Pathobiological roles of Anaplasma phagocytophilum inclusion membrane proteins

描述（由申请人提供）：病原体占据的空泡（pv）是宿主细胞衍生的内体/吞噬体，被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避难所。病原菌衍生蛋白定位于PV膜（PVM）对PV的构建和融合原性的改变至关重要。此外，病原体编码的PVM蛋白作为PV与宿主膜交通和信号通路相互作用的接口。因此，了解细菌PVM蛋白的病理生物学作用是了解孔内病原体生存机制的关键。绝大多数已鉴定/预测的细菌PVM蛋白的功能仍然不明确，这表明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认识存在相当大的差距。嗜吞噬细胞无原体是一种专性空泡细菌，侵入中性粒细胞引起新出现的潜在致命疾病，人粒细胞无原体病（HGA）。它是一种蜱传播的病原体，通过驻留在吞噬细胞占据液泡（APV）中来保护自己免受中性粒细胞的杀伤，该液泡不会沿着内体途径成熟，避免溶酶体融合，并拦截内体和高尔基基衍生的循环交通。这种细菌如何操纵囊泡交通尚不清楚。我们鉴定出了最早的2个细菌编码的APV膜蛋白——APH0032和APH1387。两者都携带大片段串联直接重复序列，真核细胞样的内吞分选基序，并装饰AVM的细胞质表面。两者都携带真核样丝氨酸/苏氨酸磷酸化基序，并在SDS-PAGE上以大于预期大小的多个同工异构体迁移。我们假设APH0032和APH1387通过与宿主膜交通调节因子和/或信号通路相互作用，参与了ApV的选择性融合性和/或嗜吞噬细胞A.的发病机制。本研究的目标是：(1)确定APH0032和APH1387阻断哪些膜交通途径，以及宿主丝氨酸/苏氨酸激酶是否使其磷酸化；(2)鉴定与APH0032和APH1387相互作用的宿主蛋白。这些探索性目标将通过解决细胞内发病机制的范例-病原体来源的PVM蛋白对宿主细胞的操纵产生高回报；进一步了解嗜吞噬芽胞杆菌如何在中性粒细胞中生存；并将为APH0032和APH1387的病理生物学作用的长期研究奠定基础。

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.