Department of Parasitology, Faculty of Science, Charles University, Vininá 7, Prague, 128 44, Czech Republic1
Author for correspondence: Jan Tachezy. Tel: +420 2 21953207. Fax: +420 2 24919704. e-mail: tachezy{at}natur.cuni.cz
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ABSTRACT |
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Keywords: Tritrichomonas foetus, iron, carbohydrate metabolism, hydrogenosome, transcription
Abbreviations: Fe-NTA, iron nitrilotriacetate; PFOR, pyruvate:ferredoxin oxidoreductase
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INTRODUCTION |
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The importance of FeS proteins in the carbohydrate metabolism of trichomonads (Gorrell et al., 1984 ; Marczak et al., 1983
; Müller, 1988
; Payne et al., 1993
) and other anaerobes (Ellis et al., 1993
; Weinbach et al., 1980
) may explain the high nutritional requirements of these organisms for iron. It has been shown that addition of 50100 µM Fe is required for optimal growth of Tt. foetus in vitro (Tachezy et al., 1996
). Not surprisingly, availability of iron in the cells environment affects metabolic activity of the hydrogenosome. Gorrell (1985)
showed that Trichomonas vaginalis had an increased EPR signal for ferredoxin, and also an increased activity of PFOR and hydrogenase if the parasite was grown in a medium with the iron concentration elevated to 200 µM. In Tt. foetus, we have demonstrated that activity of PFOR considerably decreased and activity of hydrogenase became undetectable in cells maintained in iron-restricted media in comparison with those grown in an excess of iron (Tachezy et al., 1996
). These phenomena could be explained either by a shortage of iron required for assembly of [FeS] clusters or by involvement of iron in mechanisms of hydrogenosomal protein expression, a process which is still poorly understood. The hydrogenosomes of trichomonads do not possess their own genome (Clemens & Johnson, 2000
). Thus the proteins operating in these organelles are encoded exclusively in the nucleus. Johnson et al. (1993)
showed that expression of hydrogenosomal proteins closely resembles that known for the nucleus-encoded proteins of mitochondria. It includes synthesis of nascent protein on free ribosomes (Lahti & Johnson, 1991
), protein targeting by means of an amino-terminal leader sequence, translocation to the organelle and intrahydrogenosomal maturation, including cleavage of the leader sequence (Bradley et al., 1997
). Whether and at which level (protein targeting, translation, mRNA stabilization, gene transcription) nutritional factors such as iron regulate expression of hydrogenosomal proteins has not been established.
In this paper we provide comparative biochemical data characterizing iron-dependent changes in metabolic fluxes in Tt. foetus, we show that iron is involved in expression of both FeS and non-FeS proteins operating in hydrogenosomes, and we demonstrate that a putative iron-dependent regulation of hydrogenosomal proteins takes place at the transcriptional level.
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METHODS |
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Cell fractionation.
Cytosolic and hydrogenosome-rich fractions used for enzymic studies were obtained by differential centrifugation according to Drmota et al. (1996) . To determine the activity of succinate thiokinase, the hydrogenosome-rich fraction was further subfractionated according to Jenkins et al. (1991)
with a few modifications. The fraction was resuspended in a buffer containing 0·1 M Tris/HCl, 0·175 M KCl and 100 µg leupeptin ml-1, pH 7·5, and sonicated at 10 W for 3 cycles of 5x1 s on ice. The sonicated fraction was centrifuged at 130000 g for 1 h and the supernatant was used for enzyme determinations.
Purified hydrogenosomes, used in the hydrogenosomal protein analysis, were obtained from the cell homogenate using a Percoll gradient according to Lahti et al. (1992) . At least 90% latency of malic enzyme as a hydrogenosomal marker was found immediately after isolation of the organelles.
Determination of enzyme activities.
All enzyme activities were assayed spectrophotometrically at 25 °C. Activities of PFOR (EC 1 . 2 . 7 . 1), hydrogenase (EC 1 . 18 . 99 . 1) and NADH:ferredoxin oxidoreductase (EC 1 . 18 . 1 . 3) were assayed as the rate of methyl viologen reduction monitored at 600 nm. The assays were performed under anaerobic conditions using pyruvate and H2 as substrates for PFOR and hydrogenase, respectively (Kabíková et al., 1988
), and NADH as substrate for NADH:ferredoxin oxidoreductase (Steinbuchel & Müller, 1986a
). Activities of adenylate kinase (EC 2 . 7 . 4 . 3) (Dinbergs & Lindmark, 1989
), malate dehydrogenase (EC 1 . 1 . 1 . 37) (Drmota et al., 1997
), pyruvate decarboxylase (EC 4 . 1 . 1 . 1) (
erkasovová et al., 1984
), succinate thiokinase (EC 6 . 2 . 1 . 5) (Cha & Parks, 1964
) and fumarate reductase (EC 1 . 3 . 1 . 6) were measured at 340 nm under anaerobic conditions as the rate of NADH oxidation. The assay mixture for measurement of fumarate reductase activity consisted of 100 mM sodium phosphate buffer pH 7·5, 10 mM DTT, 500 mM fumarate and 10 mM NADH. The activity of hydrogenosomal (EC 1 . 1 . 1 . 39) and cytoplasmic (EC 1 . 1 . 1 . 40) malic enzyme was measured aerobically at 340 nm as the rate of NAD+ and NADP+ reduction, respectively (Drmota et al., 1996
), whilst NADH oxidase (EC 1 . 6 . 99 . 3) was determined as the rate of NADH oxidation (Tanabe, 1979
).
Determination of metabolic end products.
To determine production of organic acids and alcohols, intact trichomonads were resuspended in Dorans medium (78 mM NaCl, 1·6 mM KCl, 0·6 mM CaCl2, 30 mM NaH2PO4, 15 mM maltose) to a density of 5x106 cells ml-1 and 1 ml aliquots were placed into 3 ml vials sealed with vaccine stoppers. The vials were then flushed with nitrogen and incubated at 37 °C for 30 min. After incubation, the cells were removed by centrifugation and the end products determined by HPLC using a PL Hi-Plex H column. HPLC analysis was performed at 65 °C; 5 mM H2SO4 was used as the mobile phase. Acetate and succinate were monitored spectrophotometrically at 205 nm, whilst ethanol and glycerol were followed by refractometry.
To determine hydrogen production, the trichomonads were incubated as above for 60 min and hydrogen concentrations measured in the gas phase using gas chromatography. The analysis was performed at room temperature using a molecular sieve 5A column connected to a thermistor detector; nitrogen was used as the carrier gas.
Time course experiment.
Trichomonads maintained for 10 passages in the presence of 180 µM 2,2-dipyridyl were resuspended in TYM or 50 mM PBS-maltose (137 mM NaCl, 2·7 mM KCl, 4·6 mM Na2HPO4, 1·5 mM KH2PO4 and 50 mM maltose, pH 7·2) supplemented with 150 µM Fe-NTA. Aliquots of 1 ml suspension containing 5x107 cells were immediately placed into 3 ml vials as above and incubated at 37 °C. Hydrogen production was determined by gas chromatography after various time intervals.
SDS-PAGE and Western blot analysis.
SDS-PAGE and Western blotting were used to analyse the protein composition of Percoll-purified hydrogenosomes. SDS-PAGE was performed on a Bio-Rad minislab gel apparatus using 12 or 18% gel, and 5% stacking gel. Electrophoretically resolved proteins were stained with silver (Swain & Ross, 1995 ) or transferred to nitrocellulose membrane to be probed with polyclonal rabbit antibodies against hydrogenosomal malic enzyme (Drmota et al., 1996
), ferredoxin and
-subunit of succinate thiokinase (the latter two antibodies were kindly provided by P. Johnson, UCLA, USA). Quantitative analysis was performed using ScanPack software (Biometra).
Amino acid sequencing.
Proteins were separated by SDS-PAGE, transblotted onto a PVDF membrane (Millipore) and stained with Coomassie brilliant blue. The bands of interest were excised and the proteins sequenced from their amino terminus by Edman degradation.
Nucleic acid isolation.
Genomic DNA and total RNA were isolated from Tt. foetus by modified guanidium thiocyanate methods as described by Bowtell (1987) and Wang & Wang (1985)
, respectively.
Northern blot analysis.
Total RNA was size-separated on 1·2% agarose/2 M formaldehyde gel and transferred to a nylon membrane (Hybond N; Amersham). Blots were hybridized with radiolabelled DNA probes for hydrogenosomal malic enzyme and PFOR. 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 means of a modified run-on assay according to Ullu & Tschudi (1990) . Briefly, trichomonads were washed and resuspended in transcription buffer (150 mM sucrose, 20 mM potassium glutamate, 3 mM MgCl2, 1 mM DTT, 10 µg leupeptin ml-1, 20 mM HEPES/Tris pH 7·9) and permeabilized in the presence of 1 mg lysolecithin ml-1. The suspension was mixed with the transcription mixture {4 mM ATP, 2 mM GTP, 2 mM CTP, 50 mM creatine phosphate, 1·2 mg creatine phosphokinase ml-1, [
-32P]UTP [3 kCi mmol-1 (111 TBq mmol-1; Amersham)], 50 mM potassium glutamate, 1·5 mM MgCl2, 5 mM DTT, 1 mM HEPES} and incubated for 20 min at 35 °C. Nascent RNA was isolated by TRIzol extraction according to the protocol of the manufacturer (Gibco-BRL). 32P-labelled transcripts were hybridized to DNA-specific probes immobilized on nitrocellulose. Blots were analysed using storage phosphor autoradiography (PhosphorImager SI; Molecular Dynamics) and signals quantified by means of Multi Analyst software (Bio-Rad).
Preparation of DNA probes.
All probes were prepared by PCR amplification using specific primer pairs (ß-tubulin: CAT CGT CCC ATC TCC AAA GG and AAT GGA ACA AGG TTG ACA GC; malic enzyme: AGG AAG AAC GTG ACC GCC and GTT GCC GAT ATC GTG GTC; PFOR: GAY GGH ACH GTN GGH GC and TCR WAD GCC CAR CCR TC; 16S rRNA: GGT GGT GCA TGG CCG and GTA GGT GAA CCT GCA GAA GGA TCA). For Northern blot analysis, PCR products were purified on agarose gels followed by phenol/chloroform extraction and labelled with [-32]dATP using a Random Primed DNA Labelling Kit (Boehringer Mannheim). In the run-on assay, PCR products were inserted into a pGEM vector (TA Cloning Kit; Stratagene) and immobilized on nitrocellulose as a probe. Nucleotide sequences of all PCR products were verified with an automatic ABI Prism 310 gene analyser (Perkin Elmer).
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RESULTS |
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Effect of iron on transcription of genes encoding PFOR and malic enzyme
Northern blot analysis was performed to determine whether chelator-treated cells possess reduced levels of steady-state mRNA encoding PFOR and hydrogenosomal malic enzyme. Consistent with the protein levels, reduced levels of PFOR and malic enzyme mRNA were found (Fig. 3). To exclude the possibility that iron starvation resulted in generalized reduction of mRNAs, the blots were stripped and rehybridized with a probe identifying ß-tubulin mRNA. A comparable signal for this mRNA was found in the organisms maintained under both iron-rich and iron-restricted conditions (Fig. 3
).
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DISCUSSION |
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Interestingly, malate-dependent formation of pyruvate in the cytosol became essential for carbohydrate metabolism of Tt. foetus maintained under iron-limited conditions (Fig. 5). In these cells, oxidative decarboxylation of malate to pyruvate catalysed by NADP-dependent malic enzyme is coupled to the conversion of pyruvate to acetaldehyde mediated by pyruvate decarboxylase. Subsequently, acetaldehyde is reduced to ethanol by alcohol dehydrogenase in a reaction accompanied by reoxidation of NADP, a critical step for maintenance of the redox balance. In addition, production of glycerol slightly increases in iron-restricted cells, which may further contribute to NADP reoxidation.
Iron-dependent modulation of cytosolic/hydrogenosomal carbon metabolism has been observed previously in T. vaginalis (Gorrell, 1985 ). Unlike Tt. foetus, the human parasite possesses a cytosolic pathway converting pyruvate to lactate, which is a major product of its carbohydrate metabolism. Gorrell (1985)
demonstrated that production of lactate decreases, whilst release of hydrogenosomal end products increases, if T. vaginalis is maintained in iron-enriched TYM medium.
It is noteworthy that changes in carbohydrate metabolism similar to those caused by iron-restricted conditions in Tt. foetus have been observed in trichomonads with in-vitro-induced resistance to metronidazole (erkasovová et al., 1984
; Kulda, 1999
). The mode of action of metronidazole, the major drug utilized for treatment of trichomoniasis, resides in its metabolic activation within hydrogenosomes (Müller, 1986
). Here, the drug is reduced by ferredoxin-donated electrons which are generated during oxidative decarboxylation of pyruvate or malate. In metronidazole-resistant strains, electrons required for drug activation are not generated as activities of the key enzymes PFOR, malic enzyme and hydrogenase disappear. As in the case of iron-restricted cells, metronidazole-resistant organisms compensate for impaired function of hydrogenosomes by increased rate of cytosolic fermentation to ethanol (
erkasovová et al., 1984
) or lactate (Kulda et al., 1993
). In the present experiments we showed that iron-dependent metabolic changes were fully reversible upon addition of excess iron. Similarly, changes in pyruvate metabolism in the early stages of drug resistance reverted if pressure of metronidazole was released (Kabí
ková et al., 1988
). In light of these observations it is tempting to speculate that mechanisms mediating iron-dependent changes in carbohydrate fluxes and those involved in development of metronidazole resistance in trichomonads may have a related molecular basis.
To determine at which level iron-dependent regulation of hydrogenosomal metabolism takes place, we compared cells of Tt. foetus grown under iron-rich and iron-restricted conditions with regard to (i) profiles of hydrogenosomal proteins, (ii) steady-state levels of mRNA encoding PFOR and hydrogenosomal malic enzyme, and (iii) synthesis of nascent mRNAs. Profound changes in protein composition were observed. The levels of two FeS proteins, PFOR and ferredoxin, as well as NAD-dependent malic enzyme (a non-FeS protein), were markedly decreased in chelator-treated cells. The decreased protein levels of both hydrogenosomal enzymes corresponded to decreased steady-state levels of pertinent mRNAs on the Northern blots. To distinguish whether the lowered steady-state mRNA levels were due to changes in mRNA stabilization or transcription we performed nuclear run-on assays. These experiments clearly showed that synthesis of nascent mRNA encoding PFOR and malic enzyme is lower in chelator-treated cells. These results indicate that the iron-dependent changes in activity of core metabolic pathways within hydrogenosomes are regulated at the transcriptional level.
Interestingly, Western blot analysis showed differences in the pattern of succinate thiokinase -subunit (
-STK) isoforms relative to availability of iron, although comparable enzyme activities were detected in cells grown under iron-rich and iron-restricted conditions. It has been shown in T. vaginalis that
-STK is encoded by three genes of identical length. However, three bands of different mobility have been recognized using anti-
-STK serum (Lahti et al., 1994
). The differences in protein mobility have been ascribed to different post-translational modifications or to conformational differences (Lahti et al., 1994
). In our experiments, presence of the lighter peptide (32 kDa) was iron-dependent, whilst the heavier peptide (38 kDa) was produced under both iron-restricted and iron-rich conditions. Alderete et al. (1998)
identified three T. vaginalis genes with either identical (AP51-2) or highly similar (AP51-1, AP51-3) nucleotide sequences known to encode the ß-subunit of succinate thiokinase (ß-STK). Northern blot analysis has shown that levels of AP51-1 and AP51-3 mRNAs depend on iron availability, whilst the level of AP51-2 mRNA was iron-independent. Thus it is plausible that in trichomonads iron regulates expression of various isoforms of both
-STK and ß-STK. Alternatively, iron could be involved in post-translational modification of the proteins, as was demonstrated for the hydrogenosomal malic enzyme (Drmota et al., 1996
).
Iron-dependent changes in enzyme activity have been extensively studied in the FeS protein aconitase present in the cytosol of higher eukaryotes. In this enzyme, the loss of enzymic activity under iron-restricted conditions is caused by disassembly of [FeS] clusters, while the level of the protein remains unchanged (Yu et al., 1992 ). Loss of [FeS] clusters is associated with an alternative function of aconitase as an iron regulatory protein (IRP) (Kim et al., 1995
). Apoaconitase mediates post-transcriptional regulation of two molecules central to cellular iron homeostasis, the transferrin receptor and ferritin (Rouault & Klausner, 1997
). Recently, a homologue of cytosolic aconitase has been found in the marine ciliate Eufolliculina uhligi, although its function as an IRP has not been proven (Markmann-Mulisch et al., 1999
). It is not known whether IRPs also operate in trichomonads; however, their presence is unlikely. Trichomonads do not posses either aconitase activity (I. Hrdý, unpublished) or a transferrin receptor (Tachezy et al., 1996
). Moreover, our experiments demonstrated the effect of iron on the transcriptional level only. Nevertheless, the presence of iron-dependent post-translational regulation cannot be ruled out. Transcriptional control of iron-regulated genes is well-known in bacteria. They possess a histidine-rich protein designated Fur (ferric uptake regulator) which acts as a repressor together with iron (Crosa, 1997
; Hantke, 1984
). The main function of Fur is to turn off the expression of genes involved in iron uptake under iron-rich conditions. To our knowledge, iron-dependent control of protein expression of the type reported here for trichomonads is different from other mechanisms of iron-dependent regulation described so far. In trichomonads, iron is involved (i) in positive transcriptional control, and (ii) in regulation of expression of proteins involved in core energy metabolism. Further studies are required to obtain detailed information on the molecular mechanisms of the iron regulatory system of trichomonads, which might be unique to these unicellular eukaryotes.
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ACKNOWLEDGEMENTS |
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Received 9 June 2000;
revised 31 August 2000;
accepted 21 September 2000.