Anaplasma regulation of host granulocyte function

 描述（由申请方提供）：人粒细胞无形体病由蜱传播的嗜吞噬细胞无形体引起，这是一种专性细胞内细菌，是嗜中性粒细胞微生物颠覆的范例，因为它直接重编程宿主细胞以改善细菌适应性，并在此过程中引起严重疾病。在过去的14年里，我们一直专注于Ephil-A。我们发现了AnkA，核调节蛋白效应蛋白，沉默宿主CYBB启动子，允许细胞内存活，CYBB和其他基因的表观遗传变化，并定义了AnkA通过组蛋白去乙酰化酶-1（HDAC 1）募集结合和调节启动子活性的分子机制。事实上，感染和AnkA介导全基因组表观遗传变化，包括DNA甲基化和组蛋白脱乙酰化，这可能掩盖了协调的转录重编程。原核生物对宿主表观基因组的如此广泛的协调是前所未有的，并且可以解释小基因组原核生物如何颠覆大基因组真核生物以获得寄生或共生生命。这些重编程事件的基础是这种竞争性更新应用的焦点，其3个具体目标是：1）HDAC-1募集到A.将用废除宿主转录并减少病原体存活的AnkA突变来研究嗜吞噬细胞菌AnkA; 2）AnkA在整个基因组的核基质附着区（MAR）结合富含AT的基因座。我们假设AnkA通过将特定染色体区域中的功能相关基因以大的基因组距离聚集在一起以协调重编程来组织三维染色质结构。在这里，我们将表明，AnkA改变了高阶粒细胞染色质的组织，影响DNA的可及性基因调控组件和协调全球转录。具有缺陷或减少的HDAC-1募集的AnkA突变体将用于辨别废除的转录和粒细胞功能;嗜吞噬细胞菌适应性和致病性与其改变细胞中影响组织和整个生物体（如在受感染的人中）的基因程序的能力有关。证明AnkA的体内功能及其与微生物适应性和致病性的关系在人类中并不容易。因此，我们将证明，顺式（目标1）和反转录调节介导的AnkA也发生在体内，并通过验证在体外或离体在人类细胞培养物中获得的结果，并通过测量如何在转录程序的变化影响病原体的繁殖和疾病的小鼠模型中的感染细胞的功能和疾病的表现。这些综合的目标将扩大原核生物的代谢与他们的主机共享的理解，并组织顺式和反式表观遗传变化的主机功能的全球重编程。这些结果将为其他微生物的研究提供新的方向，疾病干预的新方法，或利用这种和其他生物的进化适应性来创建在疾病和健康中操纵真核宿主的定制方法。

 DESCRIPTION (provided by applicant): Human granulocytic anaplasmosis is caused by a tick-transmitted Anaplasma phagocytophilum, an obligate intracellular bacterium that is the paradigm for microbial subversion of neutrophils because it directly reprograms host cells for improved bacterial fitness, and in doing so, causes significant disease. Over 14 years we focused on neutrophil-A. phagocytophilum interactions and altered functions; we discovered AnkA, the nucleomodulin effector protein that silences the host CYBB promoter allowing intracellular survival, epigenetic changes at CYBB and other genes, and defined molecular mechanisms by which AnkA binds and modulates promoter activity via histone deacetylase-1 (HDAC1) recruitment. In fact, infection and AnkA mediate genome- wide epigenetic changes, including DNA methylation and histone deacetylation that likely belie the coordinated transcriptional reprogramming. Such extensive coordination of the host epigenome by a prokaryote is unprecedented and could explain how small genome prokaryotes subvert large genome eukaryotes for parasitic or symbiotic life. The basis of these reprogramming events is the focus of this competing renewal application in 3 specific aims: 1) the molecular basis of HDAC-1 recruitment to A. phagocytophilum AnkA will be studied with AnkA mutations that abrogate host transcription and reduce pathogen survival; 2) AnkA binds AT-rich loci throughout the genome at nuclear matrix attachment regions (MARs). We hypothesize that AnkA organizes 3 dimensional chromatin structure by bringing together function-related genes in specific chromosomal territories at large genomic distances to coordinate reprogramming. Here we will show that AnkA alters higher order granulocyte chromatin organization to influence accessibility of DNA to gene regulatory components and coordinate global transcription. AnkA mutants with defective or reduced HDAC-1 recruitment will be used to discern abrogated transcription and granulocyte functions; 3) the basis for A. phagocytophilum fitness and pathogenicity relates to its ability to alter gene programs in cells impacting tissues and whole organisms, as in infected humans. Proving the in vivo functions of AnkA and its relationship to microbial fitness and pathogenicity is not readily possible in humans. Therefore, we will demonstrate that cis- (Aim 1) and trans-transcriptional regulation mediated by AnkA also occurs in vivo and impacts infected cell function and disease manifestations by validating the results obtained in vitro or ex vivo in human cell cultures, and by measuring how changes in transcriptional programs impact pathogen propagation and disease in a mouse model. These integrated aims will extend the understanding of prokaryotes whose metabolism is shared with their host and organize cis and trans epigenetic changes for global reprogramming of host functions. The results will provide new directions for investigation in other microbes, novel approaches for disease intervention, or leverage the evolutionary adaptations of this and other organisms to create custom approaches for manipulation of eukaryotic hosts in disease and health.