Degradation of p22phox and inhibition of superoxide generation by Ehrlichia chaffeensis in human monocytes.
Ehrlichia chaffeensis is an obligate intracellular bacterium which replicates in monocytes or macrophages, the primary producers of reactive oxygen species (ROS). However, effects of ROS on E. chaffeensis infection and whether E. chaffeensis modulates ROS generation in host monocytes are unknown. Here, E. chaffeensis was shown to lose infectivity upon exposure to O(2)(-) or hydrogen peroxide. Upon incubation with human monocytes, E. chaffeensis neither induced O(2)(-) generation by human monocytes, nor colocalized with nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components. Instead, it actively blocked O(2)(-) generation by monocytes stimulated with phorbol myristate acetate and caused the rapid degradation of p22(phox), a component of NADPH oxidase. These effects were not seen in neutrophil, which is another potent ROS generator, but a cell type that E. chaffeensis does not infect. Trypsin pretreatment of monocytes prevented the inhibition of O(2)(-) generation by E. chaffeensis. The degradation of p22(phox) by E. chaffeensis was specific to subsets of monocytes with bound and/or intracellular bacteria, and the degradation could be reduced by heat treatment of the bacterium, lipopolysaccharide pretreatment of monocytes, or the incubation with haemin. The degradation of p22(phox) by E. chaffeensis and its prevention by haemin or protease inhibitors also occurred in isolated monocyte membrane fractions, indicating that host cytoplasmic signalling is not required for these processes. The amount of gp91(phox) was stable under all conditions examined in this study. These findings point to a unique survival mechanism of ROS-sensitive obligate intraleucocytic bacteria that involves the destabilization of p22(phox) following the binding of bacteria to host cell surface proteins.
Lin
Mingqun
M
Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA.
Rikihisa
Yasuko
Y
D015153:Blotting, Western; D002460:Cell Line; D018469:Ehrlichia chaffeensis / Q000254:growth & development*; D005434:Flow Cytometry; D018922:HL-60 Cells; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism; D018613:Microscopy, Confocal; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism* / Q000382:microbiology; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology; D011480:Protease Inhibitors / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011480:Protease Inhibitors; D017382:Reactive Oxygen Species; D013481:Superoxides; D006427:Hemin; D006861:Hydrogen Peroxide; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human; D013755:Tetradecanoylphorbol Acetate