嗜吞噬细胞无形体四型分泌系统效应分子的系统性筛选、鉴定及功能研究

Human granulocytic anaplasmosis, an emerging tick-borne, fatal infectious disease in the United States, European, China, and other countries is caused by Anaplasma phagocytophilum,a gram-negative obligate intracellular bacterium, phylogenetically closely related to Rickettsia. In infected humans, this pathogen primarily replicates in neutrophils, the first line of host defense, and resides in a membrane-bound compartment in the cytoplasm, avoiding the fusion with host cell digestive organelle, lysosome. A. phagocytophilum has functional type IV secretion system (T4SS), as demonstrated by the presence of genes encoding the T4SS apparatus in the genome, and the translocation of AnkA and Ats-1 effectors into host cells. The T4SS plays an important role in bacterial pathogenesis by translocating effectors into host cells to disrupt host cell physiological processes. Although great progresses were made by us and our colleagues in identification and characterization of Ats-1 and AnkA, the only effectors known for A. phagocytophilum today, the current findings cannot elucidate the mechanism for all the disregulated cell functions in A. phagocytophilum-infected cells. There must be other effectors. It was reported other intracellular pathogens, such as Legionella pneumophila, secretes hundreds of T4SS effectors into host cells. Unfortunately, due to the hurdle in genetic manipulation in A. phagocytophilum, it is very difficult to identify T4SS effectors. Only two effectors were experimentally confirmed. In this proposed research project, we try to develop new strategies to identify at large scale the T4SS effectors in A. phagocytophilum, and characterize their functions. The methods we are going to employ include density-gradient centrifugation, protein identification by mass spectrometry, bioinformatics, effector translocation reporter system, yeast two-hybrid system, co-immunoprecipitation, immunofluorescence labeling, and others. The execution of this project will help better understand the underlying mechanism for A. phagocytophilum pathogenesis, and the derived knowledge will contribute to develop better vaccines, antigens, and drugs to prevent, diagnosize and treat human granulocytic anaplasmosis. We also expect that the strategies developed in this project will be applied to the identification and characterization of T4SS effectors in other Rickettsia members.

人粒细胞无形体病是一种新型蜱传可致命性传染病。现已在世界各地如美国，欧洲，中国流行。该病的病原体为嗜吞噬细胞无形体，它是一种专性活细胞内寄生的立克次体目细菌。无形体主要宿主细胞是中性粒细胞。中性粒细胞作为人体的一类专性防御细胞，本身拥有多种消灭入侵病原体的机制，但无形体成功突破宿主细胞防御机制，并在其细胞质中生长。在这一过程中，无形体的四型分泌系统被认为起着重要的作用。四型分泌系统将效应分子从病原菌体内分泌至宿主细胞中，干扰宿主细胞生理功能，导致疾病的发生。一个病原菌能分泌多达数百个效应分子至宿主细胞中，但在无形体中，目前只有AnkA和Ats-1被证实为四型分泌系统的效应分子。在本项目中，我们采用密度梯度离心、蛋白质质谱鉴定、生物信息学、酵母双杂交等技术方法系统地筛选和鉴定无形体四型分泌系统的效应分子，以此阐明此病原菌的致病机理，为人粒细胞无形体病的预防、诊断和治疗提供新的科学依据。

人粒细胞无形体病是由嗜吞噬细胞无形体感染引起的、经蜱传播的一种新发可致命性传染病。现阶段人粒细胞无形体病已在我国广泛流行，对我国人民群众生命财产有着严重威胁。嗜吞噬细胞无形体具有IV型分泌系统（T4SS）。T4SS是其重要的毒力因子。借助此分泌系统，嗜吞噬细胞无形体将效应蛋白转运至宿主细胞中，干扰宿主细胞正常生理功能，导致疾病的发生发展。此外T4SS的结构蛋白以及其分泌的效应蛋白为强免疫原，可激活机体产生免疫反应。本研究拟通过系统地鉴定嗜吞噬细胞无形体T4SS分泌的效应蛋白，阐明其致病机理，从而为人粒细胞无形体病的预防、诊断和治疗提供新的科学依据和手段。.主要研究内容。本项目主要包括两个方面的研究内容。一是构建TEM-1 β-内酰胺酶为基础的嗜吞噬细胞无形体T4SS效应蛋白易位报告系统鉴定新的效应分子，以阐明此病原体的致病机理。二是鉴定嗜吞噬细胞无形体的免疫优势抗原，建立人粒细胞无形体病血清学诊断方法。.重要结果。使用构建的嗜吞噬细胞无形体T4SS效应蛋白易位报告系统对生物信息学预测的潜在效应蛋白进行了鉴定，发现嗜吞噬细胞无形体APH0215蛋白可以被此易位报告系统转运至中国仓鼠卵母细胞CHO K1受体细胞中，表明APH0215蛋白是嗜吞噬细胞无形体T4SS效应蛋白。荧光共标记实验表明APH0215定位于宿主细胞的反面高尔基网状结构。使用人粒细胞无形体病阳性血清对14个嗜吞噬细胞无形体T4SS潜在效应蛋白的抗原性进行了鉴定，发现包括APH1384在内的九个蛋白为免疫优势抗原。.科学意义。（1）在本领域首次构建了TEM-1 β-内酰胺酶为基础的T4SS效应蛋白易位报告。（2）首次鉴定出APH0215是嗜吞噬细胞无形体T4SS效应蛋白。（3）APH0215可能在反面高尔基网状结构至嗜吞噬细胞无形体包涵体的囊泡运输过程中起作用。（4）APH1384等优势抗原的鉴定为建立人粒细胞无形体病特异性血清学诊断方法奠定了基础。