鼠伤寒沙门菌六型分泌系统效应蛋白Clpv对巨噬细胞极化的影响

Effect of T6SS effector protein Clpv of Salmonella typhimurium on macrophage polarization

摘要:

【目的】探讨VI型分泌系统(type VI secretion system, T6SS)效应蛋白Clpv在鼠伤寒沙门菌(Salmonella enterica serovar Typhimurium)致病过程中的功能。【方法】以鼠伤寒沙门菌SL1344基因组为模板克隆clpv基因，并比较与其他革兰氏阴性菌台湾假单胞菌(Pseudomonas taiwanensis)、植生拉乌尔菌(Raoultella planticola)、鳗利斯顿氏菌(Listonella anguillarum)、菠萝多源菌(Pantoea ananatis)、粘放线菌(Actinomyces viscosus)和大肠埃希菌(Escherichia coli)的同源性；将clpv基因克隆至pEGFP-N1载体构建重组质粒pEGFP-Clpv，利用Western blotting、实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction, q-PCR)、荧光显微镜以及流式细胞术检测蛋白表达、定位及诱导小鼠巨噬细胞M1型和M2型极化水平。【结果】clpv基因全长为2 637 bp，与台湾假单胞菌的同源性最高；Western blotting、qPCR和免疫荧光检测表明重组蛋白大小约120 kDa，在细胞中有明显绿色荧光并且主要定位于细胞膜；q-PCR和流式细胞术结果发现Clpv转染组巨噬细胞M1型极化显著增加(P<0.01)，M2型巨噬细胞极化显著减少(P<0.01)。【结论】成功克隆表达鼠伤寒沙门菌T6SS效应蛋白Clpv，并明确其胞内表达定位以及对巨噬细胞极化的影响。

Abstract:

[Objective] To investigate the role of type VI secretion system (T6SS) effector protein Clpv in the pathogenesis of Salmonella typhimurium.[Methods] We cloned the clpv gene from SL1344 genome of S. typhimurium and compared it with the clpv genes of other Gram-negative bacteria (Pseudomonas taiwanensis, Raoultella planticola, Listonella anguillarum, Pantoea ananatis, Actinomyces viscosus, and Escherichia coli). The clpv gene was cloned into pEGFP-N1 vector to construct the recombinant plasmid pEGFP-Clpv. The protein expression, localization, and the induced M1 and M2 polarization of mouse macrophages were detected by Western blotting, q-PCR, fluorescence microscopy, and flow cytometry. [Results] The clpv gene was 2 637 bp, showing the highest homology to clpv of P. taiwanensis. Western blotting, q-PCR, and immunofluorescence showed that the recombinant protein was about 120 kDa and apparent green fluorescence in cells was found. The protein was located primarily in cell membrane. The results of q-PCR and flow cytometry indicated that Clpv enhanced M1 polarization (P<0.01) but weakened M2 polarization of mouse macrophages (P<0.01). [Conclusion] The T6SS effector protein Clpv of S. typhimurium was cloned and expressed, and its intracellular localization and effect on macrophage polarization were clarified.