Abstract 1738 Molecular characterization of Clostridium difficile KH-domain proteins

Regulatory small RNAs (sRNAs) are non-coding RNAs that regulate gene expression at the post-transcriptional level in various bacteria, including human pathogens. Interactions between sRNAs and their target mRNAs are often facilitated by global RNA-binding proteins, such as Hfq and ProQ. However, many pathogens that encode neither Hfq nor ProQ are known to express sRNAs associated with virulence. Notably, recent discoveries highlight the involvement of KH-domain proteins, specifically KhpA and KhpB, as global RNA-binding proteins (RBPs) across diverse bacterial species, including the pathogen Clostridium difficile. These observations raise the intriguing possibility that KhpA or B proteins, or a heterodimeric complex of the two, might contribute to stabilizing sRNAs and/or facilitating their interactions with mRNAs in many bacterial species. To define the protein-protein and protein-RNA interactions of bacterial KH-domain proteins, we are employing two genetic techniques: a bacterial two-hybrid (B2H) and three-hybrid (B3H) assay. These approaches enable the heterologous expression of RNAs and proteins in E. coli, allowing for the in vivo assessment of their interactions. In both of these assays, a 'prey' protein is fused to α-NTD of RNA polymerase and a 'bait' protein or RNA is tethered upstream of a test promoter. Interaction between the prey protein and bait RNA or protein is detected through an increase in the transcription of a lacZ reporter gene. Using these assays, we have been able to detect the heterodimerization of C.difficile KhpA/B proteins, and are examining the specificity of binding to both native and non-native RNA ligands. In addition, we are developing systems to probe the RNA interactions of the heterodimeric KhpA/B complex, by co-expressing two heterologous proteins in our E.coli B3H system. We gratefully acknowledge funding support from NIH (R15GM135878), the Camille and Henry Dreyfus Foundation and Mount Holyoke College.