Molecular Mechanism Of GefA Affecting Vibrio Parahaemolyticus Motility,Biofilm Formation,and Virulence

Vibrio parahaemolyticus is a Gram-negative bacterium that lives mostly in estuaries and marine environments and can cause shrimp red body disease,fish skin ulcers and other aquatic animal diseases;and acute gastroenteritis after human infection,severe septicemia and even death.The ability of V.parahaemolyticus to adapt to different living environments renders it to become an important food-borne pathogen.In order to survive in various external environments,V.parahaemolyticus needs to change from a single-cell planktonic state to a multi-cell cluster state,such as generating colony or forming biofilm.It was found that the switching of V.parahaemolyticus in different survival ways is regulated by the signaling factor cyclic diguanosine monophosphate（c-di-GMP）.As the ubiquitous second messenger in bacteria,c-di-GMP is synthesized by the diguanylate cyclases containing the GGD（/E）EF domain and then degraded by the phosphodiesterases containing the EAL or HD-GYP domain.C-di-GMP is involved in the regulation of various physiological and biochemical processes in bacteria,such as bacterial motility,biofilm formation,bacterial virulence,etc.In this study,the function of the proteins with a single GGD（/E）EF domain in V.parahaemolyticus are investigated.1.VPA0202 and VPA1478 inhibit swarming motility of V.parahaemolyticusBioinformatics analysis shows that forty-four GGD（/E）EF domain-containing proteins are predicted in the V.parahaemolyticus RIMD 2210633 genome,and further structural domain analysis of these proteins to screen for the diguanylate cyclases containing only a single domain,including VP2076,VPA0059,VPA0068,VPA0184,VPA0202,VPA0360,VPA0739,VPA0925,VPA1457,and VPA1478.The corresponding gene deletion strains are constructed in V.parahaemolyticus RIMD 2210633 strain.The swarming abilities of these deletion strains are first examined by the motility assays,and it is found that swarming ability ofΔvpa0202 andΔvpa1478are significantly enhanced.By the complemented assays,it is proved that GGEEF is an important enzymatic active site for VPA0202 and VPA1478 to play regulatory functions.And it is found that VPA0202 and VPA1478 are functionally similar by cross-complementation experiments.This study explores the functions of VPA0202 in more depth and names VPA0202 as GefA.2.Deletion of gef A causes increasing expression of flagellar genes yet decreasing expression of extracellular polysaccharide genes in V.parahaemolyticusV.parahaemolyticus has two types of flagella:the lateral flagella and the polar flagella.To further investigate the mechanism of GefA on regulating the swarming ability of V.parahaemolyticus,the motility ofΔgef A strain is examined after the deletion of the lateral flagellum gene lfg B and the polar flagellum gene flg E,respectively,and the swarming ability ofΔgef A is significantly reduced after the deletion of lfg B.The promoter region of the lateral flagella-related gene is fused with the fluorescent reporter plasmid,and the recombinant plasmid is introduced into the strains of V.parahaemolyticus and the fluorescence value is detected.It is found that the expression of the lateral flagellum gene is significantly increased inΔgef A compared with that in WT,which indicates that GefA inhibits swarming of V.parahaemolyticus by down-regulating the expression of the lateral flagellar genes.The c-di-GMP signaling pathway has been reported to be one of the mechanisms that regulate bacterial biofilm formation.To investigate the effect of GefA on the biofilm formation of V.parahaemolyticus,the biofilm production ofΔgef A strains was examined,and it is found thatΔgef A produces significantly decreased amount of biofilm compared with that of WT.The promoter activities of the extracellular polysaccharide genes are also examined,and it is found that the transcriptional efficiency of the extracellular polysaccharide genes inΔgef A and CΔgef A^GGAAAare found to be significantly reduced,but it is returned to the WT level in the complemented strain.This suggests that GefA as a dominant guanylate cyclase is involved in regulating the formation of V.parahaemolyticus biofilm.3.Functional analysis of GefA in the context of high c-di-GMP concentration in V.parahaemolyticusIn V.parahaemolyticus,Scr ABC is a known operon involved in the regulation of c-di-GMP synthesis,and when scr ABC gene is absent,the intracellular concentration of c-di-GMP increases which causes swarming defect and enhanced biofilm formation.To explore whether there is a mutual regulation between Scr ABC and GefA,the regulation of GefA on swarming and biofilm formation inΔscr ABC strain are examined.The results show that compared withΔscr ABC,the swarming ability of theΔscr ABC/gef A is not significantly different,but the biofilm formation is significantly reduced.This suggests that GefA plays a different role in bacterial motility and biofilm formation in an environment where the intracellular c-di-GMP concentration of V.parahaemolyticus increases significantly.4.GefA plays an important role in regulating the pathogenicity of V.parahaemolyticusThe regulatory effect of c-di-GMP on bacterial virulence has been reported.To investigate whether GefA has a regulatory effect on V.parahaemolyticus pathogenicity,the survival rates of zebrafish infected with different strains of V.parahaemolyticus are compared,and the survival rate of zebrafish infected withΔgef A is found to be significantly lower,which suggests that GefA is involved in regulating the virulence of V.parahaemolyticus.It is also found that GefA has a regulatory effect on the related genes of the type III secretion system 1（T3ss1）of V.parahaemolyticus,and the deletion of T3ss1 inΔgef A causes the mutant even less toxic than that ofΔgef A,which indicates that GefA regulates the virulence of V.parahaemolyticus through T3ss1.On the other hand,since GefA also has a regulatory effect on the lateral flagella,and flagella is also an important virulence factor of bacteria.Thus,to explore whether flagella is involved in GefA-mediated regulation of V.parahaemolyticus virulence,a mutant which contains deletion of lfg B and flg E inΔgef A background is constructed.This mutant also shows less toxic than that ofΔgef A,indicating that the flagellar system is also involved in the regulation of the virulence of V.parahaemolyticus mediated by GefA.This study finds that the c-di-GMP synthase GefA of V.parahaemolyticus inhibits bacterial swarming ability and pathogenicity but enhances biofilm formation.Our results expand the knowledge of the c-di-GMP synthetases of V.parahaemolyticus and its roles in social behaviors and pathogenicity.