Characterization of Anaplasma phagocytophilum adhesins

嗜吞噬细胞粘附素无形体的表征

• 批准号: 7846689
• 负责人: Jason A Carlyon
• 金额: $ 1.23万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-05 至 2009-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7846689
• 关键词: Adherence; Adhesions; Affinity; Amino Acid Sequence; Anaplasma phagocytophilum; Anaplasmataceae; Bacteria; Bacterial Adhesins; Bacterial Adhesion; Binding; Biological Assay; Bovine Anaplasmosis; Cell Adhesion; Cell Communication; Cell surface; Cells; Complex; Cytolysis; Dependency; Development; Disease; Electrospray Ionization; Epitopes; Event; Family member; Fostering; Fucose; Genome; Glycoconjugates; Goals; HL-60 Cells; Human; Immune Sera; Individual; Inflammatory; Knowledge; Lead; Ligands; Light; Link; Mammals; Membrane Proteins; Microspheres; Modeling; N-terminal; Organism; P-selectin ligand protein; Paper; Pathogenesis; Peptides; Polysaccharides; Proteome; Recombinant Proteins; Recombinants; Reliance; Research; Selectins; Sialic Acids; Surface; Testing; Tick-Borne Infections; Ticks; Transcript; Tropism; United States; Variant; Work; base; glycosylation; human granulocytic ehrlichiosis; inhibitor/antagonist; liquid chromatography mass spectroscopy; member; neutrophil; novel; pathogen; prevent; sialyl Lewis x

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.

描述（由申请人提供）： 人粒细胞无形体病（HGA；以前称为人粒细胞埃利希体病）是一种新出现的潜在致命疾病，也是美国第二常见的蜱传感染。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趋向性。 A. 吞噬细胞粘附到中性粒细胞表面涉及对 P-选择素糖蛋白配体-1 (PSGL-1) 和唾液酸路易斯 x (sLex) 的识别，唾液酸路易斯 x (sLex) 是一种修饰 PSGL-1 和其他选择素配体 N 末端的四糖。具体来说，这种识别需要与 PSGL-1 N 端肽和 sLex 的 α 2,3-连接唾液酸和 α 1,3-连接岩藻糖相互作用。我们假设嗜吞噬细胞放线菌使用多种粘附素来协同结合这些决定簇。对嗜吞噬细胞A. 和其他无形体科成员的研究表明，嗜吞噬细胞A. 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，以实现正常功能。该提案的目的是识别和表征识别 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识别候选粘附素； (2) 将候选粘附素鉴定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。我们选择了嗜吞噬细胞粘附素变体，其粘附不依赖于 PSGL-1 和唾液酸，但仍然依赖于岩藻糖。该变体将简化岩藻糖特异性粘附素的鉴定，并通过避免野生型嗜吞噬细胞曲霉对协同结合的依赖来帮助粘附素狩猎测定。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，这些研究可能会导致开发出抑制与炎症性疾病相关的细胞粘附事件的新疗法。