Department of Molecular Genetics and Microbiology1 and Department of Pharmacy2, University of New Mexico Health Sciences Center, 915 Camino de Salud, Albuquerque, NM 87131, USA
Institute for Medical Microbiology, Medizinische Hochschule, 30625, Hannover, Germany3
Institute of Medical Microbiology, University of Zurich, CH-8028 Zurich, Switzerland4
Author for correspondence: V. Deretic. Tel: +1 505 272 0291. Fax: +1 505 272 6029. e-mail: vderetic{at}salud.unm.edu
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ABSTRACT |
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Keywords: M. tuberculosis, nitric oxide, ahpC, peroxynitrite, latency
Abbreviations: DETA nonoate, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)aminio]diazen-1-ium-1,2-diolate)
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INTRODUCTION |
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Here, we continued our investigations of the role of ahpC in M. tuberculosis biology, specifically with respect to its proposed role in resistance to reactive nitrogen species and survival in macrophages. Using knockout strains of ahpC (ahpC::Kmr) in M. tuberculosis (Springer et al., 2001 ) and M. smegmatis (Dhandayuthapani et al., 1996
), we compared the wild-type strains and their ahpC mutant derivatives for survival upon exposure to compounds producing reactive nitrogen intermediates and during infection of resting and activated macrophages.
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METHODS |
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Media and growth conditions.
The strains were grown until mid-exponential phase and/or stationary phase (as indicated) on 7H9 (Difco) or 7H11 plates, supplemented with 0·5% Tween, 0·2% glycerol and OADC (oleic acid, 10% bovine serum fraction V, glucose and catalase). Bacteria were grown at 37 °C. All manipulations of live M. tuberculosis were carried out under Biosafety Level 3 conditions.
Chemicals.
Both peroxynitrite and DETA nonoate {(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)aminio]diazen-1-ium-1,2-diolate)} were purchased from Alexis Corporation.
Detection of lipid peroxides.
Lipid peroxides were detected using FOX II reagent, which provides a sensitive colorimetric assay for peroxides measured spectrophotometrically at 560 nm. FOX II reagent contains 90% methanol, 25 mM H2SO4, 250 µM ferrous sulfate heptahydrate (Sigma) and 100 µM xylene orange (Sigma) (Jiang et al., 1992 ; Nourooz-Zadeh et al., 1994
; Wolff et al., 1994
). M. smegmatis mc2155 strains ahpC+ (wild-type), ahpC::Kmr and furA::Kmr were grown until mid-exponential phase. These cultures were then exposed to 1 mM peroxynitrite for five 3 min cycles at 37 °C. One-hundred microlitres of the treated culture was incubated for 10 min with 900 µl FOX II reagent to allow the reaction of peroxides. Experiments were carried out in triplicate and results quantified using a standard curve created with hydrogen peroxide
Sensitivity assays and survival in macrophages.
M. tuberculosis H37Rv ahpC+ and ahpC::Kmr were allowed to reach stationary phase. Similarly, M. smegmatis mc2155 strains ahpC+ (wild-type), ahpC::Kmr and furA::Kmr were grown until mid-exponential or stationary phase. These cultures were then exposed to various concentrations of peroxynitrite and DETA nonoate or used to infect J774A macrophages at an m.o.i. of 10:1 in the presence or absence of IFN (500 U ml-1) and LPS (125 ng ml-1). The results of treatment with these compounds and macrophage infections were assessed by plating and c.f.u. determination.
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RESULTS AND DISCUSSION |
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 22 March 2002;
revised 22 June 2002;
accepted 26 July 2002.