结核分枝杆菌PE_PGRS41调控LUBAC介导的NF-κB信号通路及宿主细胞命运的分子机理

We aims to elucidate the molecular basis underlying the Linear ubiquitin assembly complex (LUBAC)-mediated activation of NF-κB signaling elicited by Mycobacterium tuberculosis PE_PGRS41. PE_PGRS41 is a member of PE/PPE family, also termed aprC（acid and phagosome regulated protein C）, which can be modulated by acid and phagosome. LUBAC is the sole known E3 ligase for the linear ubiquitination. Our hypothesis is that PE_PGRS41alters the LUBAC- mediated NF-κB signaling and downstream cytokines expression and cell fates. This is based on our previous results that 1. PE_PGRS41 differentially regulates the cytokines, promoted the apoptosis of host cells and recombinant Mycobacteria survival;2.Differentially ubiquitinated the components of NF-κB, especially the NEMO and LUBAC components HOIP, HOIL-1L;3. The transcription of LUBAC components was enhanced in active tuberculosis patients peripheral blood monocytes. To test the hypothesis, truncated or site-directed mutagenesis PE_PGRS41 will be used to define the critical residues or domains for this effect, the ubiquitination of LUBAC components and linear ubiquitination of NF-κB signaling components, such as the NEMO will be monitored to test this hypothesis.This will inspire the pathogenesis quest from novel perspective.

本研究旨在阐明结核分枝杆菌PE_PGRS41调控LUBAC复合体介导的NF-κB激活与宿主细胞命运的分子机理。PE_PGRS41表达被酸和吞噬体调控，也称为aprC(acid and phagosome regulated protein C)。LUBAC是负责线性泛素化的唯一已知E3连接酶。基于我们前期PGRS41差异调控细胞因子表达，增强重组菌胞内存活、抑制宿主细胞凋亡，导致宿主细胞NF-κB信号通路多个组分泛素化，尤其是NEMO以及LUBAC复合体组分，活动性肺结核患者外周血单核细胞中LUBAC组分转录增强等结果，我们假设：PE_PGRS41调控LUBAC介导的NF-κB激活，改变细胞因子表达和细胞命运。项目将通过定量泛素化组，NF-κB-luc荧光素酶活性，截短/定点突变鉴定PE_PGRS41功能的关键残基或者功能域，LUBAC泛素化检测等验证假说，研究线性泛素化的作用和机理。

结核病仍然是危害全球公共卫生健康的重大传染病之一。结核病的致病菌-结核分枝杆菌能够与宿主相互作用，调节宿主对细菌的免疫应答，再宿主细胞内存活，逃避宿主免疫清除。结核菌PE/PPE家族由PE，PPE，PE_PGRS三个亚家族构成，约占结核分枝杆菌基因组的10%。PE/PPE家族分子可能参与细菌-宿主相互作用，是介导逃避宿主免疫清除的重要效应分子。本课题探讨分枝杆菌PE/PPE家族成员PGRS-41,并增加了PPE10、PPE60、PPE36与宿主相互作用的分子机制。研究结果显示，PPE60可引起THP-1巨噬细胞焦亡，且主要通过LUBAC依赖的NF-κB信号通路上调THP-1巨噬细胞炎症因子IL-1β、IL-6、TNF-α和IL-12p40等的表达；PPE10显著降低THP-1巨噬细胞的凋亡，下调细胞因子IL-1β，IL-6，IL-12p40和TNF-α的转录水平，上调抗炎细胞因子IL-10的转录水平，提高分枝杆菌在巨噬细胞中的存活率。使用不同通路的抑制剂处理，发现PPE10通过LUBAC和c-IAP1调节NF-κB途径，改变炎症细胞因子的表达；PPE36感染THP-1巨噬细胞后，通过抑制多种细胞极化相关因子，使巨噬细胞向M1型的极化受阻，且相关炎症因子TNF-α，IL-6的表达被下调，从而引起细菌在巨噬细胞中的存活减弱。用PPE36过表达的菌株感染小鼠后，分枝杆菌在小鼠体内的存活能力下降，且与对照组相比，小鼠体重下降程度及脏器的损伤均有所减缓，显示PPE36的过表达可能减弱了分枝杆菌的毒力。综上，本研究发现PE/PPE家族的成员PPE10，PPE60，PPE36可通过不同方式进行细菌-宿主互作，调控巨噬细胞对分枝杆菌感染后的先天免疫应答，改变分枝杆菌在宿主细胞内的命运。