伪结核棒状杆菌pld基因无痕缺失株构建及其生物学特性及致病性研究

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

伪结核棒状杆菌是一种可以感染多种动物和人的兼性胞内寄生病原,主要引起被感染动物慢性化脓性炎症反应。【目的】为进一步评价磷脂酶D基因(pld)在伪结核棒状杆菌感染致病中的作用。【方法】本研究采用同源重组技术,在不引入外源基因情况下,构建伪结核棒状杆菌pld无痕缺失株。通过比较pld缺失株和野生株的菌落形态及生长曲线、体外对巨噬细胞乳酸脱氢酶(LDH)释放及其在胞内的繁殖情况以及体内感染小鼠致死率及促炎细胞因子分泌水平,研究pld与该病原感染致病之间的关系。【结果】无痕缺失pld对伪结核棒状杆菌的菌落形态及生长无明显影响。与野生株ATCC19410和XH02相比,ATCC19410Δpld和XH02Δpld失去了与马红球菌ATCC6939的协同溶血功能,所感染巨噬细胞的LDH释放水平显著下降,胞内细菌数量显著降低;体内试验发现ATCC19410Δpld对小鼠的致死率、肝脏和脾脏载菌量以及腹水及脏器促炎细胞因子水平均低于ATCC19410。【结论】成功构建了伪结核棒状杆菌pld无痕缺失株。证实pld在该病原体外感染引发巨噬细胞死亡以及体内感染小鼠致病中具有重要作用。

[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.