Department of Parasitology, Faculty of Science, Charles University in Prague, Vininá 7, 128 44 Prague 2, Czech Republic1
Author for correspondence: Jaroslav Kulda. Tel: +42 0 2 2195 3206. Fax: +42 0 2 2491 9704. e-mail: kulda{at}natur.cuni.cz
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ABSTRACT |
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Keywords: drug resistance, hydrogenosome, pyruvate:ferredoxin oxidoreductase, ferredoxin, hydrogenosomal malic enzyme
Abbreviations: EPR, electron paramagnetic resonance; LDH, lactate dehydrogenase; ME, malic enzyme; MLC, minimal lethal concentration; NADH:FOR, NADH:ferredoxin oxidoreductase; PFOR, pyruvate:ferredoxin oxidoreductase; STK, succinyl thiokinase
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INTRODUCTION |
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Resistance to metronidazole and related 5-nitroimidazole drugs has been demonstrated both in field isolates of Trichomonas vaginalis from patients refractory to treatment (Kulda et al., 1982 ; Lossick et al., 1986
; Meingassner & Thurner, 1979
; Meri et al., 2000
; Müller et al., 1980
) and in laboratory-developed strains obtained by exposing trichomonads to sublethal pressure of the drug either in vitro (Brown et al., 1999
; Kulda et al., 1984
, 1993
; Tachezy et al., 1993
) or in vivo (de Carneri et al., 1969
; Meingassner et al., 1978
). Two distinct types of resistance have been recognized and named aerobic and anaerobic according to conditions required for demonstration of resistance by susceptibility assays in vitro (Kulda, 1999
). The aerobic resistance typically occurs in isolates from treatment-refractory patients. It is manifested only if some oxygen is present. Under anaerobiosis the resistant organisms succumb to the drug, as they possess a functional drug-activating pathway. The resistance apparently results from defective oxygen scavenging and subsequent interference of intracellular oxygen with the drug activation (Ellis et al., 1992
; Rasoloson et al., 2001
; Yarlett et al., 1986a
). Alternatively, defective redox properties of ferredoxin or decreased ferredoxin levels (Yarlett et al., 1986b
) due to altered transcription of the ferredoxin gene (Quon et al., 1992
) have been proposed as potential mechanisms. However, a causal relationship of these alterations with the aerobically resistant phenotype has not been convincingly demonstrated. The anaerobic resistance resides in elimination of pathways responsible for the reductive activation of the drug. So far it has been demonstrated only in laboratory-developed strains (Brown et al., 1999
; Kulda et al., 1984
, 1993
). It is detectable under anaerobic conditions and characterized by very high minimum lethal concentration (MLC) values in vitro (over 1000 µg metronidazole ml-1). This type of resistance has been demonstrated in Tritrichomonas foetus (
erkasovová et al., 1984
; Kulda et al., 1984
). The loss of PFOR activity holds for a key attribute of the anaerobic resistance. As shown by monitoring of the in vitro development of resistance in a Tritrichomonas foetus clone (Kabí
ková et al., 1987
), the increasing resistance was strictly paralleled by the decrease of PFOR activity and uptake of the radiolabelled drug (Kulda et al., 1989
), indicating the progressive failure of the parasite to metabolize the drug. Surprisingly, preliminary experiments with Trichomonas vaginalis (
erkasovová et al., 1987
) suggested that the development of anaerobic resistance in the human trichomonad does not follow the expected pattern. In vitro-obtained lines deficient in PFOR activity showed only low levels of anaerobic resistance to metronidazole, thus indicating involvement of an additional pathway which can provide electrons for the drug reduction.
In this study we report on changes accompanying development of resistance to metronidazole in a Trichomonas vaginalis strain exposed to increasing pressure of the drug in vitro. We monitored enzyme activities and metabolic end products of trichomonads at various stages of resistance development and investigated their correlation with the resistance phenotype of parasites. In a further step we followed expression of key hydrogenosomal proteins involved in drug activation and resistance, and tried to reveal by nucleic acid analysis at which level their expression is regulated. Preliminary results were reported at the following meetings: 2nd European Congress of Protistology, Clermont-Ferrand, France, 1995; 10th International Congress of Protozoology, Sydney, Australia, 1997; 3rd COST-B9 Meeting on Antiprotozoal Chemotherapy, Bruges, Belgium, 2000; and in a review (Kulda, 1999 ).
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METHODS |
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Susceptibility assays.
Susceptibility of trichomonads to metronidazole was determined in vitro by using a microtitre plate assay as described previously (Tachezy et al., 1993 ). Trichomonads suspended in TYM medium without agar were exposed to twofold serial dilutions of metronidazole ranging from 1600 to 0·78 µg ml-1 and incubated at 37 °C in air in a humid chamber (aerobic test) or in an anaerobic jar containing H2 with 5% CO2 (anaerobic test). Aerobic assays with strains which did not tolerate exposure to air were done under an atmosphere containing 5% O2 in a mixture of 5% CO2 and 95% N2. After 48 h incubation, the plates were examined with an inverted microscope to check for motility of trichomonads. MLC was defined as the lowest concentration of the drug at which no motile trichomonads were observed. The end points were confirmed by failure of non-motile trichomonads to grow after reinoculation into drug-free medium.
Cell fractionation.
The cells were harvested by centrifugation, washed three times in PBS (pH 7·4) and resuspended in the isolation medium (erkasov et al., 1978
) supplemented with leupeptin (10 µg ml-1), tosyl-lysine chloromethyl ketone (1 mM) and dithiothreitol (10 mM). The cell suspension was homogenized by sonication at 10 W for 3 cycles of 5x1 s on ice. Cytosolic and large-granule fractions rich in hydrogenosomes were isolated from the cell homogenate by differential centrifugation as described by
erkasov et al. (1978)
. For some experiments hydrogenosomes were purified on a Percoll gradient according to Lahti et al. (1992)
. The intactness of hydrogenosomes was assesed by measurement of at least 90% ME latency (Drmota et al., 1996
) immediately after the isolation of the organelles.
Enzyme assays.
Enzyme activities were assayed spectrophotometrically at 25 °C under aerobic conditions or in anaerobic cuvettes under an atmosphere of H2 or N2. Certified oxygen-free nitrogen (99·999% purity) passed through an Oxiclear column (Labclear) was employed. The large-granule fraction rich in hydrogenosomes was used for monitoring activities of the hydrogenosomal enzymes PFOR, hydrogenase, NADH:FOR and NAD+-dependent ME. Enzymic activities of lactate dehydrogenase (LDH), pyruvate kinase and NADP+-dependent ME were measured in the cytosolic fraction. Activities of PFOR, hydrogenase and NADH:FOR were determined under anaerobiosis at 600 nm as the rate of reduction of methyl viologen. The assays were performed using pyruvate and H2 as substrates for PFOR and hydrogenase, respectively (Kabíková et al., 1987
); NADH served as substrate for NADH:FOR (Thong & Coombs, 1987
). The activities of MEs were determined under aerobic conditions at 340 nm as the rate of NAD+ and NADP+ reduction in the presence of malate for the hydrogenosomal or cytosolic enzyme, respectively (Drmota et al., 1996
). Activity of LDH was determined at 340 nm as the rate of NADH oxidation (Bergmeyer, 1963
). Activity of pyruvate kinase was monitored by the formation of ATP, using phosphopyruvate as a substrate (Mertens et al., 1992
). Proteins were determined according to the Lowry method.
Determination of metabolic end products.
Metabolic end products were determined by HPLC and GC. Trichomonads were resuspended in a simple isotonic medium (Doran, 1959 ), adjusted to 5x107 cells ml-1 and placed in 1 ml aliquots into 3 ml vials tightly closed with vaccine stoppers. The vials were flushed with nitrogen and incubated for 30 min at 37 °C. After incubation, cells were pelleted by centrifugation, the supernatant was filtered through Whatman membrane (PVDF 0·45 µm) and the metabolic products were determined by using a PL Hi-Plex H column conditioned with 5 mM H2SO4 and thermostatted at 65 °C. Organic acids and alcohols were eluted by isocratic flow (0·6 ml min1) by the same solution and detected by UV at 205 nm (ECOM LCD 2082) and refractometry (Schodx Ri-71). The Chromatography Station for Windows (CSW) program was used for calculation of the product concentrations, according to calibration against standards. Glycerol concentration was calculated indirectly as the difference between refraction index and UV detector signals. Hydrogen production was determined by GC. Trichomonads were incubated as above for 60 min, then the gas phase was analysed. The analysis was performed at room temperature using a molecular sieve 5A column with nitrogen as carrier gas. Hydrogen was detected using a thermal conductivity detector with thermistors (Carlo Erba Fractolab-C). Each experiment was run in triplicate.
EPR spectroscopy.
EPR spectroscopy was used for detection of nitro anion radicals released during activation of metronidazole by trichomonads. The cells were washed three times in 100 mM phosphate buffer, pH 7·5, and resuspended in the same buffer with 40 mM glucose to obtain a suspension with a density of 4x108 cells ml-1. The suspension was flushed with nitrogen to maintain anaerobic conditions. After the addition of 12 mM metronidazole, 150 µl of the suspension was immediately drawn into the EPR spectrometer cavity to measure the spectrum. Spectra were recorded on a Bruker ESP 300 (Bruker Spectrospin) at 20 °C using a quartz flat cellar liquid sample holder.
SDS-PAGE and Western blot analysis.
Percoll-purified hydrogenosomes were used for the analysis of hydrogenosomal proteins. SDS-PAGE was performed on a Bio-Rad minislab gel apparatus using a 12 or 18% separating gel and a 5% stacking gel. Resolved proteins were stained by silver (Swain & Ross, 1995 ) or blotted onto nitrocellulose membrane using a semi-dry transfer unit. The blots were allowed to react with polyclonal rabbit antibodies raised against hydrogenosomal ferredoxin, the
-subunit of succinyl thiokinase (STK) (both provided by Patricia Johnson, UCLA, USA) and ME (Drmota et al., 1996
), and an mAb against Trichomonas vaginalis PFOR (provided by Guy Brugerolle, University of Clermont Ferrand, France).
Preparation of DNA probes.
Genomic DNA of Trichomonas vaginalis isolated by a modified guanidium thiocyanate method (Bowtell, 1987 ; Wang & Wang, 1985a
) was used as a template in a PCR amplification of specific DNA probes. The following primers were used: ß-tubulin forward 5'-CATCGTCCCATCTCCAAAGG-3', reverse 5'-AATGGAACAAGGTTGACAGC-3'; ME forward 5'-AGGAAGAACGTGACCGCC-3', reverse 5'-GTTGCCGATATCGTGGTC-3'; PFOR forward 5'-GAYGGHACHGTNGGHGC-3', reverse 5'-TCRWADGCCCARCCRTC-3'; 16S rRNA forward 5'-GGTGGTGCATGGCCG-3', reverse 5'-GTAGGTGAACCTGCAGAAGGATCA-3'. The PCR products were purified on agarose gels followed by phenol/chloroform extraction. For the Northern blot analysis the PCR products were labelled by [
-32P]dATP using the Random Primed DNA Labelling Kit (Boehringer). In a nuclear run-on assay, PCR products were inserted into a pGEM-T vector (TA cloning kit; Stratagene) and immobilized on nitrocellulose as a probe. Nucleotide sequences of all PCR products were verified by sequencing.
Isolation of RNA and Northern blot analysis.
Total RNA was isolated by a modified guanidium thiocyanate procedure (Wang & Wang, 1985b ). Total RNA (0·5 µg) was size-fractionated on a 1·2% agarose/2·2 M formaldehyde gel and transferred to a nylon membrane (Hybond-N; Amersham). Blots were hybridized with radiolabelled DNA probes for ME and PFOR and subsequently washed as described by Johnson et al. (1990)
. Subsequently, the membranes were stripped down and rehybridized with DNA probe for ß-tubulin as a control.
Nuclear run-on assay.
Synthesis of nascent mRNA was investigated by the permeable cells technique (Vaá
ová et al., 2001a
). Briefly, trichomonads were permeabilized with lysolecithin and incubated in presence of [
-32P]UTP. Nascent RNA was isolated by TRIzol extraction (Gibco-BRL) and 32P-labelled transcripts were hybridized to DNA-specific probes immobilized on nitrocellulose. To assure standard conditions DNA concentrations of all probes were determined spectrophotometrically and an equal amount of each probe was applied onto nitrocellulose using a Bio-Rad filtration apparatus. Probes for 16S rRNA and tubulin were used as positive controls; the probe for the pGEM vector, employed for subcloning of all probes, served as a negative control. Blots were analysed using storage phosphor autoradiography (PhosphorImager SI; Molecular Dynamics).
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RESULTS |
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In the MR-5 strain, representing the early stage of anaerobic resistance, no activity of PFOR was detected and the activity of hydrogenase decreased profoundly (14% of that found in the parent strain). Activities of ME and NADH:FOR decreased only slightly when compared with the aerobic stage (MR-3). The activity of hydrogenase continued to decline with increasing anaerobic resistance and was completely lost at the stage represented by the MR-50 strain. A marked decrease of ME activity was determined in the MR-30 strain (less than 2% of parent activity), while NADH:FOR activity still amounted to about 26% of that of the parent in the MR-50 strain. Both activities were undetectable in the MR-100 strain with fully developed anaerobic resistance (limits of detection, 0·5 nmol).
Cytoplasmic enzyme activities
The activities of three cytoplasmic enzymes, LDH, pyruvate kinase and NADP+-dependent ME, determined in the drug-susceptible strain TV 10-02 and its derivatives at different levels of resistance development, are listed in Table 4. The data show a progressive increase in LDH activity during development of anaerobic resistance, up to values exceeding those in the parent drug-susceptible strains sevenfold. The activities of pyruvate kinase increased in the resistant strains about threefold on average. The resistant strains also showed slightly increased activities of the cytosolic ME. Both activities showed minor fluctuations between the individual drug-resistant strains with a peak at the stage of early anaerobic resistance (MR-5).
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DISCUSSION |
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Aerobic resistance appeared as the earliest stage of resistance development (MR-3). The properties of in vitro-developed strains are similar to those of clinical isolates obtained from treatment of refractory patients. Both contain PFOR activity and are susceptible to the drug under anaerobic conditions. Their resistance is detectable in mice, but in vitro only if some oxygen is present (Tachezy et al., 1993 ). The strain developed in this study (MR-3) showed considerably lower activities of hydrogenosomal enzymes, except hydrogenase, in comparison with those of the parent strain. However, the pathway activating metronidazole was functional as shown by the presence of nitro free radical signals in EPR spectra and by susceptibility of the organisms to metronidazole under anaerobic conditions. The ferredoxin of this strain was expressed at a similar level as in the parent strain (Fig. 1
). Moreover, FeS clusters of this strain and of examined clinical isolates displaying aerobic resistance were intact, as shown by characteristic signals in EPR spectra (Rasoloson et al., 2001
). Thus, the reports pointing to a low specific activity of hydrogenase (Ellis et al., 1992
) or ferredoxin insufficiency (Quon et al., 1992
; Yarlett et al., 1986b
) in some aerobically resistant isolates rather reflect individual strain variability than specific attributes of aerobic resistance.
The following stage, early anaerobic resistance (MR-5), is characterized by lack of PFOR activity. Surprisingly the drug resistance of these organisms remained relatively low (MLC 15·7 µg ml-1). The metronidazole radical signal, albeit of a lower intensity, was still apparent in EPR spectra if the trichomonads were exposed to metronidazole. The ability of PFOR-deficient trichomonads to activate metronidazole indicates involvement of an alternative pathway, providing electrons for reduction of the drug. Our results strongly suggest that the hydrogenosomal NAD+-dependent ME, catalysing oxidative decarboxylation of malate to pyruvate, performs this function. By the activity of NADH:FOR, reoxidizing NADH produced in this reaction, reduced equivalents can be transferred to ferredoxin to be available for drug activation. As is evident from data presented in this paper, trichomonads at the early stage of anaerobic resistance (MR-5) possess all components of this pathway. Gradual elimination of these proteins during further development of the anaerobic resistance stresses their causal relationship with metronidazole activation at the early PFOR-deficient stage (MR-5). Consequently, acquisition of fully developed anaerobic resistance in Trichomonas vaginalis (MR-100) requires elimination of both ferredoxin-linked electron-generating systems in hydrogenosomes.
The decrease or loss of hydrogenosomal metabolism in metronidazole-resistant strains is compensated by an increased rate of glycolysis (Kulda et al., 1989 ) and by enhancement of a cytoplasmic pathway metabolizing pyruvate (Kulda et al., 1989
; Kulda, 1999
). Metronidazole-resistant strains of both Trichomonas vaginalis and Tritrichomonas foetus tune their catabolic process to production of a single dominant end product. Our metabolic data showed a four- to fivefold increase in the activity of pyruvate kinase and confirmed marked enhancement of cytosolic lactate fermentation in the resistant strains. The elevated lactate production was underlined by a progressive increase in LDH activity. While lactate is the main glycolytic end product of metronidazole-resistant Trichomonas vaginalis, resistant Tritrichomonas foetus enhances fermentation to ethanol (
erkasovová et al., 1984
). A metabolic switch to cytoplasmic pyruvate metabolism is apparently a more general strategy used by trichomonads confronted with hydrogenosomal insufficiency. We have observed a similar phenomenon in Tritrichomonas foetus exposed to iron-restricted conditions (Va
á
ová et al., 2001b
).
Some metabolic end products of trichomonads developing anaerobic resistance do not seem to be consistent with the lack of PFOR activity, but alternative metabolic processes can explain their production. Hydrogen production at the early stage of anaerobic resistance (MR-5) is apparently due to the activity of the ME pathway. It is likely that in the absence of the drug, electrons are transported by ferredoxin to hydrogenase that is still active at this stage. Accordingly, production of hydrogen is stopped at the more advanced stage (MR-30) deficient in ferredoxin. Production of acetate detected at early (MR-5) up to more advanced stages of resistance development (MR-30, MR-50) can be accounted for by the activity of alternative 2-ketoacid oxidoreductases reported by Brown et al. (1999) that are active in metronidazole-resistant Trichomonas vaginalis and do not require ferredoxin as electron acceptor. However, the fate of these activities during development of resistance has not been followed in this study.
To examine at which level down-regulation of major hydrogenosomal activities associated with drug resistance occurs, we followed changes in protein and mRNA levels by Western and Northern blotting and examined transcription of genes for PFOR and hydrogenosomal ME by nuclear run-on assay. We found that the decrease or loss of enzyme activities is due to decreased expression of pertinent proteins. We also observed loss of ferredoxin at more advanced stages of anaerobic resistance (MR-30 and higher). In contrast, the protein levels of STK, the hydrogenosomal enzyme uncommitted in metronidazole activation, remained unaffected. In parallel to the decreased protein levels, steady-state levels of PFOR and ME mRNAs decreased with increasing resistance to the drug. The nuclear run-on assays monitoring synthesis of nascent mRNA revealed that the loss of PFOR results from down-regulation of gene transcription. In contrast, transcription of the genes encoding ME was not markedly changed. Thus the expression of ME appears to be regulated at different levels, most probably at the level of mRNA stability. Whilst this manuscript was in preparation for publication, Land et al. (2001) reported decreased transcription of PFOR and loss of other hydrogenosomal proteins associated with metronidazole activation in a drug-resistant Tritrichomonas foetus. Down-regulation of PFOR and ferredoxin in strains induced for anaerobic resistance has also been observed in another Trichomonas vaginalis strain (Brown et al., 1999
) and in Giardia (Liu et al., 2000
), and apparently it is a common feature of high-level resistance to the drug. As shown in this paper, acquisition of anaerobic resistance in Trichomonas vaginalis involves in addition down-regulation of the hydrogenosomal ME.
Gradual build up of metronidazole resistance through the sequence of stages characterized here suggests that metronidazole resistance is based on stepwise accumulation of mutations that affect expression of genes for hydrogenosomal proteins involved in drug activation. Up-regulation of cytosolic pathways of pyruvate metabolism that accompany the development of resistance may be attributed to metabolic feedback mechanisms as has been observed in hydrogenosomal deficiencies of different origin (Vaá
ová et al., 2001b
). Analysis of regulatory sequences (Liston et al., 1999
; Liston & Johnson, 1999
; Quon et al., 1994
) of genes for proteins involved in metronidazole resistance may bring further insight into the molecular mechanisms of this process. Work along these lines is in progress.
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ACKNOWLEDGEMENTS |
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Received 20 December 2001;
revised 15 April 2002;
accepted 2 May 2002.