Traceless deletion of pld in Corynebacterium pseudotuberculosis and biological characteristics and pathogenicity of the mutant

Abstract:

[Objective]Corynebacterium pseudotuberculosis,a facultative intracellular pathogen,infects a variety of animals and human,causing chronic suppurative inflammation. This paper aims to further evaluate the role of phospholipase D gene (pld) in C.pseudotuberculosis infection.[Methods] We employed homologous recombination to construct the traceless pld deletion strains (Δpld) of C.pseudotuberculosis without introducing foreign genes.Then,we compared the colony morphology and growth curves of deletion strains and wild-type strains,observed the influence of pld deletion on lactate dehydrogenase (LDH) release of C.pseudotuberculosis-infected macrophages and the proliferation of the pathogen in macrophages,and determined the death rates of and levels of pro-inflammatory cytokines in mice infected with the wild type and the deletion strain,thereby dissecting the relationship between pld and pathogenicity of the bacterial species.[Results] Traceless deletion of pld had no obvious effects on colony morphology and growth of C.pseudotuberculosis.Compared with ATCC 19410 and XH02,ATCC 19410Δpld and XH02Δpld showed no synergistic hemolysis with Rhodococcus equi ATCC6939.The LDH release of the macrophages infected with ATCC 19410Δpld and XH02Δpld was significantly less than that of the macrophages infected with the wild strains,and pld deletion decreased the intracellular bacteria in macrophages.The death rate,bacterial load in the liver and spleen,and the levels of pro-inflammatory cytokines in ascites and organs of mice infected with ATCC 19410Δpld were lower than those of mice infected with ATCC 19410.[Conclusion]The traceless pld deletion strains of C.pseudotuberculosis were developed.We confirmed that pld played an important role in death of C.pseudotuberculosis-infected macrophages and pathopoiesis of C.pseudotuberculosis in mice.