TYPE IV SECRETION & SIGNAL TRANSDUCTION IN EHRLICHIOSIS

描述（由申请人提供）：嗜吞噬细胞无形体和沙菲埃立体分别引起新出现的潜在致命传染病人粒细胞无形体病（HGA）和人单核细胞埃立体病（HME）。这些病原体是挑剔的强制性细胞内细菌，感染人类白细胞，蜱是传播的生物媒介。这些细菌如何进入并继续在中性粒细胞、巨噬细胞和蜱虫细胞等敌对宿主环境中茁壮成长，在很大程度上是未知的。我们推测细菌IV型分泌系统（T4S）和双组分系统（TCS）在这一过程中起重要作用。本项目的具体目的如下：1。利用酵母双杂交系统、共免疫沉淀、双免疫荧光标记和多种药理信号抑制剂和诱导剂，对转染野生型pet4s效应物或突变型效应物进行适当修饰（截断、氨基酸替代等）的宿主细胞进行表型分析，表征T4S效应分子、相互作用宿主蛋白和下游事件。2. 通过分析chaffeensis和A. phagocytophilum在人白细胞和蜱虫细胞内复制和发育过程中CckA和CtrA的时间表达来表征CtrA的功能，并证明CtrA对上游CtrA共识结合位点基因的调控。3. 通过与活化的重组NtrX结合的基因组DNA片段亲和纯化，鉴定受NtrX调控的基因，测序DNA片段，并研究下游事件。4. 通过在人白细胞和蜱虫细胞中分析PleC和PleD的时间表达，验证PleD二胍环化酶活性，鉴定c-二gmp结合蛋白靶点及其功能，表征沙非沙蚤和嗜吞噬细胞沙非沙蚤的PleD功能。从本研究中获得的数据将继续为对细胞内细菌及其宿主之间的动态信号事件的新理解提供突破。结果可能指向治疗和预防HME和HGA的潜在靶点。

DESCRIPTION (provided by applicant): Anaplasma phagocytophilum and Ehrlichia chaffeensis cause emerging potentially fatal infectious diseases human granulocytic anaplasmosis (HGA) and human monocytic ehrlichiosis (HME), respectively. These pathogens are fastidious obligatory intracellular bacteria that infect human leukocytes, and ticks are biological vectors for transmission. How these bacteria enter and continue to thrive within hostile host milieu such as neutrophils, macrophages, and the tick cells is largely unknown. Our hypothesis is bacterial Type IV secretion (T4S) system and two-component system (TCS) play important roles in this process. The specific aims of this project are as follows: 1. Characterize T4S effector molecules, interacting host proteins, and downstream events using yeast two-hybrid system, co-immunoprecipitation, double immunofluorescence labeling, and various pharmacological signal inhibitors and inducers, and by phenotype analysis of host cells transfected with wild-typeT4S effectors or mutant effectors with appropriate modifications (truncation, amino acid substitution, etc.). 2. Characterize CtrA function by analyzing the temporal expression of CckA and CtrA during intracellular replication and development of E. chaffeensis and A. phagocytophilum in human leukocytes and tick cells, and demonstrating CtrA regulation of genes with the upstream CtrA consensus binding site. 3. Identify genes regulated by NtrX by affinity purification of genomic DNA fragments bound to the activated recombinant NtrX, sequencing the DNA fragments, and investigate the downstream events. 4. Characterize PleD function of E. chaffeensis and A. phagocytophilum by analyzing the PleC and PleD temporal expression in human leukocytes and tick cells, verifying PleD diguanyl cyclase activity and identifying c-di-GMP binding protein targets and their functions. The data to be obtained from this study will continue to provide a breakthrough to new understanding of the dynamic signaling events between obligatory intracellular bacteria and their hosts. The results may point to a potential target for treatment and prevention of HME and HGA.