Characterization of Anaplasma phagocytophilum adhesins

描述（由申请方提供）：人粒细胞无形体病（HGA;以前称为人粒细胞埃里希体病）是一种新出现的潜在致死性疾病，是美国第二常见的蜱媒感染。病原体是嗜吞噬细胞无形体，一种专性细胞内细菌，对中性粒细胞和中性粒细胞前体表现出独特的嗜性。A.嗜吞噬细胞菌对嗜中性粒细胞表面的粘附涉及P-选择素糖蛋白配体-1（PSGL-1）和唾液酸刘易斯x（sLex）的识别，唾液酸路易斯x是修饰PSGL-1和其它选择素配体的N-末端的四糖。具体而言，这种识别需要与PSGL-1 N-末端肽和sLex的α 2，3-连接的唾液酸和α 1，3-连接的岩藻糖相互作用。我们假设A.嗜吞噬细胞菌使用多种粘附素协同结合这些决定簇。研究A. phagocytophilum和其他无形体科的成员表明，A.嗜吞噬细胞菌PSGL-1/sLex靶向粘附素在发育后期被诱导，并且可能需要糖基化和多聚化成粘附素复合物以实现适当的功能。本提案的目的是鉴定和表征识别PSGL-1、唾液酸和岩藻糖的单个粘附素。具体目标是：（1）使用基于亲和力的方法鉴定候选粘附素;（2）将粘附素候选物鉴定为上调的或糖基化的外膜蛋白;（3）测试推定的粘附素与PSGL-1/ sLex糖缀合物和细胞表面的结合。我们选择了A。嗜吞噬细胞菌粘附素变体，其粘附是PSGL-1和唾液酸非依赖性的，但仍然是岩藻糖依赖性的。该变体将简化岩藻糖特异性粘附素的鉴定，并通过避免依赖野生型A来辅助粘附素狩猎测定。嗜吞噬细胞的协同结合。实现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，这些研究可能导致开发新的治疗方法，用于抑制与炎症性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.