Abstract
Vibrio parahaemolyticus, a major seafood-borne pathogen, employs quorum sensing (QS) and c-di-GMP to regulate virulence, motility, and biofilm formation. While the master QS regulator AphA promotes c-di-GMP accumulation at low cell density (LCD), the underlying mechanism remained unclear. Here, we show that AphA drives net c-di-GMP accumulation by elevating c-di-GMP production while also activating the transcription of eapA (vp0376), encoding an EAL domain-containing phosphodiesterase, revealing a complex regulatory node. RNA sequencing revealed that AphA regulates 1,542 genes, including 23 potentially linked to c-di-GMP metabolism. Among these, eapA exhibited the strongest predicted AphA-binding motif. Experimental validation confirmed AphA binds the eapA promoter to activate its transcription. The expression of eapA peaked at LCD and decreased with increasing cell density. Deletion of eapA elevated c-di-GMP levels at LCD, enhanced biofilm formation, and impaired swimming motility, while aphA deletion reduced c-di-GMP. The aphA- and eapA double mutant exhibited c-di-GMP and biofilm phenotypes resembling the eapA mutant, placing EapA downstream of AphA. Transcriptional analysis showed eapA deletion upregulated exopolysaccharide biosynthesis genes while downregulating polar flagellar genes, aligning with c-di-GMP-mediated biofilm-motility trade-offs. Our findings establish a direct AphA-EapA-c-di-GMP pathway that critically regulates the biofilm-motility switch in V. parahaemolyticus, revealing how QS integrates with second-messenger signaling to optimize environmental adaptation.IMPORTANCEVibrio parahaemolyticus (V. parahaemolyticus) poses significant threats to human health and aquaculture, yet the mechanisms linking QS to c-di-GMP signaling remain poorly understood. This work uncovers AphA as a pivotal regulator that directly activates eapA, an EAL domain phosphodiesterase (PDE), to elevate c-di-GMP levels at low cell density (LCD). We identify EapA as the LCD-specific PDE that degrades c-di-GMP and is directly activated by AphA. Deletion of eapA elevates c-di-GMP levels, enhancing biofilm formation while suppressing swimming motility; these phenotypes are epistatic to AphA. The discovery of the AphA-eapA-c-di-GMP axis provides novel insights into how QS integrates with second messengers to optimize bacterial fitness. This study underscores the complexity of c-di-GMP metabolism and highlights AphA's dual role as a global transcriptional regulator, bridging gaps in our understanding of bacterial signaling networks.