Abstract
Anaplasma phagocytophilum is an obligate intracellular bacterium that invades neutrophils to cause the potentially severe infection, human granulocytic anaplasmosis. The A. phagocytophilum invasin-host cell receptor interactions that facilitate bacterial uptake and hence disease are incompletely defined. Here, we show that EPHNCH_1611, which we refer to as A. phagocytophilum invasion protein B (AipB), mediates bacterial entry but not adhesion. The AipB host cell-interacting domain was narrowed down to amino acids 109 to 129 and is conserved among Anaplasma and Ehrlichia species. AipB(109-123) antisera reduced A. phagocytophilum infection of host cells by 28%. Combining anti-AipB(109-123) with antisera specific for the receptor binding domains of previously identified invasins AipA, Asp14, and OmpA reduced infection by 93%, suggesting a synergistic relationship among these surface proteins. Immunizing mice against AipB(109-123) alone yielded partial protection from A. phagocytophilum challenge that rivaled the protection achieved by immunizing against it together with other invasin binding domains. A yeast two-hybrid screen identified the N-terminal domain of CD18, the β2 integrin β-subunit found on the neutrophil surface, as an AipB binding partner. The interaction was validated by co-immunoprecipitation. siRNA knockdown of CD18 reduced permissiveness to A. phagocytophilum infection. Antibodies targeting the CD18 N-terminus inhibited A. phagocytophilum infection of, but not adherence to, HL-60 cells and human neutrophils. These results demonstrate that the AipB-CD18 interaction is critical for A. phagocytophilum infection in vitro and in vivo, further dissecting the multifaceted mechanism by which A. phagocytophilum invades host cells.IMPORTANCEAnaplasma phagocytophilum causes human granulocytic anaplasmosis, an emerging tick-borne infection for which there is no vaccine and limited treatments. Although A. phagocytophilum absolutely relies on invading host neutrophils to survive and cause disease, the microbe-host cell interactions that predicate infection are inadequately defined. We found that the bacterium uses its outer surface protein AipB to engage CD18 as a critical invasion step. An AipB peptide that is conserved among its homologs in other Anaplasmataceae pathogens is responsible for binding CD18 and can be targeted by antibody to inhibit infection in vitro and in vivo. However, blocking of A. phagocytophilum infection is most effectively achieved by targeting this AipB peptide together with the functional domains of other invasins. AipB is the first rickettsial invasin identified to interact with a β2 integrin, is key for pathogenesis, and could be targeted to protect against diseases caused by A. phagocytophilum and other Anaplasmataceae bacteria.