Elucidating small RNA regulation in the Vibrio cholerae Csr system

In this study, we have worked to further characterize the regulation of CsrA in Vibrio cholerae, a pathogen responsible for up to 5 million cases of cholera annually. CsrA is a global post-transcriptional regulator responsible for coordinating diverse, critical cellular processes including quorum sensing, carbon metabolism, and virulence. To study the regulation of this essential protein, we have used a mutant with a single amino acid substitution, CsrA-R6H, generating a less functional form of the CsrA protein. In V .cholerae, CsrA is negatively regulated by three small RNAs (sRNA): CsrB, CsrC, and CsrD. In the CsrA-R6H mutant, we have found significantly decreased levels of CsrB, CsrC, and CsrD. These results suggest the existence of feedback regulation wherein levels of the sRNAs are tightly controlled in order to maintain healthy levels of functional CsrA. Given the importance of CsrA, it is worthwhile to study CsrA’s upstream regulators. Homology modeling suggests that the V. cholerae protein MshH functions in the Csr system by enabling degradation of the Csr sRNAs. To test this hypothesis, the gene encoding the inner membrane protein MshH was knocked out via allelic exchange. Western blotting confirmed that the mshH mutation affected CsrA-mediated regulation of outer membrane proteins OmpT and OmpU. Next, using β-galatosidase assays, we identified markedly reduced transcription of the sRNAs in the ΔmshH strain which was dependent upon the presence of a VarA binding site motif in the promoter. Collectively, these results indicate that MshH is involved in the V. cholerae Csr system and is required for maintaining wild-type transcription rates of CsrB/C/D. Future studies will explore the mechanism of MshH’s involvement in the Csr system by differentiating whether MshH impacts transcription or stability of the sRNAs. Taken together, these results help develop our understanding of small RNA regulation at large and, more specifically, in V. cholerae.