Rab5/Rab7在查菲埃立克体抑制吞噬体溶酶体融合中的作用机制

Ehrlichia chaffeensis, an important zoonotic pathogen and obligately intracellular bacterium, causes disease of natural focus. Inhibition of fusion of phagosome with lysosomes is essential for Ehrlichia to survive inside phagocytes, but its mechanism is not clear. We have developed a novel method for obtaining highly purified ECVs (E. chaffeensis containing vacuole) from E.chaffeensis-infected DH82 cells and identified its characteristic by mass spectrometry-based proteomics. Our previous study showed that E. chaffeensis vacuole is acidified and is positive for early endosome marker Rab5 and late endosome marker Rab7, but negative to lysosomal proteins. In this study, based on the evidences that Rab5/Rab7 and their effectors play an essential role in phagosome maturation and lysosomal fusion with phagosome, we will investigate the role of activation of Rab5/Rab7 on the Ehrlichia chaffeensis phagolysosome formation. In order to illustrate the mechanism of E. chaffeensis preventing lysosomal fusion with phagosome, we also want to study on Rab5/Rab7 effectors. This study will provide new insights into the mechanism of persistent infection and find prevention and treatment targets.

查菲埃立克体作为重要的人兽共患自然疫源性疾病病原菌及专性胞内寄生菌，通过抑制吞噬体溶酶体的融合逃避宿主的杀伤作用，但其机制尚不清楚。课题组已建立从感染的巨噬细胞DH82中提取查菲埃立克体吞噬体的方法，并利用比较蛋白组学发现了其吞噬体特征：内环境酸化、具有早期吞噬体标记分子Rab5和晚期吞噬体标记分子Rab7、缺乏溶酶体标记分子。鉴于Rab5/Rab7活化及他们的效应分子在吞噬体发育成熟和吞噬体溶酶体融合过程中发挥重要作用，本研究拟通过真核转染、免疫荧光等技术方法，在DH82细胞模型中观察Rab5/Rab7活化对查菲埃立克体吞噬溶酶体形成的影响；同时以Rab5和Rab7的效应分子为切入点明确查菲埃立克体抑制吞噬溶酶体形成的效应途径，以期从宿主方面明确查菲埃立克体抑制吞噬体溶酶体融合的机制，为揭示其导致宿主持续感染的致病机理、寻找防治靶点提供线索。

本课题严格按照计划书进行，课题研究人员分工按照计划申请书进行，开展过程中无新增和退出。.本课题目前完成了下列工作：利用密度梯度离心法成功分离获得了纯度较高的查菲埃立克体吞噬体；利用比较蛋白组学结合免疫荧光分析明确了查菲埃立克体吞噬体特性，其经过了早期吞噬体阶段，获得了晚期吞噬体标记分子，但不能与溶酶体融合形成吞噬溶酶体。查菲埃立克体感染细胞后未影响宿主细胞正常的吞噬体溶酶体融合机制。此外，查菲埃立克体还可能通过逃避自噬途径，使其在宿主细胞内生存导致宿主持续感染；建立了构建过表达慢病毒载体的技术平台，构建了慢病毒载体GV287- syntaxin13、GV287- Rab7A、GV287-EEA1、GV287-RILP。通过过表达慢病毒载体转染查菲埃立克体感染的宿主细胞，利用免疫荧光技术，发现了查菲埃立克体吞噬体获得的Rab7为非活化形式。综上所述，本阶段实验在利用比较蛋白质组学联合免疫荧光分析明确了查菲埃立克体吞噬体特性的基础上，初步证实了查菲埃立克体吞噬体在成熟过程中获得了Rab7，但其为非活化形式。.部分研究结果以题为《Proteomic analysis of the Ehrlichia chaffeensis phagosome in cultured DH82 cells》已经发表在PLOSONE上。