The roles of Anaplasma phagocytophilum surface proteins in cellular invasion

描述(由申请人提供)：吞噬细胞无浆菌是一种专性细胞内细菌和立克次体病原体，它入侵中性粒细胞和内皮细胞，导致新出现的潜在致命感染--人类粒细胞无形体病(HGA)。吞噬细胞将其宿主中性粒细胞转化为特洛伊木马，从而促进病原体的复制和传播。这种细菌下调中性粒细胞的抗微生物反应，增加了对机会性感染的敏感性。阻断吞噬艾美耳球虫感染 中性粒细胞的减少将防止与病原体传播和增加机会性感染风险相关的HGA阶段。吞噬艾美耳球虫可感染心脏和肝脏的微血管内皮细胞。内皮细胞也被认为是吞噬细胞性假丝酵母菌感染的初始细胞类型。受感染的内皮细胞能够将细菌转移到中性粒细胞。消除吞噬细胞对内皮细胞的侵袭有可能防止主要器官的感染和最初感染的建立。该项目的总体目标是对促进感染的吞噬细胞菌外膜蛋白进行功能鉴定。我们确定外膜蛋白A(OmpA)和Asp14(14 kDa AP表面蛋白)是吞噬阿糖胞菌的前两个蛋白，它们对髓系细胞和内皮细胞的侵袭至关重要。靶向OmpA和Asp14几乎消除了宿主细胞的吞噬弧菌感染。在目标1中，我们将确定介导吞噬杆菌摄取的OmpA和Asp14结构域，并确定这两种蛋白是否足以入侵。在目标2中，我们将鉴定OmpA和Asp14的宿主细胞受体。在目标3中，我们将使用新型转基因吞噬细胞生物直接评估OmpA和Asp14与感染性的相关性，这些转基因生物可以被诱导表达针对OmpA或Asp14的反义RNA，从而下调OmpA或Asp14的表达。这项研究将提供对吞噬细胞菌细胞入侵的强有力的理解，并识别任何无浆体科病原体的前两个侵袭素受体对。由于OmpA和Asp14在几种立克次体病原体中保守，我们的工作将为针对导致HGA和其他衰弱疾病的细菌亚群的疫苗开发提供方向。最后，我们的工作将为评估专性细胞内细菌的基因功能提供一个有价值的工具。

DESCRIPTION (provided by applicant): Anaplasma phagocytophilum is an obligate intracellular bacterium and rickettsial pathogen that invades neutrophils and endothelial cells to cause the emerging and potentially fatal infection, human granulocytic anaplasmosis (HGA). A. phagocytophilum converts its host neutrophil into a Trojan horse that facilitates pathogen replication and dissemination. The bacterium down-regulates the neutrophil antimicrobial response, raising susceptibility to opportunistic infections. Blocking A. phagocytophilum infection of neutrophils would prevent the stage of HGA associated with pathogen dissemination and increased risk of opportunistic infections. A. phagocytophilum infects microvascular endothelial cells of heart and liver. Endothelial cells are also implicated as the initial cell type that A. phagocytophilum infects following inoculation via tick feeding. Infected endothelial cells are capable of transferring the bacterium to neutrophils. Abrogating A. phagocytophilum invasion of endothelial cells would potentially prevent infection of major organs and initial establishment of infection. The overall goal of this project is to functionally characterize A. phagocytophilum oute membrane proteins that facilitate infection. We identified outer membrane protein A (OmpA) and Asp14 (14-kDa Ap surface protein) as the first two A. phagocytophilum proteins that are critical for invasion of myeloid and endothelial cells. Targeting OmpA and Asp14 nearly abolishes A. phagocytophilum infection of host cells. In Aim 1, we will identify the OmpA and Asp14 domains that mediate A. phagocytophilum uptake and determine if either protein is sufficient for invasion. In Aim 2, we will identify the host cell receptors of OmpA and Asp14. In Aim 3, we will directly assess the relevance of OmpA and Asp14 to infectivity using novel transgenic A. phagocytophilum organisms that can be induced to express antisense RNA against ompA or asp14 to knock down OmpA or Asp14 expression. This research will provide a robust understanding of A. phagocytophilum cellular invasion and identify the first two invasin-receptor pairs for any Anaplasmataceae pathogen. As OmpA and Asp14 are conserved among several rickettsial pathogens, our work will provide direction for vaccine development against a subset of bacteria that causes HGA and other debilitating diseases. Lastly, our work will yield a valuable tool for assessing gene function in obligate intracellular bacteria.