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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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Plasmid pNO14 confers enhanced survival on E. coli in ASN.  (A) Survival of E. coli HB101 pBS (open squares) and HB101 pNO14 (solid  squares) in ASN medium. Overnight cultures were diluted 100-fold into  LB, pH 5.3, containing 10 mM NaNO2, and incubated at 37°C. CFUs  were determined at indicated times by plating serial dilutions on LB agar  containing ampicillin. In this and subsequent panels, results are means ±  SE for triplicates; some error bars fall within the symbols. (B) Growth of  M. smegmatis pOLYG (open symbols) and M. smegmatis pOLYG-NO14  (solid symbols) in 7H9 medium containing 30 mM sodium nitrite at pH  7.4 (triangles) or 30 mM sodium nitrate at pH 5.3 (squares). CFU were determined by plating on LB agar containing 50 μg/ml hygromycin, either  at onset of culture or after 12 h. (C and D) Survival of M. smegmatis  pOLYG (open squares) and M. smegmatis pOLYG-NO14 (solid squares) in  ASN medium. Bacteria were grown to an optical density of A600 = 4.5  (stationary phase cultures, C) or to A600 = 1.5 (logarithmic phase cultures,  D) and diluted 100-fold in 7H9 medium, pH 5.3, containing 30 mM  NaNO2 for stationary phase cultures and 20 mM NaNO2 for logarithmic  phase cultures. At indicated times CFUs were determined by plating appropriate dilutions on LB agar containing 50 μg/ml hygromycin.
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Figure 1: Plasmid pNO14 confers enhanced survival on E. coli in ASN. (A) Survival of E. coli HB101 pBS (open squares) and HB101 pNO14 (solid squares) in ASN medium. Overnight cultures were diluted 100-fold into LB, pH 5.3, containing 10 mM NaNO2, and incubated at 37°C. CFUs were determined at indicated times by plating serial dilutions on LB agar containing ampicillin. In this and subsequent panels, results are means ± SE for triplicates; some error bars fall within the symbols. (B) Growth of M. smegmatis pOLYG (open symbols) and M. smegmatis pOLYG-NO14 (solid symbols) in 7H9 medium containing 30 mM sodium nitrite at pH 7.4 (triangles) or 30 mM sodium nitrate at pH 5.3 (squares). CFU were determined by plating on LB agar containing 50 μg/ml hygromycin, either at onset of culture or after 12 h. (C and D) Survival of M. smegmatis pOLYG (open squares) and M. smegmatis pOLYG-NO14 (solid squares) in ASN medium. Bacteria were grown to an optical density of A600 = 4.5 (stationary phase cultures, C) or to A600 = 1.5 (logarithmic phase cultures, D) and diluted 100-fold in 7H9 medium, pH 5.3, containing 30 mM NaNO2 for stationary phase cultures and 20 mM NaNO2 for logarithmic phase cultures. At indicated times CFUs were determined by plating appropriate dilutions on LB agar containing 50 μg/ml hygromycin.

Mentions: E. coli XL1-Blue was electroporated with a genomic library of M. tuberculosis CB3.3 and exposed for 24 h to ASN (6 mM NaNO2, pH 6.0). Protonation of NaNO2 generates HNO2, whose dismutation provides NO and nitrate, and, through reaction of NO with oxygen, other RNI (35, 36). The main products of these reactions are probably dinitrogen tri- and tetra-oxides (N2O3 and N2O4) as well as S-nitrosothiols, which have profound bacteriostatic effects (37, 38, 39). ASN plays a physiologic role in the microbicidal system of the stomach (40) and the combination of RNI and low pH also mimics aspects of the intraphagolysosomal milieu of the macrophage (5, 41). The chosen conditions killed E. coli XL1-Blue efficiently (>7 log10 in 24 h). Surviving transformants were detected with a frequency of <10−6. One recombinant plasmid, called pNO14, consistently conferred upon XL1-Blue and four other E. coli hosts (DH5α, HB101, GC4468, and JTG100) as well as Salmonella typhimurium LT2 and 14028 (data not shown) an enhanced ability to resist ASN. By 12 h, transformation of HB101 with pNO14 afforded a 50-fold increase in survival above transformation of the same E. coli host with the vector alone (Fig. 1 A).


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)

Plasmid pNO14 confers enhanced survival on E. coli in ASN.  (A) Survival of E. coli HB101 pBS (open squares) and HB101 pNO14 (solid  squares) in ASN medium. Overnight cultures were diluted 100-fold into  LB, pH 5.3, containing 10 mM NaNO2, and incubated at 37°C. CFUs  were determined at indicated times by plating serial dilutions on LB agar  containing ampicillin. In this and subsequent panels, results are means ±  SE for triplicates; some error bars fall within the symbols. (B) Growth of  M. smegmatis pOLYG (open symbols) and M. smegmatis pOLYG-NO14  (solid symbols) in 7H9 medium containing 30 mM sodium nitrite at pH  7.4 (triangles) or 30 mM sodium nitrate at pH 5.3 (squares). CFU were determined by plating on LB agar containing 50 μg/ml hygromycin, either  at onset of culture or after 12 h. (C and D) Survival of M. smegmatis  pOLYG (open squares) and M. smegmatis pOLYG-NO14 (solid squares) in  ASN medium. Bacteria were grown to an optical density of A600 = 4.5  (stationary phase cultures, C) or to A600 = 1.5 (logarithmic phase cultures,  D) and diluted 100-fold in 7H9 medium, pH 5.3, containing 30 mM  NaNO2 for stationary phase cultures and 20 mM NaNO2 for logarithmic  phase cultures. At indicated times CFUs were determined by plating appropriate dilutions on LB agar containing 50 μg/ml hygromycin.
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Related In: Results  -  Collection

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Figure 1: Plasmid pNO14 confers enhanced survival on E. coli in ASN. (A) Survival of E. coli HB101 pBS (open squares) and HB101 pNO14 (solid squares) in ASN medium. Overnight cultures were diluted 100-fold into LB, pH 5.3, containing 10 mM NaNO2, and incubated at 37°C. CFUs were determined at indicated times by plating serial dilutions on LB agar containing ampicillin. In this and subsequent panels, results are means ± SE for triplicates; some error bars fall within the symbols. (B) Growth of M. smegmatis pOLYG (open symbols) and M. smegmatis pOLYG-NO14 (solid symbols) in 7H9 medium containing 30 mM sodium nitrite at pH 7.4 (triangles) or 30 mM sodium nitrate at pH 5.3 (squares). CFU were determined by plating on LB agar containing 50 μg/ml hygromycin, either at onset of culture or after 12 h. (C and D) Survival of M. smegmatis pOLYG (open squares) and M. smegmatis pOLYG-NO14 (solid squares) in ASN medium. Bacteria were grown to an optical density of A600 = 4.5 (stationary phase cultures, C) or to A600 = 1.5 (logarithmic phase cultures, D) and diluted 100-fold in 7H9 medium, pH 5.3, containing 30 mM NaNO2 for stationary phase cultures and 20 mM NaNO2 for logarithmic phase cultures. At indicated times CFUs were determined by plating appropriate dilutions on LB agar containing 50 μg/ml hygromycin.
Mentions: E. coli XL1-Blue was electroporated with a genomic library of M. tuberculosis CB3.3 and exposed for 24 h to ASN (6 mM NaNO2, pH 6.0). Protonation of NaNO2 generates HNO2, whose dismutation provides NO and nitrate, and, through reaction of NO with oxygen, other RNI (35, 36). The main products of these reactions are probably dinitrogen tri- and tetra-oxides (N2O3 and N2O4) as well as S-nitrosothiols, which have profound bacteriostatic effects (37, 38, 39). ASN plays a physiologic role in the microbicidal system of the stomach (40) and the combination of RNI and low pH also mimics aspects of the intraphagolysosomal milieu of the macrophage (5, 41). The chosen conditions killed E. coli XL1-Blue efficiently (>7 log10 in 24 h). Surviving transformants were detected with a frequency of <10−6. One recombinant plasmid, called pNO14, consistently conferred upon XL1-Blue and four other E. coli hosts (DH5α, HB101, GC4468, and JTG100) as well as Salmonella typhimurium LT2 and 14028 (data not shown) an enhanced ability to resist ASN. By 12 h, transformation of HB101 with pNO14 afforded a 50-fold increase in survival above transformation of the same E. coli host with the vector alone (Fig. 1 A).

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