葡萄糖苷转运蛋白Lmo0738介导单增李斯特菌毒力的作用研究

The glucoside transporter Lmo0738 contributes to virulence of Listeria monocytogenes

【目的】以单增李斯特菌葡萄糖苷转运蛋白Lmo0738为研究对象,研究其介导细菌毒力发挥的生物学作用。【方法】通过细菌同源重组技术构建lmo0738基因缺失株和回补株,研究它们在生长、溶血、细胞黏附与侵袭以及胞间迁移等方面与野生株的差异。同时,采用荧光定量PCR和Western blotting方法检测Δlmo0738毒力相关基因的转录水平和毒力相关蛋白的表达情况。【结果】缺失lmo0738后,李斯特菌体外生长能力、溶血能力、黏附和侵袭能力以及胞间迁移能力均显著减弱,溶血素O(listeriolysin O,LLO)蛋白毒力表达水平和关键毒力基因转录水平均显著降低。【结论】本研究证实缺失lmo0738能够降低单增李斯特菌毒力,为完善包括单增李斯特菌在内的重要食源性病原菌磷酸转移酶系统(phosphotransferase system,PTS)糖分解代谢及感染机制奠定了关键基础。

[Objective]To investigate the biological role played by the glucoside transporter Lmo0738 in the virulence of Listeria monocytogenes.[Methods]The lmo0738-deleted mutant(Δlmo0738)and complementation mutant(CΔlmo0738)were constructed by homologous recombination.The growth,hemolytic activity,cellular adhesion and invasion,and intracellular migration of the wild type strain and the mutants were assessed by the growth curves,sheep red blood cell hemolysis assay,infection of human epithelial cells(Caco-2),and infection of mouse fibroblastic cells(L929),respectively.Additionally,the mRNA and protein levels of the virulence factor listeriolysin O(LLO)were determined by real-time quantitative reverse transcription PCR(RT-qPCR)and Western blotting,respectively.[Results]The L.monocytogenes strain with the deletion of lmo0738 demonstrated weakened growth and diminished hemolytic activity.Notably,Δlmo0738 exhibited significantly reduced cell adhesion,invasion,and intracellular migration compared with the wild type strain.In addition,the mRNA and protein levels of LLO were significantly down-regulated inΔlmo0738.[Conclusion]This study provides the evidence that the absence of lmo0738 attenuates the virulence of L.monocytogenes,laying a crucial foundation to illustrate the mechanism of the phosphotransferase system(PTS)in regulating the sugar catabolism and the infection mechanism of major food-borne pathogens including L.monocytogenes.