AphA-dependent c-di-GMP production in <i>Vibrio parahaemolyticus</i> is mediated by direct regulation of <i>eapA</i> transcription encoding an EAL domain-containing protein

<i>Vibrio parahaemolyticus,</i> 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 <i>eapA</i> (<i>vp0376</i>), 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, <i>eapA</i> exhibited the strongest predicted AphA-binding motif. Experimental validation confirmed AphA binds the <i>eapA</i> promoter to activate its transcription. The expression of <i>eapA</i> peaked at LCD and decreased with increasing cell density. Deletion of <i>eapA</i> elevated c-di-GMP levels at LCD, enhanced biofilm formation, and impaired swimming motility, while <i>aphA</i> deletion reduced c-di-GMP. The <i>aphA</i>- and <i>eapA</i> double mutant exhibited c-di-GMP and biofilm phenotypes resembling the <i>eapA</i> mutant, placing EapA downstream of AphA. Transcriptional analysis showed <i>eapA</i> 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 <i>V. parahaemolyticus</i>, revealing how QS integrates with second-messenger signaling to optimize environmental adaptation.IMPORTANCE<i>Vibrio parahaemolyticus</i> (<i>V. parahaemolyticus</i>) 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 <i>eapA</i>, 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 <i>eapA</i> elevates c-di-GMP levels, enhancing biofilm formation while suppressing swimming motility; these phenotypes are epistatic to AphA. The discovery of the AphA-<i>eapA</i>-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.