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Mechanisms of Action: Physiological Effects

Enzymes Associated with Reductive Activation and Action of Nitazoxanide, Nitrofurans, and Metronidazole in Helicobacter pylori

Gary Sisson, Avery Goodwin, Ausra Raudonikiene, Nicky J. Hughes, Asish K. Mukhopadhyay, Douglas E. Berg, Paul S. Hoffman
Gary Sisson
1Department of Microbiology and Immunology
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Avery Goodwin
1Department of Microbiology and Immunology
2GlaxoSmithKline, Collegeville, Pennsylvania
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Ausra Raudonikiene
1Department of Microbiology and Immunology
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Nicky J. Hughes
2GlaxoSmithKline, Collegeville, Pennsylvania
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Asish K. Mukhopadhyay
3Department of Molecular Microbiology and Department of Genetics, Washington University Medical School, St. Louis, Missouri
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Douglas E. Berg
3Department of Molecular Microbiology and Department of Genetics, Washington University Medical School, St. Louis, Missouri
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Paul S. Hoffman
1Department of Microbiology and Immunology
2GlaxoSmithKline, Collegeville, Pennsylvania
4Division of Infectious Diseases, Department of Medicine, Faculty of Medicine, Dalhousie University, HaliFax, Nova Scotia B3H 4H7
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  • For correspondence: phoffman@tupdean2.med.dal.ca
DOI: 10.1128/AAC.46.7.2116-2123.2002
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  • FIG. 1.
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    FIG. 1.

    POR assay and competition with benzyl viologen. (A) Competitive inhibition of POR activity (benzyl viologen reduction) in H. pylori extracts as a function of NTZ concentration was monitored spectrophotometrically at 546 nm. The specific activity at each concentration of nitazoxanide was recorded. (B) POR was assayed in cell extracts of H. pylori by monitoring the pyruvate-dependent reduction of nitazoxanide at 412 nm (A) as described in the text.

  • FIG. 2.
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    FIG. 2.

    Lack of NTZ- or nitrofuran-induced DNA fragmentation in H. pylori. The MTZs strain H. pylori (Hp) 26695 was challenged with various concentrations of NTZ or nitrofurazone for 30 min as described in the text. The bacteria were suspended and lysed in agarose plugs, and agarose gels were run under alkaline conditions to display the extent of DNA fragmentation of denatured genomic DNA. Bacteria were treated with hydrogen peroxide (20 mM) for 15 min (positive controls). wt, wild type.

  • FIG. 3.
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    FIG. 3.

    Lack of drug-induced DNA fragmentation of E. coli strain CC104 carrying pBSK. The bacteria were grown in the presence of the nitrofuran drugs as described in the text. The preparation of agarose plugs is as described in Fig. 2. Hydrogen peroxide was added at a 20 mM concentration as a positive control. The distinct bands noted in each of the lanes are pBSK plasmid DNA.

  • FIG. 4.
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    FIG. 4.

    Lack of nitazoxanide-induced DNA fragmentation of E. coli strains carrying rdxA of H. pylori. E. coli strain CC104 containing either pBSK (control) or pGS950 (rdxA+) was grown in the presence of NTZ, and bacteria were suspended and lysed in agarose plugs and electrophoresed as described for Fig. 2 and detailed in the text. Hydrogen peroxide was added at a 20 mM concentration as a positive control. The distinct bands noted in the various lanes are plasmid DNA.

Tables

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  • TABLE 1.

    POR activity of PORGDAB expressed in E. colia

    CompoundSp act (nmol/min/mg of protein)
    JVQ2:pBSKPORAB1157:pPBSKPOR
    NTZ3,325 ± 4282,233 ± 117
    Nitrofurazone41.2 ± 5.437.9 ± 1.5
    Nitrofurantoin29.2 ± 4.121.4 ± 0.6
    Furazolidone25.6 ± 1.718.1 ± 0.4
    MTZ<0.1<0.1
    • ↵ a The E. coli strains used in this study are AB1157 (wild type) and JVQ2nfsAnfsB (deficient in nitroreductase activity). Plasmid pPORGDAB contains the POR operon in pBSK. Enzyme activities were determined under anaerobic conditions at the appropriate wavelength for each compound as detailed in the text.

  • TABLE 2.

    Substrate specificity of RdxA and FrxA nitroreductases

    SubstrateSp act (μmol/min/mg of protein)a
    RdxA nitroreductaseFrxA nitroreductase
    MTZ5.13<0.0002
    NADPH (MTZ)b9.01<0.0002
    NTZ13.422.2
    Nitrofurazone<0.00020.50
    Furazolidone<0.00021.50
    Nitrofurantoin<0.00022.01
    • ↵ a Specific activities are calculated as the means for five assays of each of two independent batches of enzyme for FrxA and RdxA. The error in these assays is <15%.

    • ↵ b NADPH oxidation was followed at 340 nm with MTZ as the electron acceptor.

  • TABLE 3.

    Mutagenic action of sublethal concentrations of MTZ, NTZ, and furazolidone on Mtzs and MtzrH. pylori

    Drug concn (μg/ml)Mutation frequency (no. Rifr/108)
    MetronidazoleNitazoxanideFurazolidone
    WT (EOP)ardxA (EOP)WT (EOP)rdxA (EOP)WT (EOP)rdxA (EOP)
    012 (1)8 (1)2.27 (1)6.10 (1)1.0 (1)4.35 (1)
    0.25NTbNT1.27 (1)NT2.64 (0.1)16.43 (1)
    0.5NTNT1.50 (1)2.94 (1)6.90 (0.1)64.91 (0.1)
    1.014 (1)5 (1)1.41 (0.5)4.46 (1)0 (10−3)0
    2.069 (10−2)10 (1)0.97 (0.1)2.47 (1)NTNT
    3.0144 (10−2)5 (1)NTNTNTNT
    5.00 (0)12 (1)0 (10−5)5.26 (0.5)NTNT
    10.0NTNTNT0 (10−8)NTNT
    25.0NT213 (10−2)NTNTNTNT
    • ↵ a Strains used were 26695 and the rdxA mutant. 0, insufficient survivors from which to calculate a mutation frequency. The data represent the means of three determinations each from two independent experiments. WT, wild type.

    • ↵ b NT, not tested.

  • TABLE 4.

    Frequency of rifampin resistance in E. coli tester strain CC104

    Strain, compound, and concn (μg/ml)aSurvivalbRifr frequency (per 108)cFold
    CC104
        Nitazoxanide
            014.8
            516.7
            1012.6
    CC104(pGS950)
        Nitazoxanide
            017.0
            515.5
            1016.3
    CC104
        Nitrofurazone
            013.36
            1126.47.9
            20.758.617.4
            510−375.422.4
        Nitrofurantoin
            013.36
            10.96.141.8
            20.98.592.6
            52.5 × 10−232.589.7
        Furazolidone
            013.36
            0.110−264.019
            110−30
    Controld
        Metronidazole
            010.75
            510−25370
            1010−4378504
    • ↵ a The bacteria (E. coli CC104 + pBSK or CC104 + pGS950rdxA+) were grown on LB agar with the appropriate concentration of drug and then plated on medium containing rifampin as indicated in the text.

    • ↵ b EOP − efficiency of plating (EOP of 1 = no loss of viability).

    • ↵ c The Rifr frequencies represent the means of three determinations and are reported as the number of Rifr colonies per 108 bacteria plated.

    • ↵ d Similar to data of Sisson et al. (29); included for comparison. Control, CC104(pGS950).

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Enzymes Associated with Reductive Activation and Action of Nitazoxanide, Nitrofurans, and Metronidazole in Helicobacter pylori
Gary Sisson, Avery Goodwin, Ausra Raudonikiene, Nicky J. Hughes, Asish K. Mukhopadhyay, Douglas E. Berg, Paul S. Hoffman
Antimicrobial Agents and Chemotherapy Jul 2002, 46 (7) 2116-2123; DOI: 10.1128/AAC.46.7.2116-2123.2002

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Enzymes Associated with Reductive Activation and Action of Nitazoxanide, Nitrofurans, and Metronidazole in Helicobacter pylori
Gary Sisson, Avery Goodwin, Ausra Raudonikiene, Nicky J. Hughes, Asish K. Mukhopadhyay, Douglas E. Berg, Paul S. Hoffman
Antimicrobial Agents and Chemotherapy Jul 2002, 46 (7) 2116-2123; DOI: 10.1128/AAC.46.7.2116-2123.2002
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KEYWORDS

Helicobacter pylori
Ketone Oxidoreductases
metronidazole
nitrofurans
Nitroreductases
thiazoles

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