产单核细胞李斯特菌actA/plcB缺失株的构建及其生物学特性

Construction and characterization of a mutant strain of Listeria monocyto-genes with a deletion of actA and plcB

摘要:

产单核细胞李斯特菌的毒力因子与该菌在细胞间扩散、传播有着直接的关系, 其中肌动蛋白聚集因子ActA是细菌由细胞浆扩散至相邻细胞所必须的因子, 而广谱磷脂酶C则参与具有双层膜吞噬体的裂解过程。[方法]本研究中利用同源重组技术成功构建了毒力因子ActA 和 PC-PLC双缺失的突变株, [目的]并对突变株的毒力和免疫应答潜能进行评价。[结果]Western blot 和磷脂酶活性测定实验, 分别从蛋白质水平上证实actA和plcB基因的缺失。突变株的毒力显著降低, 对小鼠半数致死剂量比野生型菌株提高约103倍, 但仍然保持较好地诱导T细胞应答的能力, 并且能完全保护野生型细菌致死剂量的攻击。实验结果不仅表明ActA 和 PC-PLC是产单核细胞李斯特菌的重要毒力因子, 而且证实安全性提高的突变株依然保持有较强地诱导细胞免疫应答的能力。[结论]因此, 该突变株的获得不仅对李斯特菌病的预防具有重要作用, 而且为构建预防人类和动物疫病的疫苗载体奠定了基础, 此外对于阐明LM毒力因子的致病机理与免疫保护作用提供了条件。

Abstract:

The virulence of Listeria monocytogenes is directly related to its ability to spread from cell to cell without leaving the intracellular milieu. Among the bacterial factors involved in cell-to-cell spread, actin-polymerizing protein ActA is required for bacterial spread to adjacent cells, while the broad-range phospholipase C (PC-PLC) contributes to bacterial escape from secondary vacuoles. [Methods] Based on homologous recombination technology, we constructed a double gene-mutant strain by deleting two virulence factors, ActA and PC-PLC, [Objective] its toxicity and immunopotency was evaluated in murine model. [Results] And then, the mutant strain was further verified the absence of ActA and PC-PLC using Western blot analysis and phospholipase activity assay. It showed highly attenuated and its 50% lethal dose in BLAB/c mice was increased at least 103-fold compared to the parent strain. Nevertheless, mice preimmunized with the mutant strain elicited strong T-cell responses and fully protected against a lethal challenge with the virulent strain. [Con-clusion] These results not only verified that ActA and PC-PLC were essential virulence factors for L. monocytogenes, but also demonstrated that the mutant strain possessed good immunogenicity with higher safety. The mutant strain could be further studied as a vaccine candidate to prevent listeriosis and used as a vector to deliver heterologous antigens. Fur-thermore, it provided the possibilities to elucidate the molecular mechanisms of pathogenesis and immune response trig-gered by L. monocytogenes.