Construction And Characterization Of Pld Deletion Of Corynebacterium Pseudotuberculosis

Pseudotuberculosis was a chronic zoonotic infectious disease caused by Corynebacterium pseudotuberculosis.The prevalence of the disease was very widespread,mainly infecting goats and sheep.It was an intracellular bacteria.The host was mainly infected through the wound,and it could be swallowed by macrophages and neutrophils.It remained active and spreads with blood and lymph to the people,forming an abscess at the site of infection and causing host cell death.Since the surface of the lesion infected by C.pseudotuberculosis was covered with a thick and dense cyst,it was difficult for many drugs to penetrate into it and the pathogenic bacteria in the abscess cannot be completely killed.Therefore,the prevention and control of the disease was difficult,and the pathogenesis of the disease needed to be further clarified.The pathogenic effect of C.pseudotuberculosis was closely related to its abscess,which was characterized by a significant increase in inflammatory cytokines at the infected site.The mechanism of inflammation was not completely clear.Phospholipase D(pld),encoded by the pld gene,was the most important virulence factor of C.pseudotuberculosis,and was also considered to be a leukocyte-toxic exoprotein that helps C.pseudotuberculosis invade local lymph nodes during infection.It was closely related to the destruction of macrophages and the pathogenesis of other tissues and organs.The pld gene-deficient strain of C.pseudotuberculosis was constructed and its biological characteristics including inflammation were evaluated to provide a scientific basis for further research on the mechanism of pld in the pathogenic infection.1.Construction of pld gene-deficient strains of C.pseudotuberculosis ATCC 19410 and XH02In this study,the classic homologous recombination technique,with the ATCC19410 and XH02 as the target bacteria,was used to try to construct the pld gene deletion strains of C.pseudotuberculosis.Three methods were used to construct pld gene-deficient strains of C.pseudotuberculosis.The first scheme was as follows:deletion strains was constructed by double exchange method after connecting homologous arms with pMD19-T vector;scheme two:deletion strains was constructed by by single exchange with ZTOPO-T vector;scheme three:deletion strains was constructed by double exchange method using pK18mobsacB as a gene knockout vector.Single colony did not grow after electrotransformation in the first two schemes,and the pld gene deletion strains were constructed successfully in the third scheme.The method is as follows:the upstream homology arm(1326 bp)and the downstream homology arm(970 bp)of the pld gene were amplified,and the upstream and downstream homology arms were connected by fusion PCR,cloned into the ZTOPO-T vector.The fusion fragment of the homologous arm of the upstream and downstream was ligated to the plasmid pK18mobsacB,and the pld gene suicide recombinant plasmid pK18mobsacB/?pld was constructed.The suicide recombinant plasmid was electrotransformed into C.pseudotuberculosis competent cells,and Kanamycin-resistant clones were screened on culture medium containing Kanamycin.The first homologous recombination conloys of C.pseudotuberculosis were spread on blood agar LB plates containing 20%sucrose,and clones that had undergone the second homologous recombination were negatively screened by the sucrose lethal gene(sacB gene).It was confirmed that a C.pseudotuberculosis with pld gene-deficient strain(with a deletion of 496 bp within the pld)was obtained by PCR identification and gene sequencing.It was found that the wild strain of C.pseudotuberculosis and Rhodococcus equinus(ATCC 6939)showed significant synergistic hemolytic activity by synergistic hemolysis test,while the pld gene-deficient strain(?pld)completely lost the synergistic hemolytic effect with Rhodococcus equinus.It showed that the pld gene-deficient strains of ATCC 19410 and XH02 strains were successfully constructed.The pld gene-deficient strains of ATCC 19410 and XH02 strains were successively subcultured,and they did not return to the wild type within 9 generations.2.Comparison of in vitro biological characteristics between pld gene-deficient strains of C.pseudotuberculosis and wild strainsThe ATCC 19410?pld and ATCC 19410 wild strain were cultured on fresh blood agar medium to observe the morphology and staining characteristics of the colony.They were cultured with different concentrations of NaCl(normal osmotic pressure and 10%NaCl)and different pH conditions(pH 5.0 and pH 9.0).The growth curves of pld gene-deficient strain(ATCC 19410?pld)and ATCC 19410 wild strain were measured to observe the effects of pld gene deletion to C.pseudotuberculosis on environmental adaptability.In addition,J774A.1 macrophages were infected with pld gene-deleted strain(ATCC 19410?pld)and ATCC 19410 wild strain in vitro,and the cell was counted by lysis and dilution.The results showed that there were no significant differences in colony morphology,staining characteristics,and adaptability to different osmotic pressures and pH between the pld gene-deficient strain(ATCC19410?pld)and the wild strain ATCC 19410.In terms of cell adhesion,compared with the ATCC 19410 wild strain,the pld gene-deficient strain(ATCC 19410?pld)had a decreased adhesion for 26.63%to J774A.1 macrophages.It is showed that the pld gene deletion had no significant effect on the growth and proliferation of C.pseudotuberculosis and its adhesion to macrophages.3.Pathogenicity comparison between pld gene-deficient strains of C.eudotuberculosis and wild strainsThe pld gene deletion strain(ATCC 19410?pld)and ATCC 19410 wild strain were inoculated into kunming mice in vivo(6×10~5 CFU/0.2mL/head).The disease deaths in mice were were observed for 2 weeks,and the mice survival curve was drawed.Further ATCC 19410 and ATCC 19410?pld were inoculated into C57BL/6mice(1×10~5 CFU/0.2mL/head),and the microbial load of the liver and spleen tissues were detectied to assess the effects of a lack of pld gene pathogenicity in mice.The results showed that compared with the death with 85.7%(C57BL/6)mice infected by ATCC 19410 wild strain,the pathogenicity of ATCC 19410?pld decreased by 71.4%,and the death time of infected mice was delayed.For ATCC 19410?pld group,the load of C.pseudotuberculosis in the liver and spleen was lower than that of the wild-type strain.The load of spleen was the most significant,and its load decreased by 84%.It was shown that the pld gene deletion can significantly extenuate the pathogenicity of C.pseudotuberculosis to mice.The ATCC 19410?pld and ATCC 19410 wild strain were inoculated into mice(6×10~5 CFU/0.2 mL/head),and organs were tested for cytokines after 12 hours.It was showed that the levels of IL-1?and TNF?in ascites,liver,spleen,lung and kidney of mice infected with ATCC 19410?pld were decreased compared with mice infected with wild-type strain ATCC 19410 at an overall level.Among them,the secretion of TNF?in lungs and ascites of mice infected with ATCC 19410?pld was significantly lower than that of wild-type strains of ATCC 19410,and the secretion of IL-1?in liver and kidney of mice infected with ATCC 19410?pld was significantly lower than that of wild-type strains of ATCC 19410.It was indicated that the pld gene deletion can extenuate the inflammation of mice infected with C.pseudotuberculosis.In a summary,The pld gene deletion strains,ATCC 19410?pld and XH02?pld,were successfully constructed.The culture characteristics,environmental adaptability and the adhesion ability of phagocytic cells of pld gene deletion strains had no significant effect.It can significantly extenuate the inflammatory effect and pathogenicity of the pathogen infection,laying a foundation for further elucidating the pathogenic mechanism of the pld gene of C.pseudotuberculosis.