Isocitrate lyase from Mycobacterium tuberculosis promotes survival of Mycobacterium smegmatis within macrophage by suppressing cell apoptosis
Isocitrate lyase from Mycobacterium tuberculosis promotes survival of Mycobacterium smegmatis within macrophage by suppressing cell apoptosis
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结核分枝杆菌的异柠檬酸裂解酶通过抑制细胞凋亡促进巨噬细胞内耻垢分枝杆菌的存活
DOI：
10.1097/00029330-200806020-00015
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发表时间：
2008-06-20
影响因子：
6.1
通讯作者：
Yang Chun
中科院分区：
文献类型：
--
作者：
Li Jun-Ming;Li Na;Yang Chun
背景 异柠檬酸裂解酶 (ICL) 先前已被证明在结核分枝杆菌 (MTB) 的细胞内代谢中发挥关键作用。目前，一些证据表明来自 MTB 的 ICL (MTB-ICL) 可能在 MTB 与宿主巨噬细胞之间的相互作用中发挥一定作用。但目前尚未有MTB-ICL与宿主巨噬细胞相互作用的研究。方法采用聚合酶链式反应(PCR)扩增MTB-icl和耻垢分枝杆菌(MS)-icl基因,克隆至大肠杆菌-分枝杆菌穿梭质粒pUV15中,获得重组穿梭质粒pMTB-icl和pMS-icl。通过电穿孔转化至MS后，通过逆转录酶(RT)-PCR验证pMTB-icl和pMS-icl的表达。还通过荧光显微镜验证了rMS被巨噬细胞吞噬时pUV15衍生的重组质粒的表达。使用Ms 1-2c、rMS-pUV15、rMS-pMS-icl和rMS-pMTB-icl感染RAW264.7细胞，并在感染后0、24和48小时通过细菌培养监测细胞内MS的存活。收集Ms 1-2c、rMS-pUV15、rMS-pMS-icl和rMS-pMTB-icl感染的巨噬细胞的培养上清液，并分别通过ELISA或Griess测定测量干扰素（IFN）-γ和一氧化氮（NO）浓度。采用原位TUNEL技术检测巨噬细胞的凋亡情况。结果RT-PCR显示pMTB-icl和pMS-icl在MS中均有表达。荧光显微镜观察表明，当MS被巨噬细胞吞噬时，pUV15衍生的重组质粒(pUV15-IG)也可以在MS中表达。细菌培养数据表明，与 Ms 1-2c、rMS-pUV15 和 rMS-pMS-icl 相比，rMS-pMTB-icl 在鼠巨噬细胞系 RAW264.7 中表现出显着增加的细胞内存活率。这种细胞内存活率的增加并不伴随宿主巨噬细胞中IFN-γ和NO的上调。但与其他MS株感染的巨噬细胞相比，rMS-pMTB-icl感染的巨噬细胞凋亡率较低。结论MTB-ICL可以促进MS的细胞内存活。抑制宿主巨噬细胞的凋亡可能是细胞内存活增加的重要机制之一。
Background Isocitrate lyase (ICL) was previously demonstrated to play a pivotal role in the intracellular metabolism of Mycobacterium tuberculosis (MTB). Presently several lines of evidence suggest that ICL from MTB (MTB-ICL) may play some roles in the interaction between MTB and host macrophage. However, there has been no research on the interaction between MTB-ICL and host macrophage.Methods MTB-icl and M. smegmatis (MS)-icl genes were amplified by polymerase chain reaction (PCR) and cloned into the E coli-mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmids pMTB-icl and pMS-icl. Following transformation into MS by electroporation, the expression of pMTB-icl and pMS-icl was verified by reverse transcriptase (RT)-PCR. The expression of recombinant plasmids derived from pUV15 when rMS was phagocytized by macrophage was also verified via fluorescence microscope. Ms 1-2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were used to infect RAW264.7 cells and the survival of intracellular MS was monitored by bacterial culture at 0, 24 and 48 hours after infection. The culture supernatants from macrophage infected by Ms 1-2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were collected and the interferon (IFN)-gamma and nitric oxide (NO) concentrations were measured by ELISA or by Griess assay, respectively. The apoptosis of macrophage was assayed by the in situ TUNEL technique.Results RT-PCR showed that both pMTB-icl and pMS-icl could be expressed in MS. Fluorescence microscopic observation showed that recombinant plasmids derived from pUV15 (pUV15-IG) could also be expressed in MS when MS were phagocytized by macrophage. Bacterial culture data demonstrated that rMS-pMTB-icl exhibited significantly increased intracellular survival in the murine macrophage cell line RAW264.7 compared with Ms 1-2c, rMS-pUV15 and rMS-pMS-icl. This increased intracellular survival was not accompanied by the upregulation of IFN-gamma and NO in host macrophage. But a lower apoptosis rate of macrophages infected with rMS-pMTB-icl was observed when compared with macrophages infected with other strains of MS.Conclusions MTB-ICL could promote the intracellular survival of MS. Suppressing the apoptosis of host macrophage may be one of the important mechanisms involved in this increased intracellular survival.