TsrA modulates type III secretion system 2 expression as a co-regulator of H-NS in Vibrio parahaemolyticus

Vibrio parahaemolyticus, a gram-negative marine bacterium, is a major cause of seafood-borne gastroenteritis worldwide. This pathogen relies on type III secretion system 2 (T3SS2), which is encoded on a pathogenicity island, for its enteropathogenicity. Expression of T3SS2 is activated by a regulatory pathway centered on the transcriptional activator VtrB, which is antagonized by the xenogeneic silencer, histone-like nucleoid-structuring protein (H-NS). However, the complete transcriptional network is not yet fully understood. In this study, we identified TsrA as a negative regulator of T3SS2 gene expression. TsrA is a small protein conserved among Vibrio species that lacks a putative DNA-binding motif but has been implicated in the regulation of virulence genes in Vibrio cholerae. In V. parahaemolyticus, deletion of tsrA increased VtrB production and T3SS2 secretion, thereby enhancing T3SS2-dependent pathogenicity. Transcription of vtrB occurs via a two-step activation process, in which TsrA affects the primary activation step, thereby modulating VtrB production. We further provide experimental evidence that TsrA physically interacts with H-NS via its C-terminal region, which correlates with its regulatory activity on vtrB expression. A systematic mutational analysis of the C-terminal 26 residues revealed several residues critical for TsrA regulatory activity. Moreover, the regulatory effect of TsrA on T3SS2 gene expression was dependent on H-NS, demonstrating that TsrA functions in concert with H-NS. Thus, our findings provide new insights into the regulatory mechanisms of virulence gene expression in V. parahaemolyticus by defining the role of TsrA in this network, while also placing TsrA among H-NS co-regulators.IMPORTANCENucleoid-associated proteins (NAPs) play key roles in virulence gene regulation in bacteria. The best-studied NAP is H-NS, which often functions with co-regulators to fine-tune gene expression. TsrA, a small protein lacking a DNA-binding motif conserved among Vibrio species, has been suggested to be functionally related to H-NS in Vibrio cholerae, although its mechanism remains unknown. Here, we demonstrate that TsrA negatively regulates the expression of type III secretion system 2 (T3SS2), a major virulence determinant of Vibrio parahaemolyticus, an important seafood-borne pathogen. TsrA modulates the transcription of vtrB, which encodes the essential activator for T3SS2 expression, through direct physical interaction with H-NS. Our findings reveal a molecular link between TsrA and H-NS, providing mechanistic insights into NAP- and TsrA-mediated regulation of virulence in Vibrio.