Abstract
ABSTRACTThe obligatory intracellular pathogen Ehrlichia chaffeensis lacks most genes that confer resistance to oxidative stress but can block reactive oxygen species (ROS) generation by host monocytes-macrophages. Bacterial and host molecules responsible for this inhibition have not been identified. To infect host cells, Ehrlichia uses the C terminus of its surface invasin, entry-triggering protein of Ehrlichia (EtpE; EtpE-C), which directly binds the mammalian cell surface receptor glycosylphosphatidylinositol-anchored protein DNase X. We investigated whether EtpE-C binding to DNase X blocks ROS production by mouse bone marrow-derived macrophages (BMDMs). On the basis of a luminol-dependent chemiluminescence assay, E. chaffeensis inhibited phorbol myristate acetate (PMA)-induced ROS generation by BMDMs from wild-type, but not DNase X−/−, mice. EtpE-C is critical for inhibition, as recombinant EtpE-C (rEtpE-C)-coated latex beads, but not recombinant N-terminal EtpE-coated or uncoated beads, inhibited PMA-induced ROS generation by BMDMs from wild-type mice. DNase X is required for this inhibition, as none of these beads inhibited PMA-induced ROS generation by BMDMs from DNase X−/− mice. Previous studies showed that E. chaffeensis does not block ROS generation in neutrophils, a cell type that is a potent ROS generator but is not infected by E. chaffeensis. Human and mouse peripheral blood neutrophils did not express DNase X. Our findings point to a unique survival mechanism of ROS-sensitive obligate intramonocytic bacteria that involves invasin EtpE binding to DNase X on the host cell surface. This is the first report of bacterial invasin having such a subversive activity on ROS generation.
Highlights
IMPORTANCE Ehrlichia chaffeensis preferentially infects monocytes-macrophages and causes a life-threatening emerging tick-transmitted infectious disease called human monocytic ehrlichiosis
Because E. chaffeensis enters host cells via DNase X to successfully establish infection [20], we first investigated whether the DNase X-mediated entry pathway prevents reactive oxygen species (ROS) generation by using a luminol-dependent chemiluminescence (LDCL) assay
We measured levels of ROS generated in response to phorbol myristate acetate (PMA) by wild-type (WT) and DNase XϪ/Ϫ bone marrow-derived macrophages (BMDMs) preincubated with isolated E. chaffeensis for 30 min
Summary
IMPORTANCE Ehrlichia chaffeensis preferentially infects monocytes-macrophages and causes a life-threatening emerging tick-transmitted infectious disease called human monocytic ehrlichiosis. Ehrlichial infection, and the disease, depends on the ability of this bacterium to avoid or overcome powerful microbicidal mechanisms of host monocytes-macrophages, one of which is the generation of ROS. Our findings reveal that an ehrlichial surface invasin, EtpE, triggers bacterial entry and blocks ROS generation by host macrophages through its host cell receptor, DNase X. Generation of reactive oxygen species (ROS) by the phagocyte NADPH oxidase (NOX2) complex upon pathogen encounter is a classic oxygen-dependent antimicrobial defense mechanism of phagocytes [4]. When phagocytes are in a resting state, the NADPH oxidase remains inactive by keeping its components dissociated Stimulating agents such as phorbol myristate acetate (PMA), invading pathogens, or latex beads [15] can induce the rapid assembly of all components of the NOX2 complex into a holoenzyme to catalyze the production of O2Ϫ from oxygen. Results from the present study revealed that, this route of entry actively impeded NOX2 complex activation on host cells in response to PMA to ensure successful E. chaffeensis colonization of macrophages
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