A DHH/DHHA1 family 3′-phosphoadenosine 5′-monophosphate (pAp) phosphoesterase Vp2835 is essential for regulating motility, biofilm formation and type III secretion system 1 in Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus, a predominant seafood-borne pathogen responsible for acute gastroenteritis worldwide, employs sophisticated regulatory networks to adapt to environmental stresses. However, the molecular mechanisms connecting environmental signals to coordinated regulation of motility, biofilm formation, and virulence effectors expression remain to be elucidated. In this study, we identified vp2835, a gene encoding a putative phosphoesterase of the DHH/DHHA1 family, from a swarming-deficient mutant library. Biochemical studies revealed that Vp2835 could specifically cleave 3′-phosphoadenosine 5′-monophosphate (pAp) into AMP and phosphate. Deletion of vp2835 significantly attenuated both swimming and swarming motility while enhancing biofilm formation. Transmission electron microscopy (TEM) revealed impaired lateral flagellar biosynthesis in the mutant strain when it was cultured on agar surfaces. RNA-seq analysis demonstrated that the deletion of vp2835 triggered differential expression of 1142 genes (|Log₂FC|≥1, p < 0.05), which was characterized by upregulation of extracellular polysaccharide (EPS) biosynthesis operons (e.g., cpsA-cpsK and scvD-scvF, Log₂FC = 1.7–3.7), downregulation of polar/lateral flagellar systems (e.g., pomAB, flgTOP, flgCEGHJKL, Log₂FC = −1.1-3.0; fliN, Log₂FC = −2.5), as well as all Type III Secretion System 1 (T3SS1) virulence components (Log₂FC = −2.8-7.0). Cytotoxicity assays corroborated these findings, showing a 90 % reduction in HeLa cell lactate dehydrogenase (LDH) release, which was associated with T3SS1 suppression in the mutant. Collectively, our findings establish vp2835 as a critical regulator of motility and behavioral transitions, biofilm development, and virulence mediated by T3SS1 in V. parahaemolyticus.