| Pasteurella multocida,a kind of gram-negative coccobacillus bacteria,the capsule and somatic form the basis for classification into serogroups(A,B,D,E,or F)and serotypes 1 ~ 16.Pasteurella multocida,which can infect many wild and domestic animal species and will cause acute and chronic infectious diseases.Diseases caused by Pasteurella multocida include haemorrhagic septicaemia in cattle and buffalo,atrophic rhinitis in pigs,fowl cholera in birds,enzootic pneumonia in cattle,snuffles in rabbit.It has been reported that Pasteurella multocida can cause local tissue and even lead to systemic disease in humans,predominantly following cat or dog inflicted injuries.It is not only a serious threat to animal and human health,but also brought serious economic losses to livestock husbandry all over the world.In recent years,with the rapid development of cattle industry,the risk of bovine infected with P.multocida is highly increased.However,most of the cattle infected enzootic pneumonia caused by capsular serotype A.Therefore,to study the pathogenesis of pneumonia caused by Pasteurella multocida plays an important role in the prevention and treatment of enzootic pneumonia in cattle.Pasteurella multocida possesses various virulence factors,including fimbriae and adhesins,capsule,lipopolysaccharide,outer membrane proteins,iron-regulated and iron acquisition proteins and toxins.Outer membrane proteins contains several protein in the surface preparations of Pasteurella multocida,lipoprotein than is a membrane protein have several roles of the host adhesion.In this study,the structure and subcellular localization of PM0979 were predicted by bioinformatics analysis generated from online software.The expression of Pm0979 were detected by real time PCR in vivo and in vitro,soluble PM0979 was purified from E.coli BL21 containing p ET-32a-PM0979,and subcellular localization of PM0979 was tested by indirect immunofluorescence;the expression of inflammatory cytokine was detected from murine macrophage after r PM0979 stimulation;To study the function of PM0979,Pm CQ2 lack of Pm0979 was constructed and the role of PM0979 in pathogenesis of Pm CQ2 was tested in a mouse model.In conclusion,these result pave the way to provide further information for study the pathogenesis of Pm.1.Bioinformatics analysis and differential expression of pm0979 in vivo and in vitroThe potential signal peptide and transmembrane domain of PM0979 were predicted by Signal P-4.1 server and TMHMM Server v2.0,respectively.The result showed that PM0979 possessed a signal peptide and without transmembrane domain.PM0979 was a putative lipoprotein which predicted by DOLOP and Lipo P 1.0 server;Subcellular localization of PM0979 was assessed by CELLOv.2.5 showed that PM0979 located in cell envelop.The sequence of PM0979 were analyzed by Clustalx1.83 for homology search,the result showed that the homology of PM0979 was highly conserved in type A and type B Pasteurella multocida,but not in type D,type F and strains isolated from sheep.According to the previous result,PM0979 located in cell envelop and highly conserved.To study the role of PM0979 in pathogenesis of Pasteurella multocida,the expression level of Pm0979 in vitro was detected using real time PCR,the result of real time PCR showed that Pm0979 was significant highly expressed in PmA,compare to Pm B and PmF.2.Prokaryotic expression of soluble recombinant PM0979(r PM0979)and subcellular localization of PM0979The PM0979 encoding gene was amplified from of Pm CQ2 genome.The PCR product was directly cloned into the p ET-32a(+)vector in frame with a six N-terminal histidine tag for expression and purification.E.coli BL21(DE3)containing p ET-32a-PM0979 was induced by IPTG,the cells were harvested by centrifugation and the lysate was sonicated to reduce viscosity.The result of indirect immunofluorescence showed that green fluorescence signal was detected from Pm CQ2 after 1% Triton 114 treatment,however,fluorescence signal of Pm CQ2 without treatment was not detected,these results suggest anti-PM0979 specifically recognize PM0979 which located in cell envelop.3.Study the pathogenic mechanism of Pm0979 in vivo and in vitroTo study the effect of r PM0979 on murine macrophage,peritoneal macrophages were isolated and then co-culture with r PM0979,the expression of TNF-?,IL-1?,IL-6,IL-18 were tested by ELISA after r PM0979 treated 24 h,the result showed that the expression of TNF-?,IL-1?,IL-6,IL-18 were increased after r PM0979 treatment,especially TNF-? and IL-6.The results indicated that PM0979 involved in the immunomodulating effects of Pm to macrophage.To construct Pm0979 mutant,the downstream and upstream homologous arm of Pm0979 was amplified from of Pm CQ2 genome,then the PCR product was directly cloned into the PUC19 ori Kan R to construct PUC19 ori KanR-? 0979.Transform PUC19 ori KanR-?0979 into Competent cells,and recombinant colonies wree selected from plate containing kanamycin.Recombinant colonies were verified by PCR and western blot.To study the role of PM0979 in Pm CQ2,biochemical reactions,growth curve and biofilm formation were tested,the result showed that biochemical characteristics and growth curve of Pm CQ2-?0979 have no significant difference,compare to wild type.However,the ability of Pm CQ2-?0979 to form biofilm was decreased.To further evaluate the role of PM0979 in pathogenesis of Pm CQ2,the virulence of Pm CQ2-?0979 was tested in vivo and in vitro,the result of challenge test showed that the lethality of Pm CQ2-?0979 and Pm CQ2 were 75% and 100%,respectively,indicated that the virulence of Pm CQ2-?0979 was attenuated.Macrophage isolate from murine infected with Pm CQ2-?0979,the expression of inflammatory cytokine was detected by ELISA,the results showed that the inflammatory cytokine,such as TNF-?,IL-1?,have no significant difference between Pm CQ2-?0979 and Pm CQ2-?0979.These above results suggest that PM0979 is the one of virulence associated factor of Pasteurella multocida,and play an important role in the pathogenesis of Pasteurella multocida. |
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