile infections due to the emergence of hypertoxinogenic C. difficile strains. Our long- term goal is to unravel pathogenic mechanisms of C. difficile, thus new strategies to prevent, treat and manage C. difficile infection can be developed.