Vibrio parahaemolyticus is a significant foodborne pathogen that causes economic and public health problems worldwide and has a high capacity to adapt to diverse environments and hosts. The second messenger cyclic diguanylate monophosphate (c-di-GMP) allows bacteria to shift from a planktonic form to a communal multicellular lifestyle and plays an important role in bacterial survival and transmission. Here, we characterized single-domain c-di-GMP synthetases in V. parahaemolyticus and identified a novel GGEEF domain-containing protein designated GefA that modulates bacterial swarming motility, biofilm formation, and virulence. GefA inhibits swarming motility by regulating the expression of lateral flagella, while it enhances biofilm formation by controlling exopolysaccharide biosynthesis. Under high-c-di-GMP conditions caused by scrABC knockout, we found that GefA is bifunctional, as it has no effect on swarming motility, but retains the ability to regulate biofilm formation. Subsequent studies suggested that GefA regulates the expression of type III secretion system 1 (T3SS1), which is an important virulence factor in V. parahaemolyticus. Here, we also revealed that the flagella participate in the infection of V. parahaemolyticus. We found that both the T3SS1 and flagella contribute to the GefA-mediated virulence of V. parahaemolyticus in the zebrafish model. Our results expand the knowledge of the V. parahaemolyticus c-di-GMP synthetases and their roles in social behaviors and pathogenicity. IMPORTANCE The c-di-GMP metabolic enzymes constitute one of the largest clusters of potential orthologues in Vibrio parahaemolyticus. However, the specific roles that these individual c-di-GMP metabolic enzymes play are largely unknown. Here, we identified a GGEEF domain-containing protein designated GefA that regulates bacterial behaviors and virulence. We also demonstrated that flagella participate in the infection of this bacterium, through which GefA regulates bacterial virulence. To our knowledge, the roles that c-di-GMP and flagella play in V. parahaemolyticus virulence have never been revealed. Our findings contribute to a better understanding of the function of c-di-GMP and its synthetases in V. parahaemolyticus.
Keywords: GGDEF; Vibrio parahaemolyticus; biofilm formation; c-di-GMP; swarming motility; virulence.