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A novel antioxidant gene from Mycobacterium tuberculosis.

Ehrt S, Shiloh MU, Ruan J, Choi M, Gunzburg S, Nathan C, Xie Q, Riley LW - J. Exp. Med. (1997)

Bottom Line: Among the major antimicrobial products of macrophages are reactive intermediates of the oxidation of nitrogen (RNI) and the reduction of oxygen (ROI).Expression of noxR1 conferred upon Escherichia coli and Mycobacterium smegmatis enhanced ability to resist RNI and ROI, whether the bacteria were exposed to exogenous compounds in medium or to endogenous products in macrophages.These studies provide the first identification of an RNI resistance mechanism in mycobacteria, point to a new mechanism for resistance to ROI, and raise the possibility that inhibition of the noxR1 pathway might enhance the ability of macrophages to control tuberculosis.

View Article: PubMed Central - PubMed

Affiliation: Division of International Medicine and Infectious Disease, Department of Medicine, Cornell University Medical College, New York 10021, USA.

ABSTRACT
Among the major antimicrobial products of macrophages are reactive intermediates of the oxidation of nitrogen (RNI) and the reduction of oxygen (ROI). Selection of recombinants in acidified nitrite led to the cloning of a novel gene, noxR1, from a pathogenic clinical isolate of Mycobacterium tuberculosis. Expression of noxR1 conferred upon Escherichia coli and Mycobacterium smegmatis enhanced ability to resist RNI and ROI, whether the bacteria were exposed to exogenous compounds in medium or to endogenous products in macrophages. These studies provide the first identification of an RNI resistance mechanism in mycobacteria, point to a new mechanism for resistance to ROI, and raise the possibility that inhibition of the noxR1 pathway might enhance the ability of macrophages to control tuberculosis.

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Survival advantage conferred by noxR1 on E. coli cultured in  GSNO and/or hydrogen peroxide. (A and B) Time course of survival of  E. coli HB101 transformed with pBS (open squares) or pNO14 (solid  squares) and cultured in LB at pH 5.0 with (A) 2 mM GSNO or (B) 0.5  mM H2O2, as determined by the dye reduction microplate assay. (C–F)  Concentration-response curves for GSNO (C and E) and H2O2 (D and F)  after 6 h incubation at a pH of 5 (C and D) or pH 7 (E and F). (G and H)  Resistance to the combination of GSNO and H2O2. (Black bars) E. coli  HB101 transformed with pBS. (Gray bars) E. coli HB101 transformed with  pNO14. (G) pH 7.0 with the following treatments: I, none; II, H2O2 (0.1  mM); III, GSNO (5 mM); IV, H2O2 (0.05 mM) + GSNO (5 mM). (H)  pH 5.0 with the following treatments: I, none; II, H2O2 (0.1 mM); III,  GSNO (1 mM); IV, H2O2 (0.05 mM) + GSNO (1 mM). Data in panels  A–F are means ± SE for triplicates; some error bars fall within the symbols. Data in panels G and H are means of duplicates.
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Figure 4: Survival advantage conferred by noxR1 on E. coli cultured in GSNO and/or hydrogen peroxide. (A and B) Time course of survival of E. coli HB101 transformed with pBS (open squares) or pNO14 (solid squares) and cultured in LB at pH 5.0 with (A) 2 mM GSNO or (B) 0.5 mM H2O2, as determined by the dye reduction microplate assay. (C–F) Concentration-response curves for GSNO (C and E) and H2O2 (D and F) after 6 h incubation at a pH of 5 (C and D) or pH 7 (E and F). (G and H) Resistance to the combination of GSNO and H2O2. (Black bars) E. coli HB101 transformed with pBS. (Gray bars) E. coli HB101 transformed with pNO14. (G) pH 7.0 with the following treatments: I, none; II, H2O2 (0.1 mM); III, GSNO (5 mM); IV, H2O2 (0.05 mM) + GSNO (5 mM). (H) pH 5.0 with the following treatments: I, none; II, H2O2 (0.1 mM); III, GSNO (1 mM); IV, H2O2 (0.05 mM) + GSNO (1 mM). Data in panels A–F are means ± SE for triplicates; some error bars fall within the symbols. Data in panels G and H are means of duplicates.

Mentions: To more fully explore the phenotype afforded by expression of noxR1, we made use of a fluorescent, dye-reduction microplate assay whose results corresponded almost perfectly (correlation coefficients r2 >0.96) to the results of the more laborious colony-forming agar-plate assay after exposing bacteria to RNI in vitro or to the intraphagosomal milieu of macrophages (29). Not only was E. coli HB101 rendered far more resistant to ASN (2.5 mM NaNO2, pH 5) by expression of noxR1 (data not shown), but the bacteria also better resisted S-nitrosoglutathione (GSNO; 2 mM, pH 5.0), a physiologic and bacteriostatic (37, 43, 44) source of several RNI, including ammonia (45; Fig. 4, A and C). By 6 h, the survival advantage was close to 100-fold. GSNO was more bactericidal at pH 5.0 (Fig. 4, A and C) than at pH 7.0 (Fig. 4 E), but noxR1 conferred protection under both conditions.


A novel antioxidant gene from Mycobacterium tuberculosis.

Ehrt S, Shiloh MU, Ruan J, Choi M, Gunzburg S, Nathan C, Xie Q, Riley LW - J. Exp. Med. (1997)

Survival advantage conferred by noxR1 on E. coli cultured in  GSNO and/or hydrogen peroxide. (A and B) Time course of survival of  E. coli HB101 transformed with pBS (open squares) or pNO14 (solid  squares) and cultured in LB at pH 5.0 with (A) 2 mM GSNO or (B) 0.5  mM H2O2, as determined by the dye reduction microplate assay. (C–F)  Concentration-response curves for GSNO (C and E) and H2O2 (D and F)  after 6 h incubation at a pH of 5 (C and D) or pH 7 (E and F). (G and H)  Resistance to the combination of GSNO and H2O2. (Black bars) E. coli  HB101 transformed with pBS. (Gray bars) E. coli HB101 transformed with  pNO14. (G) pH 7.0 with the following treatments: I, none; II, H2O2 (0.1  mM); III, GSNO (5 mM); IV, H2O2 (0.05 mM) + GSNO (5 mM). (H)  pH 5.0 with the following treatments: I, none; II, H2O2 (0.1 mM); III,  GSNO (1 mM); IV, H2O2 (0.05 mM) + GSNO (1 mM). Data in panels  A–F are means ± SE for triplicates; some error bars fall within the symbols. Data in panels G and H are means of duplicates.
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Figure 4: Survival advantage conferred by noxR1 on E. coli cultured in GSNO and/or hydrogen peroxide. (A and B) Time course of survival of E. coli HB101 transformed with pBS (open squares) or pNO14 (solid squares) and cultured in LB at pH 5.0 with (A) 2 mM GSNO or (B) 0.5 mM H2O2, as determined by the dye reduction microplate assay. (C–F) Concentration-response curves for GSNO (C and E) and H2O2 (D and F) after 6 h incubation at a pH of 5 (C and D) or pH 7 (E and F). (G and H) Resistance to the combination of GSNO and H2O2. (Black bars) E. coli HB101 transformed with pBS. (Gray bars) E. coli HB101 transformed with pNO14. (G) pH 7.0 with the following treatments: I, none; II, H2O2 (0.1 mM); III, GSNO (5 mM); IV, H2O2 (0.05 mM) + GSNO (5 mM). (H) pH 5.0 with the following treatments: I, none; II, H2O2 (0.1 mM); III, GSNO (1 mM); IV, H2O2 (0.05 mM) + GSNO (1 mM). Data in panels A–F are means ± SE for triplicates; some error bars fall within the symbols. Data in panels G and H are means of duplicates.
Mentions: To more fully explore the phenotype afforded by expression of noxR1, we made use of a fluorescent, dye-reduction microplate assay whose results corresponded almost perfectly (correlation coefficients r2 >0.96) to the results of the more laborious colony-forming agar-plate assay after exposing bacteria to RNI in vitro or to the intraphagosomal milieu of macrophages (29). Not only was E. coli HB101 rendered far more resistant to ASN (2.5 mM NaNO2, pH 5) by expression of noxR1 (data not shown), but the bacteria also better resisted S-nitrosoglutathione (GSNO; 2 mM, pH 5.0), a physiologic and bacteriostatic (37, 43, 44) source of several RNI, including ammonia (45; Fig. 4, A and C). By 6 h, the survival advantage was close to 100-fold. GSNO was more bactericidal at pH 5.0 (Fig. 4, A and C) than at pH 7.0 (Fig. 4 E), but noxR1 conferred protection under both conditions.

Bottom Line: Among the major antimicrobial products of macrophages are reactive intermediates of the oxidation of nitrogen (RNI) and the reduction of oxygen (ROI).Expression of noxR1 conferred upon Escherichia coli and Mycobacterium smegmatis enhanced ability to resist RNI and ROI, whether the bacteria were exposed to exogenous compounds in medium or to endogenous products in macrophages.These studies provide the first identification of an RNI resistance mechanism in mycobacteria, point to a new mechanism for resistance to ROI, and raise the possibility that inhibition of the noxR1 pathway might enhance the ability of macrophages to control tuberculosis.

View Article: PubMed Central - PubMed

Affiliation: Division of International Medicine and Infectious Disease, Department of Medicine, Cornell University Medical College, New York 10021, USA.

ABSTRACT
Among the major antimicrobial products of macrophages are reactive intermediates of the oxidation of nitrogen (RNI) and the reduction of oxygen (ROI). Selection of recombinants in acidified nitrite led to the cloning of a novel gene, noxR1, from a pathogenic clinical isolate of Mycobacterium tuberculosis. Expression of noxR1 conferred upon Escherichia coli and Mycobacterium smegmatis enhanced ability to resist RNI and ROI, whether the bacteria were exposed to exogenous compounds in medium or to endogenous products in macrophages. These studies provide the first identification of an RNI resistance mechanism in mycobacteria, point to a new mechanism for resistance to ROI, and raise the possibility that inhibition of the noxR1 pathway might enhance the ability of macrophages to control tuberculosis.

Show MeSH
Related in: MedlinePlus