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Antimicrobial Agents and Chemotherapy, June 2004, p . 2185-2189, Vol . 48, No . 6

Pyruvate Decarboxylase, the Target for Omeprazole in Metronidazole-Resistant and Iron-Restricted Tritrichomonas foetus

Róbert Sutak, Jan Tachezy, Jaroslav Kulda, and Ivan Hrdy*

Department of Parasitology, Charles University, Prague, Czech Republic

Received 1 August 2003/ Returned for modification 12 November 2003/ Accepted 10 February 2004


   ABSTRACT

 
The substituted benzimidazole omeprazole, used for the treatment of human peptic ulcer disease, inhibits the growth of the metronidazole-resistant bovine pathogen Tritrichomonas foetus in vitro (MIC at which the growth of parasite cultures is inhibited by 50%, 22 µg/ml [63 µM]) . The antitrichomonad activity appears to be due to the inhibition of pyruvate decarboxylase (PDC), which is the key enzyme responsible for ethanol production and which is strongly upregulated in metronidazole-resistant trichomonads . PDC was purified to homogeneity from the cytosol of metronidazole-resistant strain . The tetrameric enzyme of 60-kDa subunits is inhibited by omeprazole (50% inhibitory concentration, 16 µg/ml) . Metronidazole-susceptible T . foetus, which expresses very little PDC, is only slightly affected . Omeprazole has the same inhibitory effect on T . foetus cells grown under iron-limited conditions . Similarly to metronidazole-resistant cells, T . foetus cells grown under iron-limited conditions have nonfunctional hydrogenosomal metabolism and rely on cytosolic PDC-mediated ethanol fermentation .


   INTRODUCTION

 
Tritrichomonas foetus is a parasitic flagellate that causes sexually transmitted diseases in cattle that can lead to abortion and infertility . For decades the drugs of choice for the treatment of urogenital trichomoniasis were 5-nitroimidazoles, such as metronidazole (Fig . 1), dimetridazole, and related derivatives, until they were subjected to regulatory actions in the United States, the European Union, and some other countries . The antitrichomonad activity of metronidazole depends upon reduction of its nitro group to short-lived free radicals, which cause multiple forms of cellular damage, including DNA breakage and subsequent cell death . Reductive activation of the drug proceeds in typical trichomonad organelles called hydrogenosomes, whose main function is a substrate-level synthesis of ATP linked to the production of molecular hydrogen . Electrons required for metronidazole reduction are generated by pyruvate:ferredoxin oxidoreductase, the key hydrogenosomal enzyme catalyzing oxidative decarboxylation of pyruvate (6, 16, 18) .


FIG . 1 . Structures of thiamine (a), omeprazole (b), and metronidazole (c).

 
Prolonged cultivation of trichomonads with sublethal concentrations of metronidazole can result in the development of stable drug resistance accompanied by the loss of pyruvate:ferredoxin oxidoreductase activity (14, 15) . Metronidazole-resistant trichomonads show an altered carbohydrate metabolism . The pyruvate-oxidizing pathway in the hydrogenosomes disappears, and the loss of ATP is compensated for by an increased rate of glycolysis in the cytosol . The dominant end product of glucose breakdown becomes ethanol, the production of which is negligible in metronidazole-susceptible trichomonads . The ethanol production depends on the activities of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (2) . While the activity of the latter enzyme is not markedly changed in metronidazole-resistant strains, the activity of PDC is strongly upregulated, suggesting that PDC is the rate-limiting enzyme in the production of ethanol (2, 15) .

Metabolic changes similar to those accompanying the development of metronidazole resistance were described in trichomonads cultivated under iron-restricted conditions . Hydrogenosomal metabolism depends on iron-containing proteins, and a lack of iron in the medium results in the cessation of pyruvate breakdown in the organelles, while the activity of PDC in the cytosol markedly increases (25) . Thus, the dominant role of PDC in the metabolism of both metronidazole-resistant and iron-restricted T . foetus cells qualifies this enzyme as a suitable target for chemotherapeutic intervention .

The substituted benzimidazole omeprazole (Fig . 1) is an antiulcer proton pump inhibitor which acts on gastric H+,K+ ATPase (7) . Furthermore, omeprazole has specific activity against Helicobacter pylori in vitro (9) and is used in combination with antibiotics to treat H . pylori infections . However, the mechanism behind its activity against Helicobacter is not understood, but it is not due to the inhibition of ATPase activity (1) . Omeprazole is also effective in killing promastigotes as well as intracellular amastigotes of Leishmania donovani (10), with the likely target being the H+,K+ ATPase on the plasma membrane of the parasite .

In this work we report on the purification and characterization of the PDC from metronidazole-resistant T . foetus and demonstrate its inhibition with omeprazole . We further demonstrate the actions of omeprazole against both metronidazole-resistant and iron-restricted T . foetus cells in vitro .


   MATERIALS AND METHODS

 
Organism and cultivation. T . foetus strain Lub-1 MIP and its metronidazole-resistant derivative, Lub-MR 100, which displays stable anaerobic resistance (23, 24), were maintained in Trypticase-yeast extract-maltose (TYM) medium (pH 7.2) supplemented with 10% heat-inactivated horse serum (5) . Iron-restricted organisms were obtained by maintaining metronidazole-sensitive trichomonads in TYM medium containing 180 µM 2,2'-dipyridyl (Sigma) for 10 passages .

Enzyme assay. PDC activity was determined spectrophotometrically at 340 nm as the rate of acetaldehyde formation detected as the oxidation of NADH in a coupled reaction with yeast alcohol dehydrogenase (approximately 10 IU/ml) (8) in 50 mM morpholineethanesulfonic acid (MES; pH 6.2)-30 mM pyruvate-0.11 mM NADH at 25°C . The assay mixture also contained known cofactors of PDC: 5 mM Mg2+ (as MgCl2) and 0.5 mM thiamine pyrophosphate . The molar extinction coefficient ({varepsilon}340) of NADH was taken as 6,220 M–1 cm–1 . For inhibition experiments, the purified enzyme in a complete reaction mixture without pyruvate was preincubated with various concentrations of omeprazole (catalog no . O-104; Sigma) for 15 min at 25°C . The reaction was initiated by addition of pyruvate . Interference of omeprazole with auxiliary alcohol dehydrogenase was excluded by preincubation of alcohol dehydrogenase with 50 µg of omeprazole per ml for 15 min . To exclude the interfering activity of NADH oxidase, the PDC activities in crude cell extracts were measured under anaerobic conditions . One unit of enzyme activity was defined as the amount of protein catalyzing the decarboxylation of 1 µmol of pyruvate per min . The activities and the Km calculations were based on at least three determinations and are expressed as means ± standard deviations .

Enzyme purification. Metronidazole-resistant T . foetus cells (Lub-MR 100) in the late logarithmic phase of growth (4.5 x 106 cells/ml, 3 liters of culture) were harvested by centrifugation, washed with ST buffer (250 mM sucrose, 0.5 mM KCl, 10 mM Tris-HCl [pH 7.2]), and suspended in ST buffer with 50 µg of N{alpha}-tosyl-L-lysine chloromethyl ketone per ml and 10 µg of leupeptin per ml . The cells were disrupted by sonication, and the cytosolic fraction was obtained by centrifugation at 150,000 x g for 30 min . Ammonium sulfate was added to the supernatant at a final concentration of 1.5 M, and the solution was gently stirred on ice for 15 min, followed by centrifugation at 20,000 x g for 10 min . Liquid chromatography was performed at room temperature with a medium-pressure BioLogic HR system (Bio-Rad) . All buffers used in the chromatographic steps contained 5 mM MgCl2 and 0.5 mM thiamine PPi . After ammonium sulfate precipitation the supernatant was loaded onto a Macro-Prep butyl HIC column (Bio-Rad) equilibrated with 1.5 M ammonium sulfate-20 mM MES (pH 6.2) . Adsorbed proteins were eluted with a decreasing ammonium sulfate gradient (1.5 to 0 M) . Active fractions were pooled and loaded onto a Bio-Scale CHT 5-I hydroxyapatite column (Bio-Rad) equilibrated with 15 mM Na/K phosphate buffer (pH 6.2) . The column was washed with 1 M NaCl, and the enzyme was eluted with a linear gradient of phosphate (0.015 to 0.5 M) . The fractions containing enzyme activity were pooled and concentrated, and the buffer was changed to 20 mM Tris (pH 7.2) . The sample was injected onto an UNO Q1 column (Bio-Rad) equilibrated with the same buffer . Proteins were eluted with a linear gradient of NaCl (0 to 0.7 M) . The PDC-containing fraction was injected onto a Bio-Prep SE-1000/17 column (Bio-Rad) equilibrated with 20 mM MES (pH 6.2) . PDC was eluted with the same buffer as a single peak .

Susceptibility assays. To evaluate the susceptibilities of trichomonads to omeprazole, the MICs at which 50% growth inhibition of parasite cultures occur (MIC50s) were determined by a microtiter plate assay under anaerobic conditions in 96-well round-bottom plates . Two strains were tested on a single plate, with each strain tested in five experimental rows and one control row . To prepare a stock solution of the drug, omeprazole was dissolved in 50 mM NaOH and was immediately diluted 10 times with TYM medium to a concentration of 500 µg/ml . The TYM medium was added in 50-µl aliquots to 72 of 96 wells (rows A to F) . Then, the stock solution of omeprazole was added in 50-µl aliquots to wells 1 to 5 and 7 to 11 of row A, while wells 6 and 12 received 50 µl of the control solution (TYM with 5 mM NaOH) . Five twofold serial dilutions were made starting from row A . Finally, 150 µl of the trichomonad suspension in TYM medium (5 x 104 cells/ml) was added to each well of a pertinent strain sector . The resulting drug concentrations ranged from 100 to 6.25 µg/ml . The plates were incubated for 48 h in an anaerobic jar with an Anaerobic System BR 38 (Oxoid) to generate an anaerobic atmosphere . After incubation, the cell density was determined in five wells for each drug concentration by counting with a hemacytometer, and the counts were compared to those in the control row . The MIC50 was defined as the lowest concentration of omeprazole at which the cell density decreased to 50% or more of that of the control . The results are based on two independent experiments, each run in duplicate for each strain, and are expressed as mean ± standard deviation of the mean MIC50 .

The susceptibilities of the trichomonads to metronidazole were determined in the same assay system, with minor modifications: each strain was tested in a separate plate, and the twofold dilution series was extended (rows 1 to 12) to obtain final drug concentrations ranging from 200 to 0.098 µg/ml for the metronidazole-resistant strain (Lub-MR 100) and from 50 to 0.024 µg/ml for the iron-restricted and metronidazole-susceptible parent strain (Lub-1 MIP) . The metronidazole (Sigma) stock solution was prepared by dissolving 6.4 or 3.2 mg of the drug per ml in distilled water by autoclaving and was further diluted with TYM medium .

Analytical methods. The chromatographic fractions were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) with Coomassie staining . The Mr value of the native protein was determined by gel filtration on a Sephacryl S 300 HR column (Amersham-Pharmacia Biotech) calibrated with thyreoglobulin (669 kDa), apoferritin (443 kDa), ß-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa) (all from Sigma) . Protein concentrations were determined by the method of Lowry .

Antibody production and immunoblotting. Antibodies against the purified PDC were raised in BALB/c mice . Each injection used 50 to 100 µg of PDC that has been excised from SDS-polyacrylamide gels and homogenized with phosphate-buffered saline . Two mice were injected intraperitoneally four times at 10-day intervals, and the antiserum was obtained 1 week after the fourth immunization . The cytosolic fractions of metronidazole-sensitive, metronidazole-resistant, and iron-restricted trichomonads were transblotted onto an Immobilon P membrane (Millipore), incubated with anti-PDC antiserum, and probed with anti-mouse immunoglobulin G peroxidase conjugate (Sigma) . The proteins were visualized by a West Pico enhanced chemiluminescence assay (Pierce) .


   RESULTS

 
Enzyme purification. In order to investigate the properties of the T . foetus PDC, the enzyme was purified to apparent homogeneity from the cytosolic fraction of the metronidazole-resistant strain by ammonium sulfate precipitation and hydrophobic, anion-exchange, and gel filtration chromatography . The purification procedure is summarized in Table 1 . The purification scheme resulted in a 70-fold purification . Approximately 6.5% of the PDC activity present in the trichomonad cytosol was recovered . SDS-PAGE revealed a single protein band with an apparent molecular mass of 60 kDa (Fig . 2A) . These results indicate that PDC represents as much as about 1.4% of cytosolic proteins in metronidazole-resistant T . foetus .


TABLE 1 . Purification scheme for PDC from metronidazole-resistant T . foetus

 

FIG . 2 . (A) SDS-PAGE of PDC fractions during purification steps from metronidazole-resistant T . foetus cytosol . Lanes: 1, molecular weight standards; 2, cytosol; 3, fractions after butyl HIC column chromatography; 4, fractions obtained after hydroxyapatite column chromatography; 5, fractions obtained after UNO Q 1 column chromatography; 6, fractions obtained after Bio-Prep 1000 column chromatography . Proteins were stained with Coomassie brilliant blue . (B) Immunodetection of PDC in the cytosolic fractions of T . foetus . Lanes: 1, metronidazole-resistant strain; 2, metronidazole-sensitive strain; 3, metronidazole-sensitive strain grown under iron-limited conditions.

 
Biochemical and kinetic properties of PDC. The molecular mass of the native enzyme determined by gel filtration chromatography was about 240 kDa, suggesting that the native enzyme is a homotetramer .

The pH profile of PDC activity showed a relatively narrow optimum in the range of pH 5.75 to 6.5 . The Km for pyruvate was 0.62 ± 0.08 mM . In the presence of 100 mM NaCl, the Km for pyruvate increased to 1.25 ± 0.15 mM, indicating the inhibitory effect of Cl– . A similar effect of Cl– was observed with PDC from Oryza sativa (21) .

Comparison of PDC activity in metronidazole-sensitive, metronidazole-resistant, and iron-restricted strains. The activities of PDC in the cytosols of metronidazole-sensitive, metronidazole-resistant, and iron-restricted cells were compared . In the resistant and iron-restricted trichomonads, the specific activities of PDC were more than 30 and 20 times higher, respectively, in comparison with that in the metronidazole-sensitive strain (specific activities, 76.2 ± 3.9, 52.0 ± 5.6, and 2.06 ± 0.17 mU/mg in the cytosolic fractions of metronidazole-resistant, iron-restricted, and metronidazole-sensitive strains, respectively) . Accordingly, immunodetection of PDC on the Western blot demonstrated the overexpression of PDC in metronidazole-resistant as well as iron-restricted T . foetus (Fig . 2B) .

Inhibition of PDC by omeprazole. To investigate the inhibitory effect of omeprazole on T . foetus PDC activity, the purified enzyme in a complete assay mixture without pyruvate was preincubated for 15 min with various concentrations of the drug, and then the activity was measured by starting the reaction with pyruvate . The concentration of omeprazole required for 50% inhibition of PDC activity (IC50) was 16 ± 2 µg/ml (Fig . 3) . Since the PDC assay is based on monitoring of the consumption of acetaldehyde by alcohol dehydrogenase, we checked for the effect of omeprazole on this auxiliary enzyme . The inhibition was attributable solely to PDC, as omeprazole (50 µg/ml) had no effect on alcohol dehydrogenase .


FIG . 3 . Effect of omeprazole on activity of purified PDC from metronidazole-resistant T . foetus . The data represent the means ± standard deviations of five measurements.

 
Antitrichomonad activity of omeprazole. We observed the antiparasitic effects of omeprazole on both metronidazole-resistant and iron-restricted T . foetus cells in vitro . In the microtiter plate assay, the mean MIC50s observed were 22 ± 2.4 and 7.4 ± 1.7 µg/ml for metronidazole-resistant and iron-restricted trichomonads, respectively . For the metronidazole-sensitive strain, against which the activity of PDC as well as in which the level of expression of PDC is very low (Fig . 2B), the MIC50 surpassed 100 µg/ml (the highest omeprazole concentration used in the assay) .

Susceptibility to metronidazole. The susceptibilities of all three T . foetus strains used in this study to metronidazole were tested by the microtiter plate assay . As expected, parental strain Lub-1 MIP was sensitive to the drug, with a mean MIC50 of 0.052 ± 0.015 µg/ml, while the mean MIC50 for its resistant derivative, Lub-MR 100, was 66.7 ± 22.2 µg/ml . The iron-restricted trichomonads were fully susceptible to metronidazole, with a mean MIC50 of 0.026 ± 0.0036 µg/ml .


   DISCUSSION

 
In this report we describe the inhibitory effect of omeprazole on PDC from the bovine parasite T . foetus . We also show that in vitro omeprazole has antiparasitic effects against metronidazole-resistant trichomonads as well as against the trichomonads maintained under iron-depleted conditions, both of which are dependent on PDC-mediated ethanol fermentation . Wild-type trichomonads that express very little PDC were only slightly affected .

In order to verify the target of omeprazole, we have purified and partially characterized the PDC from metronidazole-resistant T . foetus . To our knowledge, this is the first PDC characterized from a protozoan . PDC is common in plants, yeasts, and fungi and also occurs in certain helminths; it is absent from higher animals and is rare in prokaryotes (12, 13) . The biochemical and kinetic properties (tetrameric structure, subunit size, pH optimum, and affinity for pyruvate) of the T . foetus PDC are similar to those of most PDCs purified from other organisms (12, 20) .

T . foetus PDC turned out to be quite potently inhibited by omeprazole, with IC50s of about 16 ± 2 µg/ml . The inhibition was most likely competitive due to the structural similarity of omeprazole to thiamine pyrophosphate, a cofactor of PDC and other enzymes, namely, decarboxylases and transketolases . Competitive inhibition by omeprazole has already been described for the PDC from a eubacterium, Zymomonas mobilis (19) .

In T . foetus, PDC seems to be the key cytosolic enzyme involved in the development of anaerobic resistance to metronidazole . It was shown that the activity of PDC increases about seven times in the fully resistant strain in comparison with that in the metronidazole-sensitive one, which is consistent with the fact that metronidazole-resistant trichomonads convert glucose almost completely to ethanol (2) . In this study we have used a different T . foetus strain (Lub-1 MIP) and its metronidazole-resistant derivative and detected even more profound differences in the PDC activities between the strains . PDC also appears to be indispensable for T . foetus cells cultivated under iron-restricted conditions . Similarly to metronidazole-resistant strains, trichomonads cultivated under iron-limited conditions downregulate the hydrogenosomal metabolism, which is dependent on iron-containing enzymes, and strongly upregulate the PDC-dependent pathway of ethanol formation in the cytosol (25) . In agreement with these observations, we confirmed the overexpression of PDC protein in metronidazole-resistant cells as well as iron-restricted cells by immunoblotting . In contrast, both the PDC activity and protein were barely detectable in the parent strain .

In addition to its inhibitory effect on PDC, omeprazole also has antitrichomonad activity in vitro . However, only the metronidazole-resistant trichomonads and trichomonads maintained under iron-restricted conditions were significantly affected by the drug, which is consistent with the role of PDC in these strains . Metronidazole-sensitive trichomonads that are not dependent on ethanol fermentation and that express very little PDC were not significantly inhibited in the presence of the highest omeprazole concentration used in the experiments . These data strongly suggest that the target of the antitrichomonad activity of omeprazole is PDC .

Interestingly, the cells grown under iron-limited conditions did not acquire metronidazole resistance, indicating that the pathway of metronidazole reductive activation is still functional in these cells . This is in agreement with our earlier work, in which we reported on the changes of pyruvate metabolism induced by iron limitation in the same T . foetus strains as those used in this study . In these cells, the activities of hydrogenosomal enzymes, namely, the drug-activating pyruvate:ferredoxin oxidoreductase, are present at low levels (25), which, however, are still sufficient to activate the drug (16) .

Our work demonstrates for the first time the antimicrobial activity of omeprazole due to the inhibition of an enzyme other than ATPase . Plasma membrane K+,H+ ATPase appears to be the target of omeprazole in L . donovani promastigotes and amastigotes, with the omeprazole mode of action presumably being similar to that against the gastric K+,H+ ATPase (10), in which protonation of omeprazole to sulfenamide at acidic pH is required for the inhibition of ATPase (17) . Omeprazole is also used in combination with antibiotics for the treatment of human infections caused by H . pylori; however, the drug target in H . pylori still remains to be elucidated .

PDC likely plays a role in the metabolism of various other pathogens . It was found in Candida glabrata (Q . Wang et al., unpublished data; GenBank accession number AF545432), and the PDC gene regulator was identified in Candida albicans (11) . The PDC gene was found in the genome of the intracellular human pathogen Mycoplasma penetrans (22), and the genomes of Mycobacterium leprae (4) and Mycobacterium tuberculosis (3) also contain the gene coding for pyruvate or indolepyruvate decarboxylase . Potent inhibitors of this enzyme, which is absent from mammals, could therefore prove to be useful against pathogens that are dependent on anaerobic pyruvate fermentation .

 


   ACKNOWLEDGMENTS

 
This work was performed in the framework of the COST B-9 program and was supported by grants OC.B9.10 and OC.B22.001, provided by the Ministry of Education of the Czech Republic, and by grant 204/00/156, provided by the Grant Agency of the Czech Republic .

We thank Michaela Marcinciková and Miroslava Sedinová for technical assistance .


   FOOTNOTES

 
* Corresponding author . Mailing address: Department of Parasitology, Charles University, Vinicná 7, 128 44 Prague 2, Czech Republic . Phone: 420 221951811 . Fax: 420 224919704 . E-mail: hrdy{at}cesnet.cz .


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