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FEMS Immunology and Medical Microbiology 44 (2005) 137–142

Helicobacter pylori disulphide reductases: role inmetronidazole reduction

Nadeem O. Kaakoush, George L. Mendz *

School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Biological Science Builiding, Sydney, NSW 2052, Australia

Received 26 June 2004; received in revised form 27 October 2004; accepted 22 November 2004

First published online 2 December 2004

Abstract

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 LL-cystine and ferredoxin as sub-

strates. The kinetic parameters of these activities were determined and it was demonstrated that the reductase activities were inhib-

ited 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.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Helicobacter pylori; Disulphide reductases; Metronidazole; Resistance; Redox potential

1. Introduction

Metronidazole (Mtr) is an important component oftherapeutic regimes currently used to treat many micro-

bial 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 metroni-

dazole effective against organisms of low intracellular re-

dox 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 in-

creased 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

0928-8244/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsim.2004.11.007

* Corresponding author. Tel.: +61 2 9385 2042; fax: +61 2 9385

1483.

E-mail address: G.Mendz@unsw.edu.au (G.L. Mendz).

to understand the primary causes of resistance to metro-

nidazole in this bacterium. Extensive investigations on

H. pylori established that main causes of metronidazoleresistance 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 muta-

tions in either rdxA or frxA indicated that the molecular

basis of H. pylori resistance to Mtr has not been charac-

terised completely.

Early studies showed that oxygen tensions have alarge 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 phe-

notype 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 thecell.

. Published by Elsevier B.V. All rights reserved.

138 N.O. Kaakoush, G.L. Mendz / FEMS Immunology and Medical Microbiology 44 (2005) 137–142

In the present study, ferredoxin (Fdx) oxidoreductase

and two other enzyme activities which use oxidised glu-

tathione (GSSG), LL-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 involvementof these disulphide reductases in metronidazole activa-

tion was investigated by measuring the effects of their

substrates on the rates of metronidazole reduction.

2. Materials and methods

2.1. Chemicals 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 albu-

min, copper II sulphate, LL-cystine, DTNB, Fdx, GSSG,

Cys-Cys, metronidazole, mineral oil, b-nicotinamide

adenine dinucleotide reduced form (NADH), polymixinB 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, Eng-

land). Tris base was from Amersham Biosciences (Mel-

bourne, VIC, Australia). All other reagents were of

analytical grade.

2.2. Bacterial cultures and preparation

The strains used in this study were J99 (with anno-

tated genome), NCTC 11639, N6 (Institut Pasteur,

Paris), and P10 and SS1 (University of New South

Wales collection). The isolates LC11 and LC20 were ob-

tained recently from patients with gastritis, and the Mtr

susceptible and resistant matched pairs 10593/2 andRIG 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 sus-

ceptible and resistant strains, the matched pairs have the

same genetic background [13]. Bacteria were grown on

Campylobacter selective agar supplemented with defi-brinated horse blood, 2.0 lg 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 con-

firmed 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 dis-

carded. 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 solublefraction.

Protein concentrations were estimated by the bicinch-

oninic acid method based on microtitre plate protocol

[12].

2.3. Nuclear magnetic resonance spectroscopy

Suspensions of bacterial lysates or cell-free extractswere placed in 5 or 10 mm tubes (Wilmad, Buena, NJ,

USA), the appropriate substrates added, and measure-

ments 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 spectrome-

ter, respectively, operating in the pulsed Fourier trans-form 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 ls) and relax-

ation delay with solvent presaturation 1.7 s. Spectral res-

olution was enhanced by Gaussian multiplication with

line broadening of �0.7 Hz and Gaussian broadeningfactor of 0.19. Proton spectra were acquired with presat-

uration of the water resonance. The instrumental

parameters for the DMX-500 spectrometer were: oper-

ating 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 ls) and relaxation

delay 0.1 s. Spectra were acquired with composite pulse

decoupling of protons. Exponential filtering of 15 Hzwas applied prior to Fourier transformation.

The time-evolution of substrates and products were

followed by acquiring sequential spectra of the reac-

tions. Progress curves were obtained by measuring the

integrals of compounds at different points in time. Max-

imal rates were calculated from good fits to straight lines

(correlation coefficientsP 0.95) of the data for 30 min

for GSSG reduction or 2 h for Mtr reduction. Calibra-tions of the peaks arising from substrates were per-

formed by extrapolating the resonance intensity data

to zero time and assigning to this intensity the appropri-

ate 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 employ-

ing 14N NMR spectroscopy in lysate suspensions in the

N.O. Kaakoush, G.L. Mendz / FEMS Immunology and Medical Microbiology 44 (2005) 137–142 139

same buffer as for disulphide reduction assays but with2H2O:H2O (1:10 v/v), 12 mM Mtr and 30 mM NADH.

To assay Mtr reduction, dissolved oxygen was substi-

tuted 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 withatmospheric oxygen (Fig. 1).

2.4. Spectrophotometry

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 50mM 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 coeffi-cient of molar absorbance of this ion is 13.6 · 103

mol�1 cm�1.

2.5. Calculation 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.

Fig. 1. 1H spectra of glutathione reduction in cell-free extract suspensions. Sp

Resonances arising from the substrates GSSG and NADH are indicated on

NAD+ are indicated on the top spectrum. The GSSG signals decrease with ti

by measuring this decrease with respect to time.

2.6. Statistical 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 quotedas ± 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.

3. Results

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 sub-

strates. These activities were measured in all strains

tested (data not shown). The controls below were used

to validate the enzyme assays. Chemical reduction ofDTNB, 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 reductionof GSSG, Cys-Cys or Fdx did not take place if NADH

was not present.

ectra were acquired at the time points indicated on the right-hand side.

the bottom spectrum. Resonances arising from the products GSH and

me as the compound is reduced, and the rate of reduction is calculated

140 N.O. Kaakoush, G.L. Mendz / FEMS Immunology and Medical Microbiology 44 (2005) 137–142

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 resistantstrain were smaller than in the susceptible strain. No sig-

nificant differences were observed in the kinetic parame-

ters 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 weresignificantly smaller than in the susceptible

counterparts.

The effects of Mtr on disulphide reduction were inves-

tigated by measuring the rates of reduction in the pres-

ence 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 with-out 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 sub-

strates, the inhibition constant (Ki) can be calculated

from the expression

v0=v ¼ 1þ I=K i;

where �v0� and �v� are the uninhibited and inhibited rates

of enzyme activity, respectively, and �I� is the concentra-tion 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 gi-

ven in Table 2. Larger Ki values were observed in resis-tant 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 per-

formed and the Ki mean value of the susceptible strains

for both substrates, 1.2 ± 0.3 mM, was significantly

Table 1

Disulphide reduction activities in H. pylori 10593/2 Mtr-susceptible and resi

Isolate Substrate

10593/2 Susceptible DTNB

GSSG:NADH

Fdx:NADH

10593/2 Resistant DTNB

GSSG:NADH

Fdx:NADH

DTNB concentrations ranged from 10 lM to 1 mM; GSSG concentration

NADH concentrations were 0.5 mM for DTNB assays, 50 mM for GSSG ass

13 rates. Errors were calculated using the Enzyme Kinetics program from n

lower than the mean value for the resistant strains,

3.8 ± 1.1 mM.

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 sus-ceptible 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) orFdx (p < 0.02). The reduction rates for the resistant

strains were also inhibited in the presence of the sub-

strates 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.

4. Discussion

H. pylori is one of the most prevalent bacterial infec-

tions in humans. The bacterium is susceptible to several

antibiotics including metronidazole, but it readily devel-

ops resistance in vivo and in vitro on exposure to the

drug. The prevalence of Mtr-resistant isolates varies be-tween 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 pro-

voked 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 resis-

tant strains suggested a role for the NAD(P)H flavinoxidoreductase FrxA, in Mtr resistance in H. pylori

[6,16]. Investigations of matched susceptible and resis-

tant pairs showed the existence of unchanged rdxA in

susceptible and resistant isolates, and of different rdxA

stant cells

Km Vmax (nmol mg�1 min�1)

45 ± 3 lM 17 ± 2

2.3 ± 0.2 mM 129 ± 4

3.0 ± 0.4 lg ml�1 10 ± 1

17 ± 2 lM 12 ± 1

2.8 ± 0.2 mM 166 ± 2

No reduction No reduction

s from 0.5 to 70 mM; and Fdx concentrations from 0 to 80 lg ml�1.

ays, and 0.2 mM for Fdx assays. Kinetic fits were performed using 10–

on-linear regression fits to the data.

Table 2

Metronidazole inhibition constants (Ki) of disulphide reductase

activities for Mtr-susceptible and Mtr-resistant strains

Strain Ki (mM)

Substrates Susceptible Resistant

SS1 GSSG 1.1 ± 0.2 5.5 ± 0.8

10593/2 GSSG 1.6 ± 0.1 2.8 ± 0.3

Cys-Cys 1.3 ± 0.2 3.9 ± 0.5

10827/6 Cys-Cys 0.7 ± 0.1 2.7 ± 0.4

RIG117 Cys-Cys 1.2 ± 0.2 4.1 ± 0.6

Enzyme activities were measured in lysates suspended in potassium

phosphate or TrisHCl buffer for the GSSG and Fdx assays, respec-

tively. Five rates were used for each inhibition plot. Errors were

determined from the best-fitted line in the inhibition plot.

Fig. 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 lM, 4 mM and 0.17 mg ml�1 for the susceptible

lysates and 30 lM, 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.

N.O. Kaakoush, G.L. Mendz / FEMS Immunology and Medical Microbiology 44 (2005) 137–142 141

in susceptible strains [5,13,16–21]. Similarly, it was dem-

onstrated 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 fac-

tors contributing to the resistant phenotype. For exam-

ple, 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 observa-tions that resistance to Mtr can be overcome by expos-

ing 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 oxidore-

ductase, a-ketoglutarate oxidoreductase, and pyru-

vate:flavodoxin oxidoreductase were observed in theresistant 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 ofH. pylori

Mtr-susceptible strains and Mtr-resistant mutants dem-onstrated 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 sus-

ceptibility of H. pylori to Mtr was provided by the find-

ing that the alkyl hydroperoxide reductase activity of

Mtr-susceptible strains was absent in their Mtr-resistant

counterparts [12,27]. Thus, it became important toinvestigate the role of specific disulphide reduction activ-

ities 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 disul-

phide reduction competitively in each of the three activ-

ities, and the measured Ki of Mtr for the reduction ofGSSG or Cys-Cys indicated that the effects of Mtr were

stronger in susceptible strains than in resistant ones (Ta-

ble 2). The data demonstrated that Mtr modulated di-

rectly 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 reduc-

tases 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 resistantphenotype needs to be investigated further, as well as

putative molecular mechanisms relating these disulphide

reductases to the activities of RdxA and FrxA, in partic-

ular 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 alsocontribute to the development of Mtr resistance in, for

instance, Campylobacter spp. and various parasites.

142 N.O. Kaakoush, G.L. Mendz / FEMS Immunology and Medical Microbiology 44 (2005) 137–142

Acknowledgement

This work was made possible by the support of the

Australian Research Council.

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