结核分枝杆菌PE26蛋白调节巨噬细胞免疫反应进而促进分枝杆菌胞内定殖

Mycobacterium tuberculosis PE26 regulates macrophage immune response and then promotes Mycobacterium intracellular colonization

结核分枝杆菌(MTB)PE/PPE家族蛋白介导MTB与宿主间的相互作用,调节宿主炎症反应,有助于其的存活和疾病发展。为研究PE/PPE家族中PE26蛋白的作用,本实验利用PCR扩增pe26基因片段,构建重组载体p AIN-PE26,转化耻垢分枝杆菌(Ms)构建重组菌p AIN-PE26/Ms,经western blot鉴定结果显示,p AIN-PE26/Ms正确表达重组PE26蛋白(rPE26)。利用差速离心分离p AIN-PE26/Ms亚细胞组分,经western blot鉴定结果显示,r PE26定位于p AIN-PE26/Ms细胞壁。通过p AIN-PE26/Ms的菌落形态观察和生长曲线测定,结果显示,p AIN-PE26/Ms菌体表面较p AIN/Ms对照组更粗糙,脊不规则且相对较高,褶皱不明显;p AIN-PE26/Ms生长速度较对照组极显著增加(P<0.001)。利用H37Ra感染巨噬细胞,通过q RT-PCR检测pe26转录水平,结果显示,H37Ra感染巨噬细胞后pe26转录水平极显著增加(P<0.001)。利用p AIN-PE26/Ms感染巨噬细胞,分别通过q RT-PCR、ELISA检测细胞因子转录和表达水平,采用乳酸脱氢酶(LDH)细胞毒性试验、流式细胞术和胞内定殖的方法检测细胞毒性,结果显示,p AIN-PE26/Ms促进宿主细胞炎性细胞因子(IL-1β、IL-6、IL-12和TNF-α)的转录和表达(P<0.05);r PE26促进巨噬细胞死亡,且在感染48 h时促进细胞早期凋亡(P<0.05);r PE26促进p AIN-PE26/Ms在巨噬细胞内的定殖(P<0.01)。上述结果首次表明,定位于细胞壁的r PE26在感染巨噬细胞过程中促进p AIN-PE26/Ms的生长、炎性细胞因子的转录和表达、巨噬细胞凋亡和胞内定殖。本实验为解析PE26参与分枝杆菌的致病机制和MTB调节宿主免疫反应的机制提供一定参考依据。

Mycobacterium tuberculosis(MTB)PE/PPE family proteins mediate pathogen-host interactions,regulating hostinflammatory responses and contributing to pathogen survival and disease progression.To investigate the role of PE26 protein in PE/PPE family,the recombinant vector p AIN-PE26 was constructed by PCR of pe26 fragment and transformed into Mycobacterium smegmatis(Ms)to construct recombinant p AIN-PE26/Ms.Recombinant PE26 protein(r PE26)was successfully expressed by p AIN-PE26/Ms and identified by western blot.The subcellular components of p AIN-PE26/Ms were isolated using differential centrifugation,and r PE26 was mainly localized to the cell wall of p AIN-PE26/Ms as identified by western blot.Colony morphology results and the growth curve results showed that the surface of p AIN-PE26/Ms was rougher than that of the control group,the ridges were irregular and relatively high,and the folds were not obvious,the growth rate of p AIN-PE26/Ms increased compared to the control group(P<0.001).H37Ra infected macrophages and detected pe26 transcription levels by q RT-PCR,and the results showed that the transcription levels of pe26 increased after H37Ra infection of macrophages(P<0.001).p AIN-PE26/Ms infected macrophages and detected pe26 transcription and expression levels by q RT-PCR,ELISA,detected cytotoxicity by LDH cytotoxicity assay,flow cytometry and intracellular colonization,and the results showed that p AIN-PE26/Ms promoted transcription levels and expression of host cell inflammatory cytokines(IL-1β,IL-6,IL-12 and TNF-α)(P<0.05),r PE26 promoted cell death and early apoptosis at 48 hours(P<0.05),r PE26 promoted intracellular colonization of p AIN-PE26/Ms in macrophages(P<0.01).These results suggest that r PE26 localized in the cell wall plays a role in the process of infecting macrophages,promoting the growth of p AIN-PE26/Ms,inflammatory cytokine transcription and expression,apoptosis and intracellular colonization.This study provides theoretical basis for resolving the involvement of PE26 in the pathogenesis of Mycobacterium and the mechanism of MTB regulation of host immune response.