Pathogenic Function Analysis Of Plasma Membrane Protein SecY In Edwardsiella Piscicida

Edwardsiella piscicida is an important pathogen that cannot be ignored in aquaculture industry,which seriously hinders the healthy development of aquaculture industry.The protein encoded by secY is an important component of the cell membrane of E.piscicida and an important channel for transmembrane transport and secretion of proteins in E.piscicida cells.According to existing studies,secY is a key virulence related gene when bacteria invade the host.However,the role of SecY in the stress resistance and pathogenicity of E.piscicida and its regulation have not been reported.In this study,secY of E.piscicida was selected as the research object,and the interference strain secY-i2 of secY was constructed using gene interference technology,so as to study the role of this gene in the stress resistance and pathogenicity of E.piscicida.The regulatory relationship between SecY,Ycc A and SecY was studied.The main results of this study are as follows:Two interference fragments(RNAi1 and RNAi2)with different interference sites were designed for secY sequence,and two interference strains(secY-i1 and secY-i2)of E.piscicid TH1 strain were obtained by p T3 vector.Interference efficiency detection results showed that,compared with wild strain TH1,SecY transcription levels in secYi1 and secY-i2 were down-regulated to 14.45% and 15.18%,respectively,and SecY protein expression levels were down-regulated to 70% and 60%,respectively,which proved that the interference strain was successfully constructed.In view of the fact that transcription level of secY in secY-i1 and secY-i2 is downregulated to a similar extent,and secY-i2 is more significantly down-regulated in secY protein level,secY-i2 is selected for follow-up experiments in this paper.The results of stress resistance experiment showed that the growth of secY-i2 under normal conditions was not different from that of wild strain TH1,but the growth of secY-i2 under oxidation conditions was significantly inhibited,and the survival rate of secY-i2 under oxidation conditions was 14%,which was significantly lower than that of wild strain TH1(26%).The survival rate of secY-i2 under antibiotic condition was36%,which was significantly lower than that of wild strain TH1(50%).These results indicate that SecY plays an important role in the stress resistance of E.piscicid.The results of pathogenicity related experiments showed that compared with wild strain TH1,the biofilm forming ability,adhesion rate to host cells(FG,He La)and survival ability in host cells(RAW)of secY-i2 were significantly decreased.Under the condition of host serum,the survival rate of secY-i2 was 61.83%,which was significantly lower than that of wild TH1(81.44%).In further challenge experiments,the tissue infection ability and fatality rate of secY-i2 against host(Tilapia Tilapia)were also significantly reduced compared with that of wild strain TH1.These results indicate that SecY plays an important role in the pathogenicity of E.piscicid.The results of the experiments on the regulatory relationship of SecY showed that:compared with wild strain TH1,secY transcription level and SecY protein level were down-regulated in secY-i2,Ycc A transcription level and ycc A protein level were significantly up-regulated,and fts H transcription level had no significant change;In TH1Δycc A,the transcription level of secY was down-regulated,but the SecY protein level and fts H transcription level were not significantly changed;In TH1Δfts H,the transcription levels of secY and ycc A were significantly down-regulated,while the protein levels of SecY and Ycc A were up-regulated to varying degrees;β-galactosidase assay showed that Ycc A did not affect the activity of secY promoter.The above experimental results combined with the existing studies showed that Ycc A could respond to the changes of SecY and inhibit Fts H’s degradation of SecY in response,but no excessive degradation of SecY was observed after ycc A knockout.Fts H is not affected by the changes of SecY and Ycc A,and the protein levels of Ycc A and SecY significantly increase after fts H knockout.Therefore,it can be seen that there are unknown proteins that can inhibit the activity of Fts H and maintain the stability of SecY,which needs further study.In summary,this paper preliminarily explored the function of E.piscicid membrane channel protein SecY,and found that it was involved in bacterial antibiotic tolerance,antioxidant,biofilm formation,and anti-serum,adhesion,infection and proliferation of bacteria in the host body.At the same time,it was found that the homeostasis of SecY was affected by the combination of virulence factor Ycc A and metalloproteinase Fts H.In this paper,the role of plasma membrane channel protein SecY in E.piscicid in its stress resistance and pathogenicity was described for the first time,which provided a theoretical basis for the subsequent research on pathogenesis and disease prevention and control.