沙门氏菌效应蛋白协作调控TRAF6泛素化介导细胞异噬时序调控的感染机制研究

Xenophagy is primarily regarded as a specialized autophagy system for the clearance of invading pathogens, which belongs to a kind of defensive responses within host cells. Recently, xenophagy is found to facilitate intracellular replication of Salmonella inside host cells. Untill now, the exact role of xenophagy during Salmonella infection is really unclear. In fact, to our knowledge, after the invasion of the eukaryotic cells, intracellular Salmonella are consisted with two kinds of populations with different replication rates and subcellular locations, named cytosolic and intra-vacuolar populations, respectively. Here, in order to elucidate the differential functions of xenophagy in replication of cytosolic and intra-vacuolar Salmonella, we plan to isolate and draw growth curve of these two kinds of intracellular Salmonella by differential centrifugation combined with ELISA antibody binding technology. In great possibility, this will resolve argues about previous controversial opinions about the function of xenophagy during Salmonella infection. Based on our previous results, SopB/SopE2, effectors secreted by SPI-1 (Salmonella Pathogenicity Island, SPI) T3SS (the Type III Secretion System, T3SS), abundantly activated the ubiquitination of TRAF6. Whereas, SseL, effector secreted by SPI-2 T3SS, promoted the deubiquitination of TRAF6. Therefore, firstly, we will investigate the dynamic changes of TRAF6 ubiquitination by SPI-1 and SPI-2 T3SS effectors and its subsequent regulations to xenophagy during S. Typhimurium infeciton. Next, We will uncover the role of this TRAF6-regualted xenophagy in differential replication of cytosolic and intra-vacuolar Salmonella populations, demonstrating a novel strategy of Salmonella facilitating persistent intracellular bacterial replication by temporal regulation of xenophagy by co-operation of SPI-1 and SPI-2 T3SS effectors.

异噬最初被认为属于宿主细胞免疫防御反应的一部分，清除胞内沙门氏菌。随着研究的深入，异噬被发现能够促进胞内菌体的繁殖。异噬在沙门氏菌感染过程中的生物学功能仍不清楚。本项目利用差速离心结合ELISA抗体吸附技术将SCV包被沙门氏菌以及胞浆游离沙门氏菌分离并计数，探索异噬分别对两种类型胞内菌繁殖的影响。基于已有工作基础，沙门氏菌SPI-1 T3SS效应蛋白SopB/SopE2激活TRAF6泛素化而SPI-2 T3SS效应蛋白SseL促进TRAF6去泛素化，本项目拟重点研究（一）感染进程中受SPI-1和SPI-2效应蛋白调节的TRAF6泛素化动态变化及其对细胞异噬的调控机制；（二）受TRAF6泛素化调节的细胞异噬分别对胞内SCV包被沙门氏菌和胞浆游离沙门氏菌的生物学功能。本项目旨在揭示沙门氏菌效应蛋白间通过相互合作调控宿主细胞异噬功能，进而有利于胞内菌体持续繁殖的新机制。

本项目按照计划书内容按时完成预期目标，建立了利用生物素-链霉亲合素酶联免疫吸附（Biotin-Streptavidin-Amplified Enzyme-Linked Immunosorbent Assay, BA-ELISA）结合平板计数法分离胞内沙门氏菌的方法。以人肠上皮细胞Henle-407和鼠成纤维细胞MEF为例，感染不同时间内SCV包被沙门氏菌和胞质游离沙门氏菌增殖完全不同。Henle-407细胞内SCV包被沙门氏菌占比从70%（2h）降至23%（16h），胞质游离沙门氏菌从22%（2h）增至65%（16h）；MEF细胞内SCV包被沙门氏菌随着时间延长占比稳定保持在60%左右，胞质游离沙门氏菌占胞内总沙门氏菌占比20%左右，两者繁殖方式差异大。前期研究发现，沙门氏菌感染诱导宿主细胞 TRAF6 泛素化修饰的同时发生降解，未降解片段 55 kDa，降解后片段43 kDa，该降解与异噬相关。为了研究TRAF6与异噬的关系，利用免疫印迹和实时荧光定量PCR技术发现与野生型细胞相比，在TRAF6缺失细胞（Traf6-/-）中，LC3-II蛋白大量积累，但是LC3蛋白转录水平却显著降低，即LC3-II蛋白在合成来源减小的前提下累积，表明TRAF6缺失导致沙门氏菌感染诱导的细胞异噬受阻，即TRAF6能够在沙门氏菌感染后激活异噬。通过检测异噬衔接蛋白发现，沙门氏菌通过TRAF6激活衔接蛋白NDP52的表达，进而正调控异噬相关蛋白DEPTOR，FOXO3，LAMP1和LC3表达，激活宿主细胞异噬作用，这是一种沙门氏菌感染能够在宿主细胞胞内存活的新机制，为设计抗菌药物提供分子模型，也为宿主定向疗法提供重要靶点和理论依据。.此外，采用透射电镜、Western blot和纳米粒子追踪分析三种方法对沙门氏菌感染宿主细胞后产生的外泌体进行鉴定和表征，发现外泌体中富含409种沙门氏菌蛋白，其中包括30多种效应蛋白。着重验证了沙门氏菌SPI-I 三型分泌系统效应蛋白SopB，发现其可通过外泌体途径被巨噬细胞胞吞，激活巨噬细胞Akt蛋白磷酸化，抑制巨噬细胞凋亡。同时，在沙门氏菌毒力蛋白中发现沙门氏菌转录因子SlyA第89-94位氨基酸之间拥有一段与核定位信号相似度很高的“AKRIKL”序列，该SlyA蛋白能够成功进入细胞核，但是入核后的功能尚不清楚。

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