Analysis Of The Toxicity Of Shewanella Putrefaciens On The Virulentity Of Vibrio Parahaemolyticus Based On AHLs Degradation Enzyme

Vibrio parahaemolyticus is the most harmful pathogen in marine foods.The expression of the virulence gene is often regulated by the Shewanella putrefaction in the symbiotic environment,including tdh gene expression,hemolytic activity,and biofilm formation are affected.However,the mechanism of the S.putrefaction to control the virulence of V.parahaemolyticus is not clear.This study intends to identify the type of Shewanella putrefactive through protein separation and purification,bioinformatics analysis,and heterologous expression of the target gene.How the substance regulates the virulence of V.parahaemolyticus.1.V.parahaemolyticus and commensal commensal bacteria were co-cultured.Their influence on the virulence of tdh gene expression and hemolytic activity of V.parahaemolyticus was compared.It was found that the virulence of V.parahaemolyticus was most strongly enhanced by S.putrefaction.Through the co-cultivation of Shewanella spp.and V.parahaemolyticus at different times,and the co-cultivation of the supernatant with the V.parahaemolyticus and V.parahaemolyticus signalling strains,it was found that the Shewanella spp.Signal molecules regulate the virulence expression of V.parahaemolyticus.2.S.Purification crude proteins of the degradation signal of Shewanella spp.include the three steps of ultrafiltration centrifugal tube crude extraction,Sephadex 200 column chromatography and SDS-PAGE electrophoresis.Sephadex 200 was then used to separate and purify the molecules.The absorbance was measured at a specific absorption wavelength of 276 nm and the elution curve was plotted.Three elution peaks were obtained,and the collected peaks were again passed through an ultrafiltration centrifuge tube.After filtration,concentrates F1,F2 and F3 of the protein separation peak were obtained.Comparing the degradation activities of the three eluting peaks on the signalling molecules of V.parahaemolyticus,respectively,it was found that F1 has no degrading activity,and F2 degrades the signal activity of V.parahaemolyticus.The results showed that the S.putrefaciens regulate the virulence expression of V.parahaemolyticus through secretory proteins.3.According to the ncbi and the S.putrescence gene library,the amidase acylase gene was obtained and analyzed by bioinformatics.The basic physicochemical properties of the amidase gene,the conserved and stable spatial structure of the amidase gene,are extracellular secretory proteins.The functional domain is Ntn_hydrolase.It is presumed that its function is to destroy the acyl group of a type of signal molecule(AI-I),thereby inactivating the signal molecule and once regulating the virulence expression of V.parahaemolyticus.4.Through gene synthesis,recombinant plasmids were constructed and transformed into E.coli to induce its expression,and isolated and purified to obtain relatively pure amidase.The enzymatic properties of amidase acylase were studied with the index of degradation rate of V.parahaemolyticus signalling molecule.The degradation of signal molecule activity by Shevaella spp.was affected by temperature and p H value.The optimum reaction temperature was 37�C.The optimum p H is 8.In the experiment,amidase was also found to have a close relationship with metal ions.On the one hand,when the enzyme was treated with the metal ion chelating agent EDTA,the enzyme activity decreased significantly,indicating that the metal ion is required for the protein to play a role;on the other hand,When Mg2+ and Fe2+ were added to the reaction system,the enzyme activity was significantly enhanced.This study demonstrated that the Shewanella spp.was degraded by the amidase degrading signaling molecules in the symbiotic environment and regulating the virulence expression of V.parahaemolyticus.However,the mechanisms and pathways of signalling molecules consistent with the regulation of V.parahaemolyticus virulence genes are unclear.Still need further in-depth study.However,the results of the study provide new ideas for the suppression of the virulent susceptibility of V.parahaemolyticus.Since the S.putrefaciens promote the virulence expression of V.parahaemolyticus by degrading the signaling molecules,it can be used to inhibit the production of amide by inhibiting Shewanella spp.The enzyme inhibits the activity of amidase,so as to achieve the effect of inhibiting the virulence expression of V.parahaemolyticus.In addition to this,although the enzyme properties of amidase have been initially studied,it is still unclear about the enzyme's enzymatic properties,key domains,etc.,and further studies are needed.Afterwards,key domains can be mutated and knocked out.The domain influences the regulatory function and defines the molecular mechanism of its regulation.