Chu, J, Song, HH, Zarember, KA, Mills, TA and Gallin, JI. Persistence of the bacterial pathogen Granulibacter bethesdensis in chronic granulomatous disease monocytes and macrophages lacking a functional NADPH oxidase. J Immunol 191: 3297-3307
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.The Journal of Immunology (Impact Factor: 4.92). 08/2013; 191(6). DOI: 10.4049/jimmunol.1300200
Granulibacter bethesdensis is a Gram-negative pathogen in patients with chronic granulomatous disease (CGD), a deficiency in the phagocyte NADPH oxidase. Repeated isolation of genetically identical strains from the same patient over years, and prolonged waxing and waning seropositivity in some subjects, raises the possibility of long-term persistence. G. bethesdensis resists killing by serum, CGD polymorphonuclear leukocytes (PMN), and antimicrobial peptides, indicating resistance to nonoxidative killing mechanisms. Although G. bethesdensis extends the survival of PMN, persistent intracellular bacterial survival might rely on longer-lived macrophages and their precursor monocytes. Therefore, we examined phagocytic killing by primary human monocytes and monocyte-derived macrophages (MDM). Cells from both normal and CGD subjects internalized G. bethesdensis similarly. G. bethesdensis stimulated superoxide production in normal monocytes, but to a lesser degree than in normal PMN. Normal but not CGD monocytes and MDM killed G. bethesdensis and required in vitro treatment with IFN-γ to maintain this killing effect. Although in vitro IFN-γ did not enhance G. bethesdensis killing in CGD monocytes, it restricted growth in proportion to CGD PMN residual superoxide production, providing a potential method to identify patients responsive to IFN-γ therapy. In IFN-γ-treated CGD MDM, G. bethesdensis persisted for the duration of the study (7 d) without decreasing viability of the host cells. These results indicate that G. bethesdensis is highly resistant to oxygen-independent microbicides of myeloid cells, requires an intact NADPH oxidase for clearance, and can persist long-term in CGD mononuclear phagocytes, most likely relating to the persistence of this microorganism in infected CGD patients.
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ABSTRACT: Ample resources have been dedicated to studying bacterial resistance, biofilm formation, and genetic encoding of resistance, metabolism or fitness mutations. However, less is known about bacterial persister cells that display multidrug tolerance, latency or adaptation.GERMS 09/2014; 4(3):73. DOI:10.11599/germs.2014.1059
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ABSTRACT: There are 5 genetic forms of chronic granulomatous disease (CGD), resulting from mutations in any of 5 subunits of phagocyte oxidase, an enzyme complex in neutrophils, monocytes and macrophages that produces microbicidal reactive oxygen species. We generated induced pluripotent stem cells (iPSCs) from peripheral blood CD34(+) hematopoietic stem cells of patients with each of 5 CGD genotypes. We used zinc finger nuclease (ZFN) targeting the AAVS1 safe harbor site together with CGD genotype-specific minigene plasmids with flanking AAVS1 sequence to target correction of iPSC representing each form of CGD. We achieved targeted insertion with constitutive expression of desired oxidase subunit in 70-80% percent of selected iPSC clones. Neutrophils and macrophages differentiated from corrected CGD iPSCs demonstrated restored oxidase activity and antimicrobial function against CGD bacterial pathogens Staphylococcus aureus and Granulibacter bethesdensis. Using a standard platform that combines iPSC generation from peripheral blood CD34(+) cells and ZFN mediated AAVS1 safe harbor minigene targeting, we demonstrate efficient generation of genetically corrected iPSCs using an identical approach for all 5 genetic forms of CGD. This safe harbor minigene targeting platform is broadly applicable to a wide range of inherited single gene metabolic disorders.Molecular Therapy (2014); doi:10.1038/mt.2014.195.Molecular Therapy 10/2014; 23(1). DOI:10.1038/mt.2014.195 · 6.23 Impact Factor
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