肽聚糖脱乙酰酶Rv1096对分枝杆菌与宿主细胞相互作用的影响

Effect of peptidoglycan deacetylase Rv1096 to interaction between Mycobacterium tuberculosis and host macrophage

目的探讨结核分枝杆菌(Mycobacterium tuberculosis, Mtb)肽聚糖脱乙酰酶Rv1096对分枝杆菌与宿主细胞相互作用的影响。方法利用过表达Rv1096基因的重组耻垢分枝杆菌(Mycobacterium smegmatis, MS)Rv1096,通过差速离心及胰蛋白酶消化试验,确定Rv1096蛋白的亚细胞定位;通过氨基酸定点突变联合伴刀豆凝集素A(Concanavalin A, ConA)免疫印迹确定Rv1096的O-甘露糖基化位点;运用刃天青显色法检测MSRv1096对溶菌酶的抵抗力;通过巨噬细胞感染试验,分析了Rv1096对耻垢分枝杆菌细胞内存活能力和宿主细胞炎症应答的影响。结果确定了Rv1096在重组耻垢分枝杆菌MSRv1096中的亚细胞定位在细胞壁;发现Rv1096蛋白含有三个O-甘露糖基修饰位点(265Thr,266Ser,267Ser);细胞外测试结果表明,Rv1096能增强耻垢分枝杆菌对溶菌酶的抵抗能力,最低抑菌质量浓度从1.5 mg/mL升至2.5 mg/mL,而细胞感染显示其并不能显著增强耻垢分枝杆菌在人单核细胞白血病细胞(THP-1细胞)内的存活能力;定量PCR检测结果显示,过表达Rv1096的耻垢分枝杆菌刺激THP-1细胞分泌炎症因子(TNF-α,IL-6和IL-1β)的能力显著下降。通过平行对比证明若删除O-甘露糖基化位点(265Thr,266Ser,267Ser),对Rv1096基因功能无显著影响。结论 Rv1096是一个细胞壁相关蛋白,具有三个O-甘露糖基化位点。过表达Rv1096对耻垢杆菌在宿主细胞内的存活能力无显著影响,但能够降低宿主细胞对耻垢分枝杆菌的炎症因子应答,且上述功能不受O-甘露糖基化修饰影响。

Objective To investigate the role of peptidoglycan deacetylase of Mycobacterium tuberculosis(Mtb) Rv1096 in the interaction between Mtb and host cell. Methods The Rv1096 over-expressing strain of Mycobacterium smegmatis(MS), namely MSRv1096 was utilized. The subcellular localization of Rv1096 was determined by differential centrifugation and trypsin digestion assays. The O-mannosylation sites of Rv1096 were screened and identified by site-directed mutagenesis and ConA lectin blotting. The resistance of MSRv1096 against lysozyme was subsequently detected by resazurin assay. Finally, the impact of Rv1096 on the survival of MS and the inflammatory responses of host cell were measured by cell infection experiment. Results Rv1096 protein in the recombinant strain MSRv1096 was mainly presence in the cell wall fraction. It was confirmed that Rv1096 protein contains three O-mannosylation sites(265Thr, 266Ser, 267Ser). The results of extracellular tests showed that Rv1096 could enhance the resistance of MS to lysozyme,with a minimal inhibition concentration increased from1.5 mg/mL to 2.5 mg/mL, but the cell infection assay showed that Rv1096 could not significantly enhance the viability of MS in THP-1 cells. However, qPCR data demonstrated that the over-expression of Rv1096 significantly reduced capability of MS to induce the expression of inflammatory cytokines(TNF-α, IL-6 and IL-1β) in THP-1 cells. In addition, we proved that the deletion of O-mannosylation site(265Thr,266Ser,267Ser) did not influence on the function of Rv1096. Conclusion Rv1096 is a cell wall associated protein, which has three O-mannosylation modification sites. Over-expression of Rv1096 had no a significant effect on the survival of MS in THP-1 cell but could reduce the inflammatory cytokines responses, and the mentioned functions of Rv1096 were not affected by O-mannosylation.