Characterization of Anaplasma phagocytophilum adhesins

描述（由申请人提供）：人粒细胞无形体病（HGA，原人粒细胞埃利希体病）是一种新出现的潜在致命疾病，是美国第二大最常见的蜱传感染。病原是嗜吞噬细胞无原体，一种专性细胞内细菌，对中性粒细胞和中性粒细胞前体具有独特的嗜性。嗜吞噬细胞与中性粒细胞表面的粘附涉及对p -选择蛋白糖蛋白配体1 （PSGL-1）和sialyl Lewis x （sLex）的识别，后者是一种修饰PSGL-1和其他选择素配体n端的四糖。具体来说，这种识别需要与PSGL-1 n端肽和α 2,3-链唾液酸和α 1,3-链sLex的焦点相互作用。我们假设嗜吞噬细胞芽胞杆菌使用多种粘附素来协同结合这些决定因子。对嗜吞噬细胞芽孢杆菌和其他无浆菌科成员的研究表明，嗜吞噬细胞芽孢杆菌PSGL-1/ slex靶向粘附素在发育后期被诱导，可能需要糖基化和多聚成粘附素复合物才能发挥正常功能。本提案的目的是识别和表征识别PSGL-1、唾液酸和病灶的单个粘附素。具体目标是：(1)使用基于亲和力的方法识别候选粘附素；(2)确定黏附素候选蛋白为上调或糖基化的外膜蛋白；(3)测试假定的粘附素与PSGL-1/ sLex糖缀合物和细胞表面的结合。我们选择了一种嗜吞噬细胞芽胞杆菌黏附素变体，其黏附是PSGL-1和唾液酸无关的，但仍然是聚焦依赖性的。这种变体将简化焦点特异性黏着素的鉴定，并通过避免依赖野生型嗜吞噬细胞芽胞杆菌的合作结合来帮助黏着素的寻找。完成这些目标将揭示细菌宿主细胞相互作用的新主题，并将确定治疗或预防HGA的靶点。此外，这些研究可能会导致新的治疗方法的发展，以抑制与炎症疾病相关的细胞粘附事件。

DESCRIPTION (provided by applicant): Human granulocytic anaplasmosis (HGA; formerly human granulocytic ehrlichiosis) is an emerging and potentially fatal disease and the second most common tick-borne infection in the United States. The etiologic agent is Anaplasma phagocytophilum, an obligate intracellular bacterium that displays a unique tropism for neutrophils and neutrophil precursors. A. phagocytophilum adhesion to neutrophil surfaces involves recognition of P-selectin glycoprotein ligand-1 (PSGL-1) and sialyl Lewis x (sLex), a tetrasaccharide that modifies the N-terminus of PSGL-1 and other selectin ligands. Specifically, this recognition requires interactions with a PSGL-1 N-terminal peptide and alpha 2,3-linked sialic acid and alpha 1,3-linked fucose of sLex. We hypothesize that A. phagocytophilum uses multiple adhesins that cooperatively bind these determinants. Studies of A. phagocytophilum and other Anaplasmataceae family members suggest the A. phagocytophilum PSGL-1/ sLex-targeting adhesins are induced late in development and may require glycosylation and multimerization into adhesin complexes-for proper function. The objective of this proposal is to identify and characterize the individual adhesins that recognize PSGL-1, sialic acid, and fucose. The specific aims are: (1) identify candidate adhesins using affinity-based approaches; (2) identify adhesin candidates as upregulated or glycosylated outer membrane proteins; (3) test binding of putative adhesins to PSGL-1/ sLex glycoconjugates and cell surfaces. We have selected for an A. phagocytophilum adhesin variant, the adhesion of which is PSGL-1- and sialic acid-independent, but remains fucose-dependent. This variant will simplify identification of the fucose-specific adhesin and will aid adhesin hunting assays by circumventing the reliance of wild-type A. phagocytophilum on cooperative binding. Accomplishing these goals will shed light onto novel themes of bacteria-host cell interactions and will identify targets for treating or preventing HGA. Furthermore, these studies may lead to the development of new treatments for inhibiting cellular adhesion events associated with inflammatory disorders.