Helicobacter pylori disulphide reductases: role in metronidazole reduction


  • Nadeem O. Kaakoush,

    1. School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Biological Science Builiding, Sydney, NSW 2052, Australia
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  • George L. Mendz

    Corresponding author
    1. School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Biological Science Builiding, Sydney, NSW 2052, Australia
      *Corresponding author. Tel.: +61 2 9385 2042; fax: +61 2 9385 1483, E-mail address: G.Mendz@unsw.edu.au
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*Corresponding author. Tel.: +61 2 9385 2042; fax: +61 2 9385 1483, E-mail address: G.Mendz@unsw.edu.au


Disulphide reductases play an important role in maintaining intracellular redox potential. Three disulphide reductase activities were identified in Helicobacter pylori, which used dithiobis-2-nitrobenzoic acid, glutathione or l-cystine and ferredoxin as substrates. The kinetic parameters of these activities were determined and it was demonstrated that the reductase activities were inhibited by the presence of metronidazole. Substrate competition experiments served to show inhibition of metronidazole reduction by dithiobis-2-nitrobenzoic acid, glutathione and ferredoxin in lysates from metronidazole susceptible and resistant matched pairs of strains. The study demonstrated that the activities of three disulphide reductases were modulated by the presence of metronidazole, and that metronidazole reduction was inhibited by the presence of disulphide reductase substrates.


Metronidazole (Mtr) is an important component of therapeutic regimes currently used to treat many microbial pathogens. This 5-nitroimidazole is activated via interactions with redox systems capable of reducing the low potential (−415 mV) nitro group in position 5 of the imidazole ring [1]. This property makes metronidazole effective against organisms of low intracellular redox state, such as anaerobic bacteria and protozoa, as well as some microaerophiles, such as Campylobacter spp. and Helicobacter pylori[2].

The frequent use of metronidazole has resulted in increased resistance to the antibiotic by many pathogens including H. pylori. The emergence of resistant isolates that do not respond to the drug fostered a keen interest to understand the primary causes of resistance to metronidazole in this bacterium. Extensive investigations on H. pylori established that main causes of metronidazole resistance are mutations in the genes rdxA or frxA[3–6]. However, insufficient data correlating RdxA and/or FrxA with the resistant phenotype, and the fact that a small percentage of resistant strains do not have mutations in either rdxA or frxA indicated that the molecular basis of H. pylori resistance to Mtr has not been characterised completely.

Early studies showed that oxygen tensions have a large impact on the resistance of H. pylori to Mtr [7–10], and several investigations have linked the activities of specific oxidoreductases to the Mtr-susceptible phenotype of the bacterium [10–12]. These results suggested a possible role in Mtr activation of enzymes catalysing redox reactions which modulate the intracellular redox status. A type of such enzymes are disulphide reductases whose reactions contribute to the redox balance of the cell.

In the present study, ferredoxin (Fdx) oxidoreductase and two other enzyme activities which use oxidised glutathione (GSSG), l-cystine (Cys-Cys) and dithiobis-2-nitrobenzoic acid (DTNB) as substrates were identified in H. pylori, and the effects of metronidazole on their activities were characterised. The potential involvement of these disulphide reductases in metronidazole activation was investigated by measuring the effects of their substrates on the rates of metronidazole reduction.

2Materials and methods

2.1Chemicals and reagents

Blood agar base No. 2, brain heart infusion (BHI), defibrinated horse blood and horse serum were from Oxoid (Heidelberg West, VIC, Australia). Amphotericin (Fungizone®), bicinchoninic acid, bovine serum albumin, copper II sulphate, l-cystine, DTNB, Fdx, GSSG, Cys-Cys, metronidazole, mineral oil, β-nicotinamide adenine dinucleotide reduced form (NADH), polymixin B and trimethoprim were from Sigma (Castle Hill, NSW, Australia). Vancomycin was from Eli Lilly (North Ryde, NSW, Australia). Deuterium oxide was from Cambridge isotope laboratories (Cambridge, England). Tris base was from Amersham Biosciences (Melbourne, VIC, Australia). All other reagents were of analytical grade.

2.2Bacterial cultures and preparation

The strains used in this study were J99 (with annotated genome), NCTC 11639, N6 (Institut Pasteur, Paris), and P10 and SS1 (University of New South Wales collection). The isolates LC11 and LC20 were obtained recently from patients with gastritis, and the Mtr susceptible and resistant matched pairs 10593/2 and RIG 117 were obtained before and after therapy from patients treated unsuccessfully with metronidazole. The SS1 resistant strains were constructed by sequential passing on plates containing increasing concentrations of Mtr. In the clinical isolates as well as in the SS1 susceptible and resistant strains, the matched pairs have the same genetic background [13]. Bacteria were grown on Campylobacter selective agar supplemented with defibrinated horse blood, 2.0 μg ml−1 Amphotericin, 5.0 mg ml−1 vancomycin, 1250 U ml−1 polymixin B, and 2.5 mg ml−1 trimethoprim. Cultures were incubated at 37 °C under the microaerobic conditions 5% CO2, 5% O2 and 90% N2. The purity of the cultures was confirmed as H. pylori by positive urease and catalase tests, and motility and morphology observed under phase contrast microscopy.

H. pylori cells were harvested in 150 mM sodium chloride (NaCl) and centrifuged at 16,000g at 4 °C for 10 min. The pellet was collected and the supernatant discarded. The pellet was resuspended in 150 mM NaCl solution and washed three times. Cells were lysed by thrice freezing in liquid nitrogen and thawing. Cell-free extracts were obtained by centrifuging lysates at 16,000g at 4 °C for 20 min and collecting the soluble fraction.

Protein concentrations were estimated by the bicinchoninic acid method based on microtitre plate protocol [12].

2.3Nuclear magnetic resonance spectroscopy

Suspensions of bacterial lysates or cell-free extracts were placed in 5 or 10 mm tubes (Wilmad, Buena, NJ, USA), the appropriate substrates added, and measurements of enzyme activities were carried out at 37 °C. Proton nuclear magnetic resonance spectroscopy (1H NMR) and nitrogen-14 nuclear magnetic resonance (14N NMR) free induction decays were collected using a Bruker DMX-600 or a Bruker DMX-500 spectrometer, respectively, operating in the pulsed Fourier transform mode with quadrature detection. The instrumental parameters for the DMX-600 spectrometer were: operating frequency 600.13 MHz, spectral width 6009.61 Hz, memory size 16 K, acquisition time 1.36 s, number of transients 64, pulse angle 50° (3 μs) and relaxation delay with solvent presaturation 1.7 s. Spectral resolution was enhanced by Gaussian multiplication with line broadening of −0.7 Hz and Gaussian broadening factor of 0.19. Proton spectra were acquired with presaturation of the water resonance. The instrumental parameters for the DMX-500 spectrometer were: operating frequency 36.14 MHz, spectral width 19,841 Hz, memory size 8 K, acquisition time 0.21 s, number of transients 2488, pulse angle 90° (30 μs) and relaxation delay 0.1 s. Spectra were acquired with composite pulse decoupling of protons. Exponential filtering of 15 Hz was applied prior to Fourier transformation.

The time-evolution of substrates and products were followed by acquiring sequential spectra of the reactions. Progress curves were obtained by measuring the integrals of compounds at different points in time. Maximal rates were calculated from good fits to straight lines (correlation coefficients ≥ 0.95) of the data for 30 min for GSSG reduction or 2 h for Mtr reduction. Calibrations of the peaks arising from substrates were performed by extrapolating the resonance intensity data to zero time and assigning to this intensity the appropriate concentration value.

Reduction rates of GSSG or Cys-Cys were measured employing 1H NMR spectroscopy in lysates or cell-free extracts suspended in 2H2O:H2O (1:5 v/v), 10 mM KCl, 25 mM NaCl and 50 mM potassium phosphate buffer, pH 7.2. Metronidazole reduction was measured employing 14N NMR spectroscopy in lysate suspensions in the same buffer as for disulphide reduction assays but with 2H2O:H2O (1:10 v/v), 12 mM Mtr and 30 mM NADH. To assay Mtr reduction, dissolved oxygen was substituted by argon in the samples by bubbling them with the inert gas for 30 min at 4 °C. Mineral oil was layered on top of the samples to stop argon exchange with atmospheric oxygen (Fig. 1).

Figure 1.

1H spectra of glutathione reduction in cell-free extract suspensions. Spectra were acquired at the time points indicated on the right-hand side. Resonances arising from the substrates GSSG and NADH are indicated on the bottom spectrum. Resonances arising from the products GSH and NAD+ are indicated on the top spectrum. The GSSG signals decrease with time as the compound is reduced, and the rate of reduction is calculated by measuring this decrease with respect to time.


Ferredoxin and DTNB reduction were measured in a Cary-100 UV–Vis spectrophotometer using 1 cm path-length cuvettes. The reaction mixture contained cell-free extracts and the appropriate substrates suspended in 50 mM Tris–HCl, pH 7.2 buffer in a final volume of 1 ml. Ferredoxin and NADH or DTNB were added just prior to measuring activities, and the change in absorbance at 340 nm (Fdx reduction) or 412 nm (DTNB reduction) over 2 min was recorded. NADH oxidase background activity was determined in assay mixtures with no Fdx. At 340 nm, the coefficient of molar absorbance of NADH is 6.22 ×103 mol−1 cm−1. At 412 nm, the coefficient of molar absorbance of this ion is 13.6 ×103 mol−1 cm−1.

2.5Calculation of kinetic parameters

Michaelis constants (Km) and maximal velocities (Vmax) were calculated by non-linear regression using the Enzyme Kinetics® program (Trinity Software, Compton, NH). The errors in these calculations are determined by the program as ± standard deviation.

2.6Statistical analyses of results

Statistical analyses of the inhibition constant data were performed by determining the mean values and standard deviations for all the susceptible and resistant strains with respect to all substrates. Errors are quoted as ± standard deviation. The mean percentile values and standard deviations for metronidazole reduction rates in assays with specific substrates were determined independently for susceptible and resistant strains using the values obtained in all the assays performed. Errors are quoted as ± standard deviation.


Three disulphide reduction activities were detected in H. pylori cell-free extracts which used DTNB, GSSG or Cys-Cys and NADH, and Fdx and NADH as substrates. These activities were measured in all strains tested (data not shown). The controls below were used to validate the enzyme assays. Chemical reduction of DTNB, GSSG, Cys-Cys or Fdx under the conditions of the assays was ruled out by observing no reduction of any of the substrates in the absence of lysates or cell-free extracts. The enzymatic origin of the reactions was established by determining that no activity was present in suspensions of lysates or cell-free extracts which had been denatured by heating at 80 °C for 2 h. Negative controls of the assays showed that reduction of GSSG, Cys-Cys or Fdx did not take place if NADH was not present.

Matched pairs of Mtr susceptible and resistant strains were employed to investigate the relationships between disulphide reduction and Mtr reduction. The kinetic parameters of DTNB, GSSG and Fdx reduction for the 10593/2 matched pairs of isolates are given in Table 1. The Km and Vmax of DTNB reduction in the resistant strain were smaller than in the susceptible strain. No significant differences were observed in the kinetic parameters of GSSG reduction for the pair of susceptible and resistant isolates. Ferredoxin reduction was observed in the susceptible isolate but was absent in the resistant one (Table 1). In the other matched pairs of isolates, Fdx reduction was observed in the resistant strain but the Km of Fdx reduction in the resistant isolates were significantly smaller than in the susceptible counterparts.

Table 1.  Disulphide reduction activities in H. pylori 10593/2 Mtr-susceptible and resistant cells
IsolateSubstrateKmVmax (nmol mg−1 min−1)
  1. DTNB concentrations ranged from 10 μM to 1 mM; GSSG concentrations from 0.5 to 70 mM; and Fdx concentrations from 0 to 80 μg ml−1. NADH concentrations were 0.5 mM for DTNB assays, 50 mM for GSSG assays, and 0.2 mM for Fdx assays. Kinetic fits were performed using 10–13 rates. Errors were calculated using the Enzyme Kinetics program from non-linear regression fits to the data.

10593/2 SusceptibleDTNB45 ± 3 μM17 ± 2
GSSG:NADH2.3 ± 0.2 mM129 ± 4
Fdx:NADH3.0 ± 0.4 μg ml−110 ± 1
10593/2 ResistantDTNB17 ± 2 μM12 ± 1
GSSG:NADH2.8 ± 0.2 mM166 ± 2
Fdx:NADHNo reductionNo reduction

The effects of Mtr on disulphide reduction were investigated by measuring the rates of reduction in the presence of different concentrations of Mtr. The three disulphide reduction activities were inhibited by Mtr. The mode of inhibition was determined by measuring the kinetic parameters of the reductions with and without 0.5 mM Mtr. In the presence of Mtr, larger Km and similar Vmax values were measured, indicating that the inhibition of these activities by Mtr was competitive.

At concentrations well below the Km of the substrates, the inhibition constant (Ki) can be calculated from the expression


where ‘v0’ and ‘v’ are the uninhibited and inhibited rates of enzyme activity, respectively, and ‘I’ is the concentration of inhibitor which results in a ‘v’ rate of reaction [14]. The Ki values for the inhibition of GSSG and Cys-Cys reduction activities of the matched pairs are given in Table 2. Larger Ki values were observed in resistant strains than in their susceptible counterparts, suggesting stronger effects of Mtr on the latter. Similar observations were made for the other matched pairs of strains. Statistical analyses of these results were performed and the Ki mean value of the susceptible strains for both substrates, 1.2 ± 0.3 mM, was significantly lower than the mean value for the resistant strains, 3.8 ± 1.1 mM.

Table 2.  Metronidazole inhibition constants (Ki) of disulphide reductase activities for Mtr-susceptible and Mtr-resistant strains
StrainKi (mM)
  1. Enzyme activities were measured in lysates suspended in potassium phosphate or TrisHCl buffer for the GSSG and Fdx assays, respectively. Five rates were used for each inhibition plot. Errors were determined from the best-fitted line in the inhibition plot.

SS1GSSG1.1 ± 0.25.5 ± 0.8
10593/2GSSG1.6 ± 0.12.8 ± 0.3
Cys-Cys1.3 ± 0.23.9 ± 0.5
10827/6Cys-Cys0.7 ± 0.12.7 ± 0.4
RIG117Cys-Cys1.2 ± 0.24.1 ± 0.6

Metronidazole reduction was measured in H. pylori lysate suspensions employing 14N NMR spectroscopy. The rate of Mtr reduction in the 10593/2 Mtr-resistant isolate was significantly lower (p < 0.02) than in the susceptible isolate (Fig. 2), correlating with the phenotype of the isolates. Similar results were obtained for the RIG 117 and SS1 matched pairs of strains (data not shown). The potential roles of disulphide reductases in Mtr reduction were investigated by performing substrate competition experiments. Metronidazole reduction rates for the susceptible strains were inhibited in the presence of the substrates DTNB (p < 0.03), GSSG (p < 0.02) or Fdx (p < 0.02). The reduction rates for the resistant strains were also inhibited in the presence of the substrates DTNB (p < 0.03), GSSG (p < 0.03) or Fdx (p < 0.03). The results for the 10593/2 matched pair of isolates are shown in Fig. 2. Similar data were obtained for Mtr-Fdx competition experiments for the other matched pairs of strains.

Figure 2.

Metronidazole reduction activities of H. pylori 10593/2 susceptible and resistant lysates from cells grown under microaerobic conditions. Lysates were suspended in phosphate buffer and subjected to argon treatment for 30 min. Initial substrate concentrations were 12 mM Mtr and 30 mM NADH. DTNB, GSSG and Fdx were added in concentrations of 15 μM, 4 mM and 0.17 mg ml−1 for the susceptible lysates and 30 μM, 6 mM and 0.17 mg ml−1 for the resistant lysates. Errors were calculated from the straight line fitting of the values used to determine the Mtr reduction rates.


H. pylori is one of the most prevalent bacterial infections in humans. The bacterium is susceptible to several antibiotics including metronidazole, but it readily develops resistance in vivo and in vitro on exposure to the drug. The prevalence of Mtr-resistant isolates varies between 10% and 80% for different parts of the world [15].

It was discovered that Mtr resistance in H. pylori can result from the loss of activity of RdxA, an oxygen insensitive NADPH nitroreductase. This finding provoked renewed interest to achieve a full understanding of the causes of resistance to this drug [4]. Later, the finding of frameshift mutations in the gene frxA in resistant strains suggested a role for the NAD(P)H flavin oxidoreductase FrxA, in Mtr resistance in H. pylori[6,16]. Investigations of matched susceptible and resistant pairs showed the existence of unchanged rdxA in susceptible and resistant isolates, and of different rdxA in susceptible strains [5,13,16–21]. Similarly, it was demonstrated that unchanged frxA are found in susceptible and resistant isolates [20–23]. Thus, both RdxA and FrxA are involved in resistance to Mtr by H. pylori, but the available data suggest that there are other factors contributing to the resistant phenotype. For example, other enzymes capable of reducing metronidazole, mechanisms which modulate the expression of rdxA and/or frxA, etc.

Several studies on H. pylori demonstrated that the intracellular oxygen status and redox potential play a role in metronidazole resistance [7–10]. The observations that resistance to Mtr can be overcome by exposing H. pylori to short periods on anaerobiosis [7,8], and that susceptible strains have greater cytosolic NADH-oxidase activities that their respective resistant mutants [7,9], suggested that the oxygen status of the cell is a major contributor to susceptibility of H. pylori the Mtr. Also, changes in the activities pyruvate oxidoreductase, α-ketoglutarate oxidoreductase, and pyruvate:flavodoxin oxidoreductase were observed in the resistant phenotype [10], implicating oxidoreductases in the resistance of H. pylori to Mtr.

Disulphide reductases catalyse oxireduction reactions which contribute to the redox balance of the cell. They have been shown to be virulence and invasive factors, and involved in drug resistance in different pathogenic bacteria [24–26]. A study with matched pairs of H. pylori Mtr-susceptible strains and Mtr-resistant mutants demonstrated that susceptible strains have higher levels of disulphide reduction, and that the total disulphide reduction activity of the cell is modulated by Mtr [10]. Evidence for a role of disulphide reductases in the susceptibility of H. pylori to Mtr was provided by the finding that the alkyl hydroperoxide reductase activity of Mtr-susceptible strains was absent in their Mtr-resistant counterparts [12,27]. Thus, it became important to investigate the role of specific disulphide reduction activities in H. pylori resistance to Mtr.

Three disulphide reduction activities which use DTNB, GSSG or Cys-Cys and NADH, or Fdx and NADH as substrates, were identified and characterised in H. pylori (Table 1). Metronidazole inhibited disulphide reduction competitively in each of the three activities, and the measured Ki of Mtr for the reduction of GSSG or Cys-Cys indicated that the effects of Mtr were stronger in susceptible strains than in resistant ones (Table 2). The data demonstrated that Mtr modulated directly the disulphide reductases and suggested a role for these reductases in Mtr reduction.

Confirmation of this role was found by examining the involvement of disulphide reductases in Mtr reduction. The presence of the substrates DTNB, GSSG or Fdx, inhibited Mtr reduction in situ, indicating that these disulphide reductases competed with Mtr as acceptors in redox reactions, and suggesting that they participated in the reduction of Mtr. Together with previous findings these results provided evidence that disulphide reductases play a role in the activation of Mtr, and thus, in the susceptibility of H. pylori to this antibiotic.

The role of these reductase activities in the resistant phenotype needs to be investigated further, as well as putative molecular mechanisms relating these disulphide reductases to the activities of RdxA and FrxA, in particular the effects of the intracellular redox potential on the expression of the latter two enzymes. Metronidazole resistance is found in other pathogenic microaerophilic organisms besides H. pylori. It will be important to ascertain whether disulphide reductase activities also contribute to the development of Mtr resistance in, for instance, Campylobacter spp. and various parasites.


This work was made possible by the support of the Australian Research Council.