Pathobiological roles of Anaplasma phagocytophilum inclusion membrane proteins

描述（由申请方提供）：病原体占据的空泡（PV）是宿主细胞来源的内体/吞噬体，由专性空泡病原体转化为独特的细胞器，作为与正常内吞连续体隔离的安全港。定位于PV膜（PVM）的病原体衍生蛋白对于PV的构建和改变的融合性是必不可少的。此外，病原体编码的PVM蛋白作为PV与宿主膜交通和信号传导途径相互作用的界面。因此，了解细菌PVM蛋白的病理生物学作用是了解液泡内病原体生存机制的关键。绝大多数鉴定/预测的细菌PVM蛋白的功能仍然不确定，这代表了我们对专性空泡病原体如何操纵其宿主细胞以逃避破坏的知识存在相当大的差距。嗜吞噬细胞无形体是一种专性空泡细菌，侵入中性粒细胞引起的新兴和潜在的致命疾病，人粒细胞无形体病（HGA）。它是一种蜱传播的病原体，通过驻留在A。嗜吞噬细胞占据的空泡（APV），其不沿着内体途径沿着成熟，避免溶酶体融合，并拦截再循环内体和高尔基体衍生的运输。这种细菌如何操纵囊泡交通尚不清楚。我们已经鉴定了前两种细菌编码的APV膜（AVM）蛋白-APH 0032和APH 1387。两者都携带大部分串联直接重复序列，真核细胞样内吞分选基序，并装饰AVM的细胞质面。两者都携带真核生物样的Ser/Thr磷酸化基序，并在SDS-PAGE上以大于预期大小的多种同种型迁移。我们假设APH 0032和APH 1387有助于ApV的选择性融合性和/或A.嗜吞噬细胞的发病机制通过与宿主膜交通调节剂和/或信号传导途径相互作用。本提案的目的是（1）确定APH 0032和APH 1387拦截哪些膜运输途径以及宿主Ser/Thr激酶是否磷酸化它们;（2）鉴定APH 0032和APH 1387相互作用的宿主蛋白。这些探索性的目标将通过解决细胞内发病机制的范例产生高回报-病原体衍生的PVM蛋白操纵宿主细胞;嗜吞噬细胞菌在中性粒细胞内生存;并将为建立APH 0032和APH 1387病理生物学作用的长期研究奠定基础。 公共卫生相关性：人粒细胞无形体病是一种新出现的、使人衰弱的和潜在致命的传染病。我们的研究旨在确定细菌蛋白，使细菌病原体，导致HGA生存在一个特定的白色血细胞。这样做可能会揭示新的治疗或疫苗治疗或预防HGA的目标。

DESCRIPTION (provided by applicant): Pathogen-occupied vacuoles (PVs) are host cell-derived endosomes/phagosomes that are converted by obligate vacuolar pathogens into unique organelles that serve as safe-havens sequestered from the normal endocytic continuum. Pathogen derived proteins that localize to the PV membrane (PVM) are essential for the construction and altered fusogenicity of PVs. Furthermore, pathogen-encoded PVM proteins serve as interfaces for PV interactions with host membrane traffic and signaling pathways. Thus, understanding the pathobiological roles of bacterial PVM proteins is key to understanding the survival mechanisms of intravacuolar pathogens. The functions of the vast majority of identified/predicted bacterial PVM proteins remain undefined, which represents a considerable gap in our knowledge of how obligate vacuolar pathogens manipulate their host cells to escape destruction. Anaplasma phagocytophilum is an obligate vacuolar bacterium that invades neutrophils to cause the emerging and potentially deadly disease, human granulocytic anaplasmosis (HGA). It is a tick-transmitted pathogen that protects itself from neutrophil killing by residing within the A. phagocytophilum-occupied vacuole (APV), which does not mature along the endosomal pathway, avoids lysosomal fusion, and intercepts recycling endosome- and Golgi-derived traffic. How this bacterium manipulates vesicular traffic is unknown. We have identified the 1st 2 bacterial-encoded APV membrane (AVM) proteins - APH0032 and APH1387. Both carry large sections of tandem direct repeats, eukaryotic-like endocytic sorting motifs, and decorate the cytoplasmic face of the AVM. Both carry eukaryotic-like Ser/Thr phosphorylation motifs and migrate as multiple isoforms of larger than expected sizes upon SDS-PAGE. We hypothesize that APH0032 and APH1387 contribute to the ApV's selective fusogenicity and/or A. phagocytophilum pathogenesis by interacting with host membrane traffic regulators and/or signaling pathways. The goals of this proposal are (1) to determine which membrane traffic pathways APH0032 and APH1387 intercept and whether host Ser/Thr kinases phosphorylate them; and (2) to identify the host proteins with which APH0032 and APH1387 interact. These exploratory aims will yield a high payoff by addressing a paradigm of intracellular pathogenesis - host cell manipulation by pathogen-derived PVM proteins; furthering our understanding of how A. phagocytophilum survives within neutrophils; and will establish a foundation for building long-term studies of the pathobiological roles of APH0032 and APH1387. PUBLIC HEALTH RELEVANCE: Human granulocytic anaplasmosis is an emerging, debilitating, and potentially fatal infectious disease. Our research seeks to identify bacterial proteins that enable the bacterial pathogen that causes HGA to survive inside a particular white blood cell. Doing so may reveal novel therapeutic or vaccine targets for treating or preventing HGA.