TYPE IV SECRETION & SIGNAL TRANSDUCTION IN EHRLICHIOSIS

描述(申请人提供)：吞噬细胞无形体和查菲埃利希氏菌分别导致新出现的人类粒细胞无形体病(HGA)和人类单核细胞埃立克体病(HME)，这两种疾病可能是致命的。这些病原体是感染人类白细胞的挑剔的强制性细胞内细菌，而扁虱是传播的生物媒介。这些细菌是如何进入并继续在中性粒细胞、巨噬细胞和扁虱细胞等敌意宿主环境中茁壮成长的，在很大程度上是未知的。我们的假设是细菌IV型分泌系统(T4S)和双组分系统(TCS)在这一过程中发挥着重要作用。本项目的具体目标如下：1.利用酵母双杂交系统、免疫共沉淀、双免疫荧光标记和多种药理信号抑制剂和诱导剂，以及通过适当修饰(截断、氨基酸替代等)的野生型T4S效应子或突变效应子的宿主细胞的表型分析，对T4S效应物分子、相互作用的宿主蛋白及其下游事件进行表征。2.通过分析查菲氏艾美耳球虫和吞噬艾美耳球虫细胞内复制和发育过程中Cck A和CtrA的表达，以及CtrA对CtrA上游共有结合位点基因的调控，来研究CtrA的功能。3.通过亲和纯化与激活的重组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，从而识别NtrX调控的基因。4.通过分析Plec和Ped在人白细胞和扁虱细胞中的时间表达，验证二鸟苷环化酶的活性，确定c-di-GMP结合蛋白的靶点及其功能，从而表征查菲和吞噬艾美耳球虫的Ped功能。从这项研究中获得的数据将继续为新的理解细胞内必需细菌与其宿主之间的动态信号事件提供突破。这一结果可能为HME和HGA的治疗和预防提供一个潜在的靶点。与公共卫生相关：人类粒细胞无形体病和人类单核细胞埃立克体病是新出现的硬体传播传染病。拟议的研究项目将调查无浆体和埃立克病原体如何感知人类粒细胞、单核细胞和扁虱细胞环境，并颠覆它们的抗菌防御。这些知识不仅有助于为这些疾病开发新的有效的化疗、化学预防和疫苗策略，而且还可能揭示专性细胞内寄生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.