Human granulocytic anaplasmosis (HGA) is a potentially fatal tick-borne infection. The steep rise in the number of reported cases in recent years, evidence that the true number is grossly underreported, and its potential for severe outcome make HGA an emerging disease and serious health concern. The etiologic agent is Anaplasma phagocytophilum (Ap), an obligate intracellular bacterium that invades neutrophils and endothelial cells. Like all obligate intracellular bacteria, Ap must enter host cells to survive. Identifying the bacterial adhesins and host cell receptors that mediate this essential process is fundamental to understanding Ap pathogenesis and for preventing infection. We have made great progress on this front during the previous funding period. We identified three Ap adhesins called OmpA, Asp14, and AipA that are sufficient and necessary for optimal invasion of myeloid and endothelial cells. Ap upregulates each of these at two critical stages of the infection cycle that make them ideal targets for neutralizing antibodies: when it converts to the infectious form that invades host cells and during the tick bloodmeal that transmits Ap into mammals. The three adhesins are highly conserved among Ap strains. We delineated the adhesins’ essential binding domains as OmpA residues 59 to 74 (OmpA59- 74), Asp14 residues 113 to 124 (Asp14113-124), and AipA residues 9 to 21 (AipA9-21). An antibody cocktail targeting only these three binding domains blocks Ap infection of host cells in vitro. In terms of comprehensively dissecting each adhesin’s role in invasion, we achieved this first for OmpA by identifying its receptor and delineating the two OmpA amino acids that mediate the interaction. We are now focused on Asp14 and AipA. We identified the Asp14 receptor as a host cell surface localized enzyme and confirmed that this interaction requires Asp14113-124. While the Asp14 receptor contributes to Ap adhesion, its cell surface enzymatic activity is important for invasion. The mechanism by which it does so is undefined. We also identified two promising AipA receptor candidates. For our competitive renewal, we will build on these exciting data. In Aim 1, we will dissect the Asp14-receptor interaction and its role in Ap infection in vitro and in vivo. In Aim 2, we will pinpoint the AipA receptor and define its relevance to infection. In Aim 3, we will establish the importance of OmpA, Asp14, and AipA for Ap infectivity in vivo using the mouse model of granulocytic anaplasmosis. Specifically, we will determine if immunizing against the adhesins’ binding domains protects against syringe- and tick-transmitted Ap challenge. Doing so will yield a comprehensive understanding of Ap infection and establish a sound rationale for developing approaches for preventing HGA.