分枝杆菌细胞壁脂质PDIM调控SUMO修饰介导肉芽肿形成的分子机制

Mycobacterium tuberculosis (MTB) cell wall lipids play a key role in the invasion and pathogenic process. PDIM is an important lipid composition of cell wall of MTB and induce the formation of tuberculosis granuloma, however the mechanism still remains mysterious. Numerous studies confirm that the pathogens can regulate the SUMO modification pathways to facilitate its infection. Our previous results show that the lipid PDIM not only induce SUMO protein expression, but also promote the granuloma formation, which indicate that the PDIM maybe mediate the granuloma formation by SUMO modification pathway. In this project, the integrated methods such as molecular cloning, flow cytometry, proteomics and gene chip technology will be performed. Firstly, we will study the effects of PDIM on SUMO modification and granuloma formation; Secondly, PDIM mediates the downstream signaling pathways by SUMO modification will be studied with quantitative proteomics, which can help us to clarify the mechanism of secretion of inflammatory factor during PDIM infection. Finally, we will mutate the sumo gene through Cas9 system, and combinate with gene chip to identify the genes involved in granuloma formation. The overall results will elucidate the molecular mechanism that PDIM induce host inflammation and granuloma formation by mediating the SUMO modification. Moreover, the results will further reveal the pathogenesis of PDIM, and provide a new idea for study on the pathogenic mechanism of MTB.

结核分枝杆菌(MTB)细胞壁脂质在细菌侵袭和致病等环节发挥关键作用。PDIM是MTB的重要脂质成分，能诱导形成肉芽肿，但机制尚不明确。研究证实病原体能调控SUMO修饰通路介导感染和致病。前期研究发现，PDIM 既能诱导SUMO修饰蛋白表达，也能促进肉芽肿形成，推测PDIM可能通过调控SUMO修饰通路介导肉芽肿形成。本项目综合运用分子克隆、流式细胞、蛋白质组学和基因芯片等技术，首先研究PDIM对肉芽肿形成及SUMO修饰蛋白的影响；然后通过定量蛋白质组学研究PDIM通过调控SUMO修饰引发的下游信号通路，阐明引起炎性因子分泌的机制；最后用Cas9靶向突变sumo基因，并结合基因芯片鉴定参与肉芽肿形成的基因，明确其对肉芽肿形成的意义与机制。研究成果将阐明PDIM通过调控SUMO修饰介导宿主炎性损伤和肉芽肿形成的分子机制，进一步揭示PDIM的致病机制，为深入研究MTB的致病机制提供新思路。

结核分枝杆菌(MTB)细胞壁脂质在细菌侵袭和致病等环节发挥关键作用。PDIM是MTB的重要脂质成分，能诱导形成肉芽肿，但机制尚不明确。研究证实病原体能调控SUMO修饰通路介导感染和致病。前期研究发现，PDIM 既能诱导SUMO修饰蛋白表达，也能促进肉芽肿形成，推测PDIM可能通过调控SUMO修饰通路介导肉芽肿形成。在本研究中，通过建立海分枝杆菌金鱼感染模型，发现含有PDIM脂质的WT菌株能导致金鱼肝脏、脾脏和肾脏等多个组织中形成典型的肉芽肿样病变，抗酸染色发现在这些脏器中有大量红色细菌分布，而PDIM缺失突变株不能诱导肉芽肿形成，也没有检测到细菌；免疫组化染色发现相对PDIM突变组，WT感染后能诱导组织中Sumo修饰相关分子如SUMO-1和Ubc-9的表达明显高于PDIM突变组，并且通过RT-PCR和western blot得到进一步证实WT感染组中SUMO-1和Ubc-9的mRNA与蛋白表达水平明显高于PDIM 突变组（P<0.01）；同时检测了去Sumo修饰的关键酶—Senp1的表达，检测发现相对PBS对照组，WT组与PDIM突变组中Senp-1表达水平均降低，且WT组表达降低相对于PDIM突变组更明显（P<0.01）；最后，我们检测了参与肉芽肿形成的关键细胞因子如TNF-α、IL-6和IL-12等表达，发现这些细胞因子在WT组比PDIM突变组中表达量明显增加（P<0.01）。.上述结果提示我们PDIM脂质可能通过调控Sumo修饰的关键分子如Sumo1和Ubc9的表达，从而调控炎症介质的表达并介导肉芽肿的形成。该结果是首次研究Sumo修饰在分枝杆菌感染及肉芽肿形成过程中的动态变化及其与细胞因子释放之间的关联，研究成果将阐明PDIM通过调控SUMO修饰介导宿主炎性损伤和肉芽肿形成的分子机制，进一步揭示PDIM的致病机制，为深入研究MTB致病机制提供新思路。