Pathobiological roles of Anaplasma phagocytophilum inclusion membrane proteins

描述(申请人提供)：病原体占据的空泡(PV)是宿主细胞衍生的内小体/吞噬小体，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避风港。病原菌来源的蛋白定位于PV膜(PVM)，对于PV的构建和改变融合活性是必不可少的。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空泡内病原体生存机制的关键。绝大多数已识别/预测的细菌PVM蛋白的功能仍未确定，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了解存在相当大的差距。吞噬无浆菌是一种专性空泡细菌，它入侵中性粒细胞，导致人类粒细胞无浆症(HGA)这种新出现的、可能致命的疾病。它是一种壁虱传播的病原体，通过驻留在吞噬细胞所占据的空泡(APV)中保护自己免受中性粒细胞的杀伤，APV不沿着内体途径成熟，避免溶酶体融合，并拦截循环的内体和高尔基体衍生的交通。这种细菌如何操纵囊泡运输尚不清楚。我们已经鉴定了前两个细菌编码的APV膜蛋白-APH0032和APH1387。两者都携带大片段的串联直接重复序列，即真核细胞样的内吞分选基序，并装饰着AVM的细胞质表面。两者都携带真核生物样的丝氨酸/苏氨酸磷酸化基序，并在SDS-PAGE上以多种大小超过预期的异构体迁移。我们推测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，在APV的选择性融合和/或吞噬细胞性致病中起作用。该方案的目的是(1)确定APH0032和APH1387截获哪些膜信号转导通路，以及宿主丝氨酸/苏氨酸蛋白激酶是否使它们磷酸化；(2)确定APH0032和APH1387相互作用的宿主蛋白。这些探索性目标将产生高回报，解决细胞内致病的范例-病原体衍生的PVM蛋白对宿主细胞的操纵；进一步了解吞噬细胞性假丝酵母菌如何在中性粒细胞内生存；并将为建立APH0032和APH1387的病理生物学作用的长期研究奠定基础。 公共卫生相关性：人类粒细胞无形体病是一种新出现的、使人衰弱的、可能致命的传染病。我们的研究试图识别能够使导致HGA的细菌病原体在特定的白细胞内生存的细菌蛋白。这样做可能会揭示治疗或预防HGA的新的治疗或疫苗靶点。

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.