TetR- and LysR-type transcriptional regulators mediate multilayered control of T3SS1 by <i>Vibrio parahaemolyticus</i> quorum sensing

The gram-negative pathogen <i>Vibrio parahaemolyticus</i> employs a sophisticated regulatory network to control virulence factors, including type III secretion system 1 (T3SS1), a key mediator of cytotoxicity. While the quorum sensing (QS) cascade LuxQ-ArcB-LuxO and master regulators AphA and OpaR are known to modulate T3SS1, the full scope of transcriptional regulation remains incompletely defined. Here, we combined the transcriptomic profiling of Δ<i>luxQ</i>, Δ<i>arcB</i>, and Δ<i>luxO</i> mutants with functional genetics to identify two novel transcription factors (TFs) that fine-tune T3SS1 activity. We demonstrate that TftR, a TetR family regulator, represses T3SS1 by binding the <i>opaR</i> promoter and enhancing OpaR production, thereby linking QS signaling to virulence suppression. Conversely, VltR, a LysR-type TF, directly activates <i>exsBA</i> transcription, independent of AphA/OpaR, to potentiate T3SS1-mediated cytotoxicity. Strikingly, VltR autoregulates its own expression, whereas TftR is reciprocally controlled by AphA (repressor) and OpaR (activator), revealing cross-talk between QS and accessory regulators. These findings delineate a multilayered regulatory hierarchy governing T3SS1, where TftR and VltR serve as opposing rheostats to calibrate virulence in response to cellular density and environmental cues. This work expands our understanding of <i>V. parahaemolyticus</i> pathogenicity and highlights potential targets for antivirulence therapies.IMPORTANCE<i>Vibrio parahaemolyticus</i> is a major global cause of seafood-associated gastroenteritis, relying on its tightly controlled T3SS1 for virulence. While the quorum sensing regulators AphA and OpaR are known to modulate T3SS1, the full regulatory network remains incompletely understood. This study identifies two novel transcription factors, TftR (TetR family) and VltR (LysR family), that fine-tune T3SS1 activity through distinct mechanisms. These findings reveal a multilayered regulatory hierarchy that enables <i>V. parahaemolyticus</i> to precisely calibrate virulence in response to cell density and environmental cues. Understanding these regulatory interactions provides new insights into bacterial pathogenesis and may guide the development of targeted antivirulence strategies against this clinically important pathogen.