The Regulation Of Vp0610 On Vibrio Parahaemolyticus Biofilm Formation

Vibrio parahaemolyticus,a Gram-negative bacterium,prefers warm coastal waters and infects the host through different ways.And the human infection is mainly caused by ingestion of the pathogen V.parahaemolyticus in consuming contaminated seafood.V.parahaemolyticus can also be detected in seafood,although antibiotic and antibacterial treatment are applied to prevent and control the infections in aquaculture.One key factor that is predicted to be important for the infection cycle is its ability to form biofilms,with which the cells can attach to the biological and non-biological surfaces,causing cross-contamination,protecting bacteria from various environmental stress,and presenting resistance to conventional antibiotics,thereby contaminating food and food processing equipment even threatening human health.Studying the biofilm formation of V.parahaemolyticus provides theoretical support for follow-up research on prevention and control methods based on pathway blockade,but the mechanism of biofilm formation is unclear now.Here,the potential biofilm-related gene vp0610,which was screened using proteomics in the previous study,was deleted to determine the function of vp0610 in biofilm formation.Then both pull down experiment and transcriptomics analysis were applied to further explore the biofilm formation related regulation mechanism of vp0610 in Vibrio parahaemolyticus.The main discoveries are as follows:(1)Bioinformatics analysis revealed that VP0610 is annotated as a hypothetical protein,consisting of 204 amino acids and a molecular weight of 22.3 kDa.VP0610 belongs to the YfgM family protein,but there is no report on YfgM family proteins in Vibrio spp.Homologous recombination was applied to construct mutant ?vp0610 and complementary strain C-?vp0610,both of which were confirmed to be successful with sequencing.The crystal violet staining demonstrated that biofilm formation was faster of the WT than that of the mutant after 24 h,meaning that this difference gradually decreased and disappeared completely by 72 h.Therefore,quantitative analysis revealed that vp0610 exerted a clear inhibitory effect during the early stages of biofilm formation.Moreover,the biofilm formation at 24 h was further confirmed by laser confocal microscope and scanning electron microscope.(2)To further explore how VP0610 affects biofilm formation,we determined the ability of motility,EPS production,and c-di-GMP production that could affect biofilm formation.The swimming ability of mutants is significantly stronger than that of the WT,indicating that vp0610 negatively regulates swimming ability,which is mediated by polar flagella,our results suggested that vp0610 has an inhibitory effect on the motility of polar flagella.Deleting vp0610 resulted in the relieving of inhibition,causing the polar flagella to move quickly,and increasing the chance of cells to contact with surfaces.However,all strains displayed similar swarming abilities,suggesting that vp0610 does not affect swarming.Additionally,the mutant strain produced significantly less water-soluble EPS than the WT strain but produced considerably more water-insoluble EPS.Thus,water-soluble EPS usually acts as energy sources,indicating that the large amount of bacteria in the biofilm of the mutant means that energy consumption is also large.Water-insoluble polysaccharides are the structural substances of the biofilm,which is consistent with the results of the biofilm measurement.Although the loss of vp0610 did not alter c-di-GMP production at 12 h,a significant decrease was observed at 24 h.So,vp0610 makes no difference at 12 h,but brings a significant decrease at 24 h,which partly explained why the biofilm appeared a slow formation after 24 h.(3)Having established that vp0610 can affect biofilm formation,we sought to find the partner proteins of VP0610.Pull-down experiments revealed that 180 proteins can interact with His-VP0610.(Gene Ontology,GO)enrichment analysis indicated that these proteins could be divided into three groups:biological process,cellular component,and molecular function,while KEGG pathway analysis revealed that many of the proteins participated in five main significant enrichment pathways,including RNA degradation,metabolic pathways,riboflavin metabolism,secondary metabolite biosynthesis,antibiotic biosynthesis,and lysine biosynthesis.Particularly,Hfq,CyaA,and PTS-related protein components(VP0710 and VP0793)were identified as key proteins involved in biofilm formation via quorum sensing and cAMP/CRP pathways.(4)RNA-Seq was applied to analyze the effect of vp0610 deletion on gene transcription regulation,showing that there were 598 genes up-regulated and 639 genes down-regulated at 12 h compared with WT,while 197 genes up-regulated and 115 genes down-regulated at 24 h.Then the GO function enrichment analysis suggested that at 12 h,20 GO Terms were significantly enriched,including redox process,cellular amino acid biosynthesis process,organic acid biosynthesis process,carbohydrate metabolism and transport,electron transfer,etc.,which involves a PTS system.PTS plays an important role in the biofilm formation network,affecting the production and degradation of extracellular polysaccharides,c-di-GMP,and type ? pili.While at 24 h,there is no significant enrichment of GO Term,but it involves flagella-dependent cell movement.Flagella mediates adhesion and colonization at the initial stage of the biofilm,and acts as a biofilm scaffold at the mature stage.Further analysis of the KEGG pathway demonstrated that there are 11 pathways that are significantly enriched at 12 h,which mainly include quorum sensing,fructose and mannose metabolism,fatty acid degradation,carbon metabolism,and secondary metabolite biosynthesis.Carbon metabolism is often related to the extracellular polysaccharides;there are 5 pathways that are significantly enriched at 24 h,mainly including valine,leucine and isoleucine degradation,propionate metabolism,starch and sucrose metabolism,fatty acid metabolism and arginine biosynthesis.In short,VP0610 can affect the motility,EPS production and c-di-GMP production of V.parahaemolyticus by acting on the phosphotransferase system,quorum sensing system and other pathways,thereby negatively regulating the early biofilm formation.