| Pasteurella multocida(P.multocida)is Gram-negative facultative anaerobic pathogenic bacteria,which can be divided into five serotypes,A,B,D,E and F,according to the different capsular antigen.Among them,bovine serotype A P.multocida is a normal resident bacteria of bovine respiratory tract,which can cause bovine respiratory disease syndrome under stress conditions,resulting in huge economic losses to the cattle industry.It has been reported that various virulence factors play an important role in the pathogenesis of P.multocida.Bacterial hemolysin is a type of exotoxin secreted by bacteria and is one of the important virulence factors of bacteria.Previous study in our laboratory found that P.multocida did exhibited hemolytic properties when cultured under anaerobic conditions rather than aerobic conditions,but its hemolysin gene,hemolytic mechanism,and whether it can be used as a virulence factor were still uncharted.Therefore,this study first evaluated the hemolytic properties of P.multocida under anaerobic conditions.Then,TMT and Label free proteomics technology combined with bioinformatics were used to analyze and scree hypothetical hemolysis candidate factors.Finally,the final screening hypothesis factors were expressed by prokaryotic expression system and analyzed its hemolytic activity.The main research methods and results are as follows:1.Analysis of hemolytic properties of Pasteurella multocidaFirstly,bovine P.multocida serotype A CQ2 strain(PmCQ2)in Martin agar plate with rabbit blood were cultured under aerobic and anaerobic conditions for 3-5 days.Another group was cultured under aerobic conditions for 12 hours,then transferred to anaerobic conditions for 3-5days to verify whether it has hemolytic properties.Similarly,verify the hemolytic characteristics of bovine type A(PmCQ1?PmCQ7),B and F type P.multocida on Martin rabbit blood and chicken blood plates,LB rabbit blood and chicken blood plates.The results showed that the P.multocida type A,B and F of bovine could have hemolytic characteristics after culturing for 3-5days under anaerobic conditions(aerobic culture may also be used),but not hemolytic characteristics under aerobic conditions.2.TMT proteomics analysis and screening of putative hemolytic factors based on PmCQ2 hemolytic phenotypeUsing TMT proteomics technology to analyze the protein expression of PmCQ2 in the middle and late stages of hemolytic phenotype(anaerobic condition,sampling time point 65 h)relative to the control group(aerobic condition,sampling time point 12 h),combined with bioinformatics to analysis of the function of differentially expressed proteins,we began to screen potential hemolytic factors from protein annotation,differential expression folds,protein domains,subcellular localization,and gene family main functions.Simultaneously,qRT-PCR and WB methods were used to verify the proteome results from mRNA level and protein level,respectively.(1)There are total 1639 proteins were identified,of which 1603 proteins were quantifiable(quantitative protein indicates that at least one comparison group had quantitative information),and the differential expression threshold was 1.2 times(ratio>1.2 or ratio<0.83,and P<0.05),Of which 164 proteins were up-regulated and 282 proteins were down-regulated.(2)GO functional annotation shows that the differential proteins are mainly involved in 8 biological processes including metabolic processes,cellular processes and single organism processes;4 cellular components such as cells and membranes;and 6 molecular functions including catalytic activity and binding activity.(3)COG function annotation shows that the differential proteins are mainly involved in 21 items including energy generation and transformation,carbohydrate transport and metabolism,translation and ribosomal structure and biosynthesis,and cell wall/membrane biosynthesis.(4)Differential proteins are mainly enriched in KEGG pathways such as ribosome,peptidoglycan biosynthesis,fatty acid metabolism and biosynthesis pathway,butyrate metabolism,oxidative phosphorylation,citric acid cycle(TCA cycle)and pyruvate metabolism.(5)The verification results of differential proteins at the mRNA level and protein level are basically consistent with the proteome measurement results,indicating that the proteomics measurement results are reliable.(6)A total of 12 candidate factors for presumed hemolysis(Pm1384,Pm0040,Pm2119,Pm0696,Pm2109,Pm2214,Pm806,Pm509,Pm1169,Pm769,Pm1333 and Pm1211)were screened based on protein annotation,differentially expressed multiple,protein domain,subcellular localization,and major gene family functions.TMT proteomic analysis results indicate that P.multocida may regulate the expression of related proteins by inhibiting the synthesis of ribosomes in the middle and late stages of the hemolytic phenotype,thereby inhibiting the synthesis and metabolism of peptidoglycan and fatty acid to be weakened bacterial material energy activities;At the same time,it promotes the expression of metabolic related enzymes such as mismatch repair,pyruvate metabolism,oxidative phosphorylation and other pathways to maintain the normal metabolic activity of the bacteria,and ultimately its growth and metabolism are significantly inhibited.3.Label free proteomics analysis based on Pm CQ2 hemolytic phenotype and screening of putative hemolytic factorsSince TMT technology can only detect relatively differentially expressed proteins,therefore,label free quantitative proteomics(Label free)technology was used to analyze the protein expression of PmCQ2 in the early stage of hemolytic phenotype relative to the control group(both sampling time points were 12 h),combined with bioinformatics analysis of the function of differentially expressed proteins,analyzeing the absolute differentially expressed proteins under anaerobic conditions(expressed only under anaerobic conditions)from protein annotations,subcellular localization,and the main functions of gene families,and selecting hypothetical hemolysis candidates from them.(1)There were total 1612 proteins were identified,of which 1399were quantifiable.Taking 1.5 times as the differential expression threshold(ratio>1.5 or ratio<0.5,and P<0.05),39 proteins were up-regulated and 16 proteins were down-regulated.(2)GO functional annotation shows that the differential proteins are mainly involved in 11 biological processes,including cell metabolism,organic matter metabolism and nitrogen compound metabolism;12 cell components,such as intracellular,cell periphery and plasma membrane;14types,including transmembrane transport and iron binding activity Molecular function.(3)In the COG functional classification,the differential proteins are mainly distributed in 12 items,such as energy generation and conversion,carbohydrate transport and metabolism,inorganic ion transport and metabolism.(4)Differential proteins in KEGG are mainly enriched in the pathways of butyrate metabolism,pyruvate metabolism,citrate cycle(TCA cycle),carbon fixation,fatty acid degradation and oxidative phosphorylation.(5)After protein annotation,gene family function and subcellular localization analysis,the absolute differentially expressed proteins were selected and 6proteins including Pm1196,Pm1271,Pm2075,Pm0440,Pm0774 and Pm1154 were selected as putative hemolysis candidates.Label free proteomics results showed that in the early stage of the hemolytic phenotype,the growth and metabolism of P.multocida's are still slightly inhibited.In general,the enzymes involved in the synthesis of ATP are basically regulated,and energy consumption related enzymes were significantly down-regulated,which was basically consistent with the results of TMT technology.4.Hemolytic activity analysis of putative hemolytic factors and immuneprotective study of potential antigensProkaryotic expression,purification and renaturation of inclusion body proteins were performed on the 18 putative hemolytic factors screened,and 0.05%Tween 20 was used as a positive control to determine the recombinant protein of hemolytic factor(concentration 2-3 mg/mL))hemolytic activity under anaerobic,aerobic and 5%CO2 conditions.Selecting rPm1333,rPm1211,rPm806,rPm509,rPm0040 and rPm2214 immunized mice,each mouse immunized 100?g each time,on the 14th day after the second immunization,collect blood from 5 mice in each immunized group by means of tail-collecting serum was separated,antibody titer was determined,and the protective effect was explored with 3.5×10~6 CFU(2LD50)muscle challenge.The results showed that a total of 12 putative hemolytic factors have been successfully expressed,and these12 putative hemolytic factors recombinant proteins had no hemolytic activity after hemolysis analysis.The antibody measurement results showed that the antibody titers produced by rPm1333,rPm1211,rPm806,rPm509,r Pm0040,and rPm2214 were between 1:6400 and 1:404800,which showed that has a good immunogenicity,of which rPm1333,rPm1211 and rPm806 are 20%,10%,and 10%protective against Pm infection in mice,respectively. |
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