RNA-based control of the expression of the type III secretion system of Yersinia pseudotuberculosis

Abstract

Expression of Ysc-Yop type III secretion system requires a tight regulation, which is conferred by two RNA-binding proteins YopD and CsrA. The underlying study demonstrated that expression of the transcriptional master regulator lcrF of the Ysc-Yop T3SS is controlled in intertwined feedback loops by these RNA-binding proteins. CsrA is a global acting post-transcriptional regulator that coordinates metabolic and virulence gene expression. This study demonstrated that expression of csrA is precisely controlled in an autoregulatory feedback loop, indirectly on the transcriptional and directly on the post-transcriptional level. CsrA was found to directly bind to two sites in the 5'-UTR of its transcript, from which one site is located within the ribosomal binding site. By this mechanism, CsrA was shown to block translational initiation and restrict CsrA synthesis when a certain CsrA amount is reached. Under non-secretion conditions, the RNA-binding protein YopD was shown to bind to the csrA transcript thereby masking a CsrA binding site and preventing the direct autoinhibition of csrA. Analysis of the role of CsrA and YopD in the lcrF regulation demonstrated a direct interaction of both RNA-binding proteins with the yscW-lcrF transcript. The 5'-UTR of lcrF contains an RNA-thermometer structure, which prevents ribosomal access through a base-pairing mechanism. Furthermore, this study demonstrated, CsrA interacts with the ribosomal binding site of lcrF and modulates the opening state of the RNA thermometer. Both regulators modulate the stability of the lcrF transcript. CsrA stabilizes and YopD destabilizes the lcrF mRNA. This is caused by the YopD- and CsrA-dependent control of the RNA degradosome. Furthermore, new putative CsrA targets could be identified by an interactome analysis with FLAG-tagged CsrA protein. At body host temperature, CsrA specifically interacts with tRNAs and transcripts of tRNA processing proteins. In summary, this study revealed the complex interplay of YopD and CsrA in controlling the expression of the T3SS master regulator lcrF. Both regulators are in involved in the post-transcriptional control of lcrF expression under non-secretion conditions and confer an antagonistic regulation.