Anaplasma phagocytophilumUtilizes Multiple Host Evasion Mechanisms To Thwart NADPH Oxidase-Mediated Killing during Neutrophil Infection

Abstract

Anaplasma phagocytophilum, the etiologic agent of human anaplasmosis, is a bacterial pathogen that specifically colonizes neutrophils. Neutrophils utilize the NADPH oxidase complex to generate superoxide (O₂⁻) and initiate oxidative killing of microorganisms.A. phagocytophilum's unique tropism for neutrophils, however, indicates that it subverts and/or avoids oxidative killing. We therefore examined the effects ofA. phagocytophiluminfection on neutrophil NADPH oxidase assembly and reactive oxygen species (ROS) production. Following neutrophil binding,Anaplasmainvasion requires at least 240 min. During its prolonged association with the neutrophil plasma membrane,A. phagocytophilumstimulates NADPH oxidase assembly, as indicated by increased cytochromeb₅₅₈mobilization to the membrane, as well as colocalization of Rac and p22^phox. This initial stimulation taxes the host neutrophil's finite oxidase reserves, as demonstrated by time- and bacterial-dose-dependent decreases in secondary activation byN-formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA). This stimulation is modest, however, and does not diminish oxidase stores to nearly the extent thatEscherichia coli, serum-opsonized zymosan, FMLP, or PMA do. Despite the apparent activation of NADPH oxidase, no change in ROS-dependent chemiluminescence is observed upon the addition ofA. phagocytophilumto neutrophils, indicating that the bacterium may scavenge exogenous O₂⁻. Indeed,A. phagocytophilumrapidly detoxifies O₂⁻in a cell-free system. Once internalized, the bacterium resides within a protective vacuole that excludes p22^phoxand gp91^phox. Thus,A. phagocytophilumemploys at least two strategies to protect itself from neutrophil NADPH oxidase-mediated killing.