The transcriptional regulator AphB^vp is involved in virulence gene expression and biofilm formation in Vibrio parahaemolyticus

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease impacting shrimp farming worldwide. The disease was originally found to be caused by a specific strain of Vibrio parahaemolyticus harboring a unique ∼70-kbp plasmid encoding the binary PirA^vp and PirB^vp toxins. We have previously shown that AphB^vp regulates the expression of these toxins. In this study, we investigated the role of AphB^vp in regulating key genes associated with V. parahaemolyticus virulence and survival by using next-generation sequencing (NGS) of wild-type (3HP) and aphB^vp-deleted mutant (ΔaphB^vp) strains. Our RT-qPCR validation of NGS data showed that 6 genes, hns^vp, dgc^vp, t6ss^vp, tssB^vp, ifp^vp and DNA mtase^vp were significantly downregulated in the ΔaphB^vp compared to the 3HP, while vcrH^vp was upregulated. Electrophoretic mobility shift assays (EMSA) confirmed that AphB^vp directly binds to the promoter regions of hns^vp, dgc^vp and t6ss^vp. Notably, AlphaFold structural prediction indicates that DGC^vp is a diguanylate cyclase involved in producing cyclic-di-GMP (c-di-GMP), a key second messenger for bacterial biofilm formation and persistence. An intracellular c-di-GMP assay further showed that the concentration of c-di-GMP in the ΔaphB^vp was significantly lower than in the 3HP, while crystal violet staining and scanning electron microscopy revealed significant changes in both the biomass and structure of the biofilm formed by the ΔaphB^vp. Collectively, our findings suggest that AphB^vp could be a key regulator for V. parahaemolyticus virulence and biofilm formation. This study of the integrated AphB^vp regulatory network deepens our understanding of the pathogenesis of AHPND and offers potential targets for disease control strategies.