Construction And Biological Characterization Of Riemerella Anantipestifer SprA Gene Deletion Mutant Strain

Riemerella anatipestifer is the pathogen of infectious serositis aiming at ducklings,geese,turkeys and other poultry.As this bacterium has many serotypes,poor cross protection and resistance to antibacterial agents,it becomes more and more difficult to control.Now it has turned into one of the bacterial diseases which cause serious damage to the global water industry.The pathogenicity of pathogen is closely related to protein secretion syetem.Through the protein secretion syetem pathogenic bacteria can secrete extracelluar enzyme,protease,virulence factors and so on to destuct the host cells and then cause the pathological damage and tissue necrosis.Por protein secretion system（PorSS）,also named type IX protein secretion system（T9SS）,is common in the members of the phylum Bacteroidetes but are apparently not found outside this phylum and it can mediate motility,secretion of protein and pathogenic mechanism of the phylum Bacteroidetes bacteria.In the serum of type 1 R.anatipestifer strain YM（RA-YM）,complete encoding genes of PorSS are found by bioinformatics analysis.Therefore,this study constructed a deletion mutant of sprA which was one of components of PorSS and then studied its biological characteristics and pathogenicity.Outermembrane proteins of RA-YM and ΔsprA were extracted respectively,and identified by Tandem Mass Tags technique.With the development of bioinformatics,differential outermembrane proteins secretd by PorSS would be found,and then help us to understand the relationship of proteins secreted by Por SS and pathogenicity of Riemerella anatipestifer.The study obtained the results as follows: 1.Construction and biological characteristics of R.anatipestifer sprA gene deleted mutant strainThe homologous leftarm,rightarm and spectinomycin resistance gene were respectively amplified.With the method of overlap PCR,LSR was linked together,and then subcloned into suicide plasmid pRE112 to construct recombination suicide vector pRE-LSR.Transform pRE-LSR into the E.coli strain X7213 and pick out positive clones.While E.coli strain X7213 transformed with the pRE-LSR was as the donor strain,and RA-YM was as the recipient strain,combined transfer experiment was carried out.After transconjugation,spectinomycin-resistant transconjugants were analyzed by PCR.The candidate strain was streaking in TSA plates with spectinomycin resistance to cultivate 5 generations to test its genetic stability.Meanwhile growth and biochemical characteristics were detected,and the results showed in the early logarithmic phase,ΔsprA growed significantly faster than RA-YM in TSB medium（P<0.05）,but in the late phase there was no significant difference in the growth speed;the basic biochemical properties of RA-YM and ΔsprA were the same.To be specific,they did not ferment sugars and could utilize urea,and RA-YM could liquefy gelatin while ΔsprA not.In the concentration of 12.5% duck serum,the survival rate of ΔsprA was significantly lower than that of wild strains of RA-YM（P < 0.01）2.Researches on pathogenicity of wild strain RA-YM and mutant strain ΔsprA for ducklingsChallenge experiment of 13-day old Cherry Valley Ducks was carried out with leg muscle injection.The results showed the the median lethal dose（LD50）of wild strain RA-YM was 7.21×104 CFU while the LD50 of mutant strain ΔsprA was 3.61×107 CFU.The virulence of mutant strain ΔsprA was 501 times attenuated compared with that of RA-YM.Detection of bacterial loadings of blood and tissues revealed after infection 24 h,the bacterial loadings of ΔsprA in blood and spleen was significangtly lower than those of RA-YM（P<0.05）.Similarly and furthermore after infection 48 h the bacterial loadings of ΔsprA in blood,liver,spleen and brains was obviously lower than of RA-YM（P<0.05）.During autopsy and histological examination,pathological changes in the liver,heart,spleen and brain samples of mutant strain ΔsprA were less serious than those of RA-YM.3.Gelatin zymography assay and LC-MS/MS sequencing analysis of wild strain RA-YM and mutant strain ΔsprABiochemical experiments showed wild strain RA-YM could liquify gelatin and mutant strain ΔsprA could not,so gelatin zymography assay was conducted.Three different stripes were obtained and then sequenced.The protein sequencing and bioinformatics screened 7 proteins which were probably secreted through PorSS.Among them,RAYM₀4099 and RAYM₀1812 which included peptidase S8/S53 subtilisin kexin sedolisin conserved domain and RAYM₀3382 with phosphohydrolases conserved domain may be responsible for the hydrolysis of gelatin and lower virulence.4.Tandem Mass Tags（TMT）technique relative quantitative outermenbrane proteomics analysis of Riemerella anatipestiferOutermenbrane proteins were extracted between wild strain RA-YM and mutant strain ΔsprA.After these proteins digested,small peptides were labeled by TMT reagent.Chromatographic fractionation with High PH HR,marked peptides entered the mass spectrum to be detected.Proteomics test results showed 1387 proteins with 213 outermenbrane proteins were identified.There were 65 differential expression outermenbrane proteins,with up-regulated proteins 43 and down-regulated proteins 22.Gene Ontology analysis displayed these proteins were involved in signal transduction,proteolysis,cell redox homeostasis,DNA metabolism and so on biological processes and were related to protein binding,receptor activity,DNA binding,transporter activity,transporter activity and so on molecular functions.Analyze the conservative domain of identified proteins,12 proteins secreted through PorSS were digged out with up-regulated proteins 4 and down-regulated proteins 1.