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

作者：Nan Zhang, Wu Xu, Xue Li, Miaomiao Zhang, Xi Luo, Bin Ni, Renfei Lu, Yiquan Zhang · 发表于：Journal of Bacteriology · 年份：2025 · DOI：10.1128/jb.00104-25 · 被引用次数：4 · 研究领域：Vibrio bacteria research studies、Bacterial biofilms and quorum sensing、Aquaculture disease management and microbiota

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 ...