Abstract

Many pathogenic bacteria regulate gene expression in response to the surrounding environment to establish 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. Type III secretion system 2 (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 switching its secretory substrates. The switching of secretory substrates is regulated by proteins called gatekeepers. Mutants deficient in the gatekeeper genes lose the ability to switch secretory substrates and lock their secretory state into a host-cell contact-dependent manner. Transcriptomic analysis of these mutants revealed the upregulation of Vp-PAI genes, which was dependent on T3SS2 secretory activity, suggesting the presence of a negative regulator secreted by T3SS2. Comparative proteomic analyses identified a previously unrecognized T3SS2 secretory substrate, VPA1369 (VtrN), that negatively regulates Vp-PAI gene transcription. Secretion of VtrN was promoted under conditions that mimic host-cell contact. vtrN gene deletion specifically upregulated Vp-PAI gene expression, independent of T3SS2 secretory activity, indicating its role as a repressor. VtrN interacts with VtrB, a key transcription factor for Vp-PAI genes, suppressing its transcriptional activity. This mechanism illustrates how V. parahaemolyticus enhances virulence gene expression upon host-cell contact through the T3SS2 recognition system, highlighting an adaptive strategy for establishing infection.