| Pasteurella multocida(PM)is one of the important pathogens causing bovine respiratory disease syndrome(BRD),which can be divided into five serotypes:A,B,D,E and F.At present,pneumonia caused by P.multocida type A is relatively common and serious,which has brought great losses to the production and breeding industry all over the world.Therefore,it is of great significance to study its pathogenic mechanism.Secretory proteins are transported or secreted outside of bacteria and play critical roles in nutrient acquisition,adaptation,virulence,intraspecific or interspecific signaling.Since the whole genome of Pasteurella multocida PM70 isolated from poultry in the United States in 2001 was reported,many scholars have analyzed and predicted the secreted proteins according to genome,but the actual secreted proteins of bovine P.multocida type A are still to be identified.Therefore,this study first applicated data-independent acquisition(DIA)LC-MS/MS to obtain more comprehensive and accurate secreted proteins of P.multocida.The proteins were obtained from the supernatants of two bovine P.multocida serotype A isolates(high virulent Pm CQ2 and low virulent Pm CQ6)cultured in Martin and BHI media.Then,the putative secreted proteins were screened through bioinformatics analysis and experiments.Finally,the selected secreted proteins were prokaryotic expressed and their immune protection was analyzed.Based on this,potential protective protein antigens were mined to provide theoretical support for the preparation of subunit vaccines.The main research methods and results were as follows:1.Identification P.multocida secretome by DIA LC-MS/MS proteomicsFirst,bovine P.multocida serotype A strains Pm CQ2 and Pm CQ6 were incubated in Martin and BHI media to explore the differences in their biological characteristics under two culture conditions,the results showed that both strains grew well in Martin medium,and Pm CQ2 cultured on BHI medium was more lethal than Martin medium.Extracted the secretory proteins in the middle and late stage of logarithmic growth,it was found that the protein types and concentrations of the two strains in the two media were different through SDS-PAGE,which suggested that culture conditions may affect the secretion of proteins related to growth metabolism and virulence.Therefore,DIA mass spectrometry was used to measure the secreted proteins in different culture supernatants and to determine the reliability of DIA data.A total of 154 proteins were identified in the supernatant,among which 135 were identified in Pm CQ2 and 147 in Pm CQ6.There was no significant difference in the number of proteins identified in the two strains(P>0.05).When identificated of proteins obtained from different media,152 proteins were detected in BHI medium and 99 proteins in Martin medium,with significant difference.In addition to ribosomal proteins,the proteins detected only in BHI culture medium also included virulence proteins,enzymes related to bacterial growth and metabolism,transporters and adhesion proteins.To a certain extent,BHI medium can simulate the host environment compared to Martin medium.2.Bioinformatics analysis and verification of Pasteurella multocida secretomeAccording to different protein secretion pathways,414 secretory proteins were predicted according to Pm CQ2 genome,among which 210 were secreted by the Sec pathway,14 were secreted by the Tat pathway,and 190 were secreted by the non-classical secreted pathway.To reduce intracellular proteins outflow caused by cell lysis,the same bioinformatics analysis was performed on the experimental data,and 50 secretory proteins were predicted,among which 26 were secreted by the Sec pathway,3 were secreted by the Tat pathway,and 21 were secreted by the non-classical secreted pathway,there were also 9 moonlighting proteins.In addition to these 50 proteins predicted according to genome as well,we identified extra 104 secretory proteins in the experiment,which indicated that the results of bioinformatics prediction were not comprehensive enough,some proteins might be secreted in an unknown way.These experimental data made up for the deficiency of genome prediction to some extent.After bioinformatics prediction,40 and 49 putative secretory proteins were identified from the supernatant of CQ2 and CQ6 respectively.The 9 proteins that only CQ6 expressed included ribosomal proteins,cyclophilin B,fad L,Rlp A,pal,as well as unknown function proteins.Pm CQ2up-expressed 3 proteins compared to Pm CQ6(Foldchange>1.5,P<0.05),including Omp A,Lpo A,sugar ABC transporter substrate-binding protein.50 and 32 putative secretory proteins were identified from BHI and Martin media,respectively.The additionally-expressed 18 proteins were mainly ribosomal proteins,metabolism related proteins and virulence proteins.Compared with Martin medium,8 proteins were up-regulated in BHI medium,namely 50S ribosomal protein L1,PCK,Omp A,Osm Y,Omp H,dpp A,DNA-binding protein HU,glutathione amide-dependent peroxidase.The results showed that Pm expressed more anti-stress proteins and virulence factors in BHI medium to resist the external environmental pressure,while protein secretion required energy and nutrition,which was consistent with the previous results of slow growth and increased virulence in BHI medium.After subcellular localization prediction,proteins located in periplasmic space or outer membrane or extracellular were screened,and combined with protein secretion level,CQ2GL000317(pyruvate kinase),CQ2GL000495(C4 dicarboxylate ABC transporter substrate binding protein),CQ2GL000675(type I glycoldehyde-3-phosphate dehydrogenase,GAPDH),CQ2GL001229(peptidase C58)and CQ2GL001603(enhancement factor Tu,EF-Tu)were selected and validated by immunohistochemistry,which indicated that the above proteins were indeed secreted in the lung tissue of mice infected with Pm.The localization of CQ2GL001603 protein was further studied by immunoelectron microscopy,and it was found that CQ2GL001603 protein could enter into the alveolar epithelial cells and capillaries of the host.3.Protective study of secretory proteinsIn accordance with the protein research in other microorganisms,and explore the possibility of unknown proteins as protective antigens,we selected 10 proteins as candidate protective antigens.Prokaryotic expression,purification and renaturation were performed on these proteins,then mixed with adjuvant to produce subunit vaccine to immune mice.Indirect ELISA was used to determine the antibody titer of mice serum,the result showed that the antibody levels were all above 1:12800,the titers of r Pm2029 were 1:102400,the titers of r Pm089,r Pm092,r Pm495,r Pm317 and r Pm1603were 1:51200,which indicated that the putative protective proteins had good reactivity.Intramuscularly challenged with 1×10~6CFU Pm CQ2,the mice immunized with recombinant protein vaccine were protected in different degrees,including r Pm696 protection rate of 62.5%,r Pm495 and r Pm317 protection rate of 37.5%,r Pm2029 and r Pm1229 protection rate of 25%,r Pm1603 and r Pm999 protection rate of 12.5%.These results suggested that it was feasible to screen protective antigens from secretory proteins.r Pm696(cyclophilin B)protein can be used as a candidate factor for subunit vaccine,but it still needs further verification. |
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