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* Inflammation Program, Department of Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, and Veterans Administration Medical Center, Iowa City, IA 52240;
Medical Scientist Training Program, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52240; and
Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52240
Successful host defense against bacteria such as Staphylococcus aureus (SA) depends on a prompt response by circulating polymorphonuclear leukocytes (PMN). Stimulated PMN create in their phagosomes an environment inhospitable to most ingested bacteria. Granules that fuse with the phagosome deliver an array of catalytic and noncatalytic antimicrobial peptides, while activation of the NADPH oxidase at the phagosomal membrane generates reactive oxygen species within the phagosome, including hypochlorous acid (HOCl), formed by the oxidation of chloride by the granule protein myeloperoxidase in the presence of H2O2. In this study, we used SA-expressing cytosolic GFP to provide a novel probe of the fate of SA in human PMN. PMN bleaching of GFP in SA required phagocytosis, active myeloperoxidase, H2O2 from the NADPH oxidase, and chloride. Not all ingested SA were bleached, and the number of cocci within PMN-retaining fluorescent GFP closely correlated with the number of viable bacteria remaining intracellularly. The percent of intracellular fluorescent and viable SA increased at higher multiplicity of infection and when SA presented to PMN had been harvested from the stationary phase of growth. These studies demonstrate that the loss of GFP fluorescence in ingested SA provides a sensitive experimental probe for monitoring biochemical events within individual phagosomes and for identifying subpopulations of SA that resist intracellular PMN cytotoxicity. Defining the molecular basis of SA survival within PMN should provide important insights into bacterial and host properties that limit PMN antistaphylococcal action and thus contribute to the pathogenesis of staphylococcal infection.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 The work was supported by National Institutes of Health Grants AI 18571 (to J.P.W.) and AI 070958 (to W.M.N.), by a Merit Review Grant (to W.M.N.) from the Veterans Administration, and by facilities and resources at the Veterans Administration in Iowa City, IA (to W.M.N.).
2 Address correspondence and reprint requests to Dr. William M. Nauseef, Inflammation Program and Department of Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, D160 MTF, 2501 Crosspark Road, Coralville, IA 52241. E-mail address: william-nauseef{at}uiowa.edu
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; HOCl, hypochlorous acid; MPO, myeloperoxidase; SA, Staphylococcus aureus; CFU, colony forming unit; DPBS, Dulbecco’s PBS; TSB, tryptic soy broth; DPI, diphenylene iodonium; MPO-D, MPO-deficient patient; GGO, glucose-glucose oxidase; MOI, multiplicity of infection.
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