分泌性PLA2 Rv3177通过水解磷脂分子进而促进结核分枝杆菌免疫逃逸的机制研究

Intracellular bacteria (Such as legionella pneumoniae etc.) use secreting phospholipase A2 (PLA2) protein to hydrolyze phospholipid molecules and then inhibit the phagocyte maturation of macrophages, leaving bacteria in a state of persistent infection, which is an important way to resist host immune defense. Tuberculosis which a caused by intracellular bacteria Mycobacterium tuberculosis (Mtb). Mtb is an important zoonotic infectious disease and severely threatens human health However, the study of Mtb PLA2 is only limited to the activity and toxicity analysis up to now, weather or not it promotes Mtb retention infection has not been reported. Our previous studies showed that Rv3177 is secreted PLA2 protein from Mtb, which has the activity of hydrolyzing phospholipid molecules and significantly increasing the retention infection of recombinant M. smegmatis in macrophages. Based on this, we speculate that Rv3177 may inhibit phagocytic maturation of macrophages and promote the escape of Mtb. Our project will take Mtb Rv3177 protein to study its effects on the various stages of macrophage phagocytosis maturation. Screen for its target phospholipid molecule of interferes with phagocyte maturation, decipher the role of Rv3177 in the phagosomes and endosomal fusion as well as the phagosome mature signaling pathway, elucidate the molecular mechanism of Mtb immune escape, which will provide a reference for the prevention and control of tuberculosis as well as the development of new anti-tuberculosis preparations.

胞内寄生菌（如嗜肺军团菌等）通过分泌磷脂酶A2（PLA2）水解磷脂分子以抑制巨噬细胞吞噬体成熟，从而使细菌处于持留感染状态是其抵抗宿主免疫防御的重要手段。结核病是由胞内菌结核分枝杆菌（Mtb）引起的严重危害人类健康的重要传染病。然而，目前关于Mtb PLA2的研究仅限于活性和毒力分析，是否有助于Mtb的持留感染还未见报道。我们前期研究发现，分泌性Rv3177是Mtb的PLA2蛋白，具有显著水解磷脂分子的活性，可明显提高重组耻垢分枝杆菌在巨噬细胞内的持留感染能力。为进一步揭示Mtb PLA2抵抗巨噬细胞免疫杀伤的机制，本项目以Rv3177为研究对象，系统评价其对巨噬细胞吞噬体成熟各阶段的影响，筛选Rv3177水解的促进吞噬体成熟相关的磷脂靶分子，解析吞噬体膜融合及其成熟的信号通路，从而阐明Rv3177抑制吞噬体成熟进而促进Mtb免疫逃逸的分子机制，为结核病防控和开发新型抗结核病制剂奠定基础。

结核病是由结核分枝杆菌（Mtb）引起的严重危害人类健康的重要传染病。Mtb能够逃避宿主免疫防御，寄生在宿主细胞中长期存在。因此，明确Mtb免疫逃逸机制对结核病的预防和治疗具有重要意义。.Mtb侵入宿主后被巨噬细胞识别，经一系列过程形成Mtb吞噬溶酶体，在此过程中磷脂酰肌醇及其磷酸化分子作为靶分子招募重要信号传递功能的效应分子，在杀伤Mtb过程中发挥重要作用。然而，现今仍然广泛存在Mtb在巨噬细胞内持留感染，它们是如何逃逸巨噬细胞的免疫杀伤，是否存在某种毒力因子抑制吞噬体成熟，是否和其中重要的磷酯分子相关，仍迫切需要更深入的探索。.本研究首先通过生物信息学分析发现在Mtb中的Rv3177蛋白具有保守的磷脂酶A2（PLA2）结构域，提示其为Mtb的磷脂酶。随后应用分光光度法验证了重组过表达纯化的Rv3177具有PLA2活性，并确定其酶活性位点为Ser79。后利用多克隆抗体明确Rv3177是主要存在于分枝杆菌菌壁上的胞外蛋白，并可明显改变重组耻垢分枝杆菌（rMs）的单菌落形态。经菌落计数发现其可显著增强rMs在小鼠腹腔巨噬细胞内的存活能力，进而通过透射电镜观察，Rv3177促进了rMs从吞噬体囊泡中逃逸。随后，经免疫荧光分析，证实Rv3177可降低rMs与吞噬溶酶体共定位，明确Rv3177在促进rMs从巨噬细胞吞噬溶酶体逃逸过程中发挥重要作用。经Biacore蛋白与小分子互作系统发现Rv3177可以与吞噬体膜相关磷脂酰肌醇结合，影响吞噬体成熟相关信号通路及其对关键下游因子的传递。此外，经感染小鼠动物模型分析， Rv3177显著提高了rMs的存活率，促进了小鼠肺脏的炎症病理反应。 .综上所述，本研究证实了Rv3177是Mtb的胞外PLA2，并阐明Rv3177通过结合磷脂酰肌醇促进分枝杆菌从巨噬细胞吞噬溶酶体中逃逸，从而提高其在宿主体内的存活率，最终达到菌体持续感染的致病机制，为结核病防控和开发新型抗结核病制剂奠定基础。