伪结核棒状杆菌lpxtg270敲除株的构建及其生物学特性与对小鼠的致病性评价

An lpxtg270 knockout strain of Corynebacterium pseudotuberculosis: construction, biological characterization, and evaluation of pathogenicity in mice

摘要:

目的 构建伪结核棒状杆菌(Corynebacterium pseudotuberculosis, Cp)宣汉株(XH02) LPXTG基序蛋白编码基因敲除菌株(XH02Δlpxtg270)，探究lpxtg270在XH02生长、生物被膜形成及感染致病过程中的作用。方法 采用CRISPR/Cas9技术构建XH02Δlpxtg270，比较该敲除菌株与野生菌株XH02在生物学特性、对J774A.1巨噬细胞的侵袭能力、体内感染小鼠致病力等方面的差异。结果 与XH02相比，XH02Δlpxtg270的菌落形态、生长曲线、黏附J774A.1巨噬细胞的能力及胞内增殖能力均无明显变化，但XH02Δlpxtg270的生物被膜形成能力、对J774A.1的侵袭能力、感染引发J774A.1释放乳酸脱氢酶(lactate dehydrogenase, LDH)及分泌IL-1β的能力显著降低，XH02Δlpxtg270体内感染小鼠的致病力及在肝脏、脾脏、肾脏、肺脏和脑中的载菌水平显著下降，引起小鼠上述脏器的病理变化较轻。结论 本研究证实LPXTG270与Cp形成生物被膜、感染侵袭巨噬细胞的能力相关，在Cp感染小鼠致病过程中发挥重要作用。

Abstract:

Objective To construct a strain XH02Δlpxtg270 with knockout of the LPXTG motif protein-coding gene from Corynebacterium pseudotuberculosis XH02 and explore the role of lpxtg270 in the growth, biofilm formation, and infection of XH02.Methods CRISPR/Cas9 was employed to construct XH02Δlpxtg270. The knockout strain and the wild strain XH02 were compared in terms of biological characteristics, invasion into J774A.1 macrophages, and pathogenicity in mice.Results Compared with XH02, XH02Δlpxtg270 did not change significantly in the colony morphology, growth curve, adhesion to J774A.1 macrophages, or intracellular proliferation, while it demonstrated reductions in the biofilm formation and invasion into J774 A.1 cells. Moreover, the release of lactate dehydrogenase and secretion of interleukin-1β from J774 A.1 cells infected with the knockout strain decreased compared with those infected with the wild strain. Compared with XH02, XH02Δlpxtg270 showed weakened pathogenicity in mice and decreased loads in the liver, spleen, kidney, lung, and brain, causing milder pathological changes of above organs in mice.Conclusion LPXTG270 of C. pseudotuberculosis is related to the biofilm formation and invasion into macrophages, playing a key role in the pathogenicity of this bacterium in mice.