CdgH-mediated c-di-GMP signaling orchestrates biofilm development through VpsT paralogues and VpsR in Vibrio alginolyticus

Vibrio alginolyticus is an opportunistic pathogen that significantly affects both aquaculture and public health. The second messenger cyclic-di-GMP (c-di-GMP) plays a central role in regulating key bacterial behaviors such as motility, biofilm formation, and secretion systems expression, thereby enhancing environmental persistence and virulence. However, the c-di-GMP regulatory network in V. alginolyticus remains poorly defined, limiting our understanding of its pathogenicity and adaptive strategies. We demonstrate that overexpression of the diguanylate cyclase CdgH increases intracellular c-di-GMP levels, which in turn promotes biofilm formation, rugose colony morphology, and Congo Red binding. RNA-seq and molecular analyses revealed that the extracellular polysaccharide synthesis locus rbr ( r egulation of b iofilm formation and r ugose morphology) is essential for the development of c-di-GMP-dependent biofilms and associated rugose colony phenotypes. Across Vibrio species, VpsT-like proteins—most of which contain a conserved c-di-GMP-binding motif, W[F/L/M][T/S]R—are important transcriptional regulators. V. alginolyticus encodes three VpsT-like proteins (VA3545, VA3546, and VA2703). Here, we identified VA3545 (RbrT) and another regulator, VpsR, as key positive regulators of biofilm formation. Genetic and expression analyses indicated that VpsR likely functions downstream of RbrT in the biofilm regulatory pathway, whereas VA3546 (RbrN) and VA2703 (RbrO) exhibited opposing effects. Surface plasmon resonance (SPR) confirmed direct binding of c-di-GMP to both RbrT and VpsR, with the conserved arginine residue within the WLSR motif being essential for RbrT function. Furthermore, elevated c-di-GMP levels suppressed swarming motility by downregulating flagellar synthesis genes. Notably, high c-di-GMP also significantly altered the transcription of several core conserved genes associated with type II and type VI secretion systems (T2SS/T6SS) in V. alginolyticus . These findings suggest distinct roles for VpsT homologs and VpsR in biofilm development and show transcriptional regulation of key secretion system genes by c-di-GMP, offering new insights into the pathogenicity and environmental adaptation of V. alginolyticus .