Regulatory mechanism of host cell contact-dependent T3SS gene expression in Vibrio parahaemolyticus

Many pathogenic bacteria have mechanisms that regulate gene expression in response to the surrounding environment to establish an infection. One such mechanism is the regulation of gene expression in response to contact with host cells. Here, we show that Vibrio parahaemolyticus, a causative agent of foodborne gastroenteritis, has a host cell contact-dependent regulatory mechanism for virulence gene expression. T3SS2, an essential virulence determinant for acute gastroenteritis encoded by V. parahaemolyticus pathogenicity island (Vp-PAI), recognizes host cell contact by sensing high intracellular K+ levels and switches its secretory substrates. This switching of secretory substrates is regulated by proteins called gatekeepers. Mutants deficient in the gatekeeper gene lose the ability to switch secretory substrates and lock their secretory state into contact with the host cell. Transcriptome analysis of T3SS2 gatekeeper gene-deficient strains showed that the genes encoded by Vp-PAI were specifically upregulated, and this upregulation was dependent on T3SS2 secretory activity, implying the presence of a negative regulator secreted by T3SS2. Comparative proteomic analyses identified a previously unidentified T3SS2 secretory substrate, VtrN, that negatively regulates Vp-PAI gene transcription. Secretion of VtrN was promoted under conditions that mimic host cell contact as well as other T3SS2 effectors. vtrN gene deletion specifically upregulated Vp-PAI gene expression, but unlike gatekeeper gene deletion, it was independent of T3SS2 secretory activity. Furthermore, VtrN interacted with VtrB, a transcription factor essential for Vp-PAI-encoded gene expression and repressed its transcriptional activity. Thus, V. parahaemolyticus has a mechanism to upregulate virulence gene expression in response to host-cell contact by utilizing the host-cell recognition mechanism of T3SS2. To investigate the effect of gatekeeper dysfunction (vpa1360 and vpa1359) on T3SS2 gene expression, the gene transcription profiles of the gatekeeper deletion mutants and parental strain were examined under a general culture condition (RIMD2210633_vpa1360 deletion mutant and RIMD2210633_vpa1359 deletion mutant vs RIMD2210633_WT). The experiment was repeated 3 times. To investigate the function of a T3SS2 regulatory gene (vpa1369), the gene transcription profiles of the deletion mutant and parental strain were compared when culture in a general condition (RIMD2210633_vpa1369 deletion mutant vs RIMD2210633). The experiment was repeated 3 times.