TYPE IV SECRETION & SIGNAL TRANSDUCTION IN EHRLICHIOSIS

描述（由申请方提供）：嗜吞噬细胞无形体和查菲埃立克体分别引起新出现的潜在致死性传染病人粒细胞无形体病（HGA）和人单核细胞埃立克体病（HME）。这些病原体是挑剔的专性细胞内细菌，感染人类白细胞，蜱是传播的生物载体。这些细菌如何进入并继续在敌对的宿主环境中茁壮成长，如中性粒细胞，巨噬细胞和蜱细胞在很大程度上是未知的。我们的假设是，细菌IV型分泌（T4 S）系统和双组分系统（TCS）在这一过程中发挥了重要作用。本项目的具体目标如下：1.使用酵母双杂交系统、免疫共沉淀、双重免疫荧光标记和各种药理学信号抑制剂和诱导剂，并通过对野生型T4 S效应子或经适当修饰（截短、氨基酸取代等）的突变型效应子转染的宿主细胞进行表型分析，表征T4 S效应子分子、相互作用的宿主蛋白和下游事件。2.通过分析CckA和CtrA在E. chaffeensis和A.在人白细胞和蜱细胞中的嗜吞噬细胞的CtrA调节，并证明了CtrA调控基因与上游CtrA共有结合位点。3.通过亲和纯化与活化的重组NtrX结合的基因组DNA片段，对DNA片段进行测序，并研究下游事件，鉴定受NtrX调控的基因。4.对E. chaffeensis和A.通过分析PleC和PleD在人白细胞和蜱细胞中的时间表达，验证PleD双胍基环化酶活性和鉴定c-di-GMP结合蛋白靶点及其功能，鉴定嗜吞噬细胞菌。从这项研究中获得的数据将继续提供一个突破，以新的认识专性细胞内细菌和它们的宿主之间的动态信号事件。这些结果可能为HME和HGA的治疗和预防提供了一个潜在的靶点。公共卫生相关性：人粒细胞无形体病和人单核细胞埃立克体病是新出现的蜱媒传染病。拟议的研究项目将调查无形体和埃立克体病原体如何感知人类粒细胞，单核细胞和蜱细胞环境，并破坏其抗菌防御。这些知识不仅有助于开发新的有效的化学疗法，化学预防和疫苗策略，这些疾病，但也可能揭示在专性细胞内寄生虫和媒介传播的人畜共患病病原体的共同主题。

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. PUBLIC HEALTH RELEVANCE: Human granulocytic anaplasmosis and human monocytic ehrlichiosis are emerging tick- borne infectious diseases. The proposed research project will investigate how anaplasma and ehrlichia pathogens sense human granulocyte, monocyte, and tick cell environment and subvert their antimicrobial defense. The knowledge may not only contribute to developing new efficacious chemotherapy, chemoprevention, and vaccine strategies for these diseases, but also may uncover common themes in obligate intracellular parasitism and vector-borne zoonotic pathogens.