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PubMed2001

Characterisation of an ATP diphosphohydrolase (Apyrase, EC 3.6.1.5) activity in Trichomonas vaginalis.

In the present report the enzymatic properties of an ATP diphosphohydrolase (apyrase, EC 3.6.1.5) in Trichomonas vaginalis were determined. The enzyme hydrolyses purine and pyrimidine nucleoside 5'-di- and 5'-triphosphates in an optimum pH range of 6.0--8.0. It is Ca(2+)-dependent and is insensitive to classical ATPase inhibitors, such as ouabain (1 mM), N-ethylmaleimide (0.1 mM), orthovanadate (0.1 mM) and sodium azide (5 mM). A significant inhibition of ADP hydrolysis (37%) was observed in the presence of 20 mM sodium azide, an inhibitor of ATP diphosphohydrolase. Levamisole, a specific inhibitor of alkaline phosphatase, and P(1), P(5)-di (adenosine 5'-) pentaphosphate, a specific inhibitor of adenylate kinase, did not inhibit the enzyme activity. The enzyme has apparent K(m) (Michaelis Constant) values of 49.2+/-2.8 and 49.9+/-10.4 microM and V(max) (maximum velocity) values of 49.4+/-7.1 and 48.3+/-6.9 nmol of inorganic phosphate x min(-1) x mg of protein(-1) for ATP and ADP, respectively. The parallel behaviour of ATPase and ADPase activities and the competition plot suggest that ATP and ADP hydrolysis occur at the same active site. The presence of an ATP diphosphohydrolase activity in T. vaginalis may be important for the modulation of nucleotide concentration in the extracellular space, protecting the parasite from the cytolytic effects of the nucleotides, mainly ATP.

Adenosine Diphosphate↗
PubMed2002

The crystal structure of Trichomonas vaginalis ferredoxin provides insight into metronidazole activation.

Crystallographic studies revealing the three-dimensional structure of the oxidized form of the [2Fe-2S] ferredoxin from Trichomonas vaginalis (TvFd) are presented. TvFd, a member of the hydrogenosomal class of ferredoxins, possesses a unique combination of redox and spectroscopic properties, and is believed to be the biological molecule that activates the drug metronidazole reductively in the treatment of trichomoniasis. It is the first hydrogenosomal ferredoxin to have its structure determined. The structure of TvFd reveals a monomeric, 93 residue protein with a fold similar to that of other known [2Fe-2S] ferredoxins. It contains nine hydrogen bonds to the sulfur atoms of the cluster, which is more than the number predicted on the basis of the spectroscopic data. The TvFd structure contains a large dipole moment like adrenodoxin, and appears to have a similar interaction domain. Our analysis demonstrates that TvFd has a unique cavity near the iron-sulfur cluster that exposes one of the inorganic sulfur atoms of the cluster to solvent. This cavity is not seen in any other [2Fe-2S] ferredoxin with known structure, and is hypothesized to be responsible for the high rate of metronidazole reduction by TvFd.

Animals↗
PubMed2002

Visualization of new virus-like-particles in Trichomonas vaginalis.

In the present work, we demonstrate virus-like particles (VLPs) with various morphological variations in Trichomonas vaginalis. The VLPs were distinct based on size, shape and electron density, with VLPs being either electron-dense or electron-lucent. We used electron microscopy thin sections of several T. vaginalis strains virus-infected, and also negative staining of fractions obtained after purification by CsCl buoyant density gradient centrifugation. The particles observed in fractions are identical to those previously described, but by thin sections, we found new forms. The shapes found were icosahedral, spherical and oblong, and the sizes varied from 33 to 120nm in diameter with the most common VLP being spherical and having a size range from 83 to 104nm. The VLPs were found in the cytoplasm closely associated with the Golgi complex, with some VLPs budding from the Golgi, and other VLPs were detected adjacent to the plasma membrane. Unidentified cytoplasmic inclusions were observed in the region close to the VLPs and Golgi. Clusters of the already described icosahedral virus were also observed in the cytoplasm, although less frequently. These results indicate that T. vaginalis organisms may be infected with different dsRNA viruses simultaneously.

Animals↗
PubMed1995

The cDNA sequence of Trichomonas vaginalis virus-T1 double-stranded RNA.

Trichomonas vaginalis virus (TVV) is a nonsegmented double-stranded (ds) RNA virus that infects the pathogenic protozoan T. vaginalis. To study the virus, we cloned the genomic ds RNA of a TVV-T1 isolate and obtained a contiguous 4647-bp cDNA sequence. Two overlapping genes separated by a + 1 reading frame shift were identified on the plus strand but none on the complementary strand RNA in this sequence. The upstream gene probably encodes a 75-kDa capsid protein, and the downstream gene encodes an 86-kDa polypeptide which is probably the viral RNA-dependent RNA polymerase (RDRP). A potential ribosomal slippage heptamer (C CUU UUU) was identified within the 14-nt overlap of the two genes. The genomic organization and RDRP sequence in TVV-T1 exhibit similarity to those of Saccharomyces cerevisiae virus and Leishmania RNA virus, suggesting that these viruses originate from common ancestry, but are only distantly related to Giardia lamblia virus.

Amino Acid Sequence↗
PubMed1998

NMR spectroscopic studies of the hydrogenosomal [2Fe-2S] ferredoxin from Trichomonas vaginalis: hyperfine-shifted 1H resonances.

The hyperfine-shifted 1H NMR resonances of oxidized and reduced Trichomonas vaginalis ferredoxin, a functionally unique [2Fe-2S] ferredoxin, have been studied. The oxidized protein spectrum displayed a pattern of six broad upfield-shifted resonances between 13 and 40 ppm with chemical shifts distinct from those of other [2Fe-2S] ferredoxins. All hyperfine 1H resonances of the oxidized ferredoxin displayed anti-Curie temperature dependences. Reduced T. vaginalis ferredoxin displayed hyperfine resonances both upfield and downfield of the diamagnetic region. These resonances showed Curie temperature dependences. Overall the hyperfine-shifted NMR spectrum of T. vaginalis ferredoxin, along with other spectroscopic properties, suggested different structural properties for the active center of oxidized hydrogenosomal ferredoxins from those of other [2Fe-2S] ferredoxins.

Animals↗
PubMed2003

Adenosine is the primary precursor of all purine nucleotides in Trichomonas vaginalis.

Trichomonas vaginalis, a parasitic protozoan and the causative agent of trichomoniasis, lacks de novo purine nucleotide synthesis and possesses a unique purine salvage pathway, consisting of a bacterial type purine nucleoside phosphorylase and a purine nucleoside kinase. It is generally believed that adenine and guanine are converted to their corresponding nucleosides and then further phosphorylated to form AMP and GMP, respectively, as the main as well as the essential pathway of replenishing the purine nucleotide pool in the organism. Formycin A, an analogue of adenosine, inhibits both enzymes as well as the in vitro growth of T. vaginalis with an estimated IC(50) of 0.27 microM. This growth inhibition was reversed by adding adenine to the culture medium but not by adding guanine or hypoxanthine. Furthermore, T. vaginalis can grow in semi-defined medium supplemented with only adenine but not with guanine or hypoxanthine. Radiolabeling experiments followed by HPLC analysis of the purine nucleotide pool in T. vaginalis demonstrated incorporation of [8-14C]adenine into both adenine and guanine nucleotides, whereas [8-14C]guanine was incorporated only into guanine nucleotides. Substantial adenosine deaminase activity and significant IMP dehydrogenase and GMP synthetase activities were identified in T. vaginalis lysate, suggesting a pathway capable of converting adenine to GMP via adenosine. This purine salvage scheme depicts adenosine the primary precursor of the entire purine nucleotide pool in T. vaginalis and the purine nucleoside kinase one of the most pivotal enzymes in purine salvage and a potential target for anti-trichomoniasis chemotherapy.

Adenosine↗
PubMed2003

Tetracycline-inducible gene expression in Trichomonas vaginalis.

Transient and stable gene delivery systems are available for Trichomonas vaginalis, however, they do not allow regulated expression of target genes. To study essential genes or proteins that are toxic to the cells when over expressed, we have developed an inducible/repressible gene expression system in this parasite, which is driven by the tet-operator (tetO) and regulated tetracycline-responsive Tet repressor (TetR). Inducible chloramphenicol acetyl transferase (CAT) gene expression is observed using a concentration of tetracycline (Tc) as low as 0.1 microg x ml(-1). Expression increases with drug dose with a maximum level of CAT induction achieved in stable transfectants using 5 microg x ml(-1) Tc. CAT protein expression is detectable within 12 h and reaches a maximum level at 48 h, demonstrating that inducible expression is time and dose-dependent. In an inverse experiment, parasites previously cultivated with 1 microg x ml(-1) of Tc for 48 h, were grown in the absence of drug to determine the kinetics of repression. A significant decrease in protein concentration is detected after 48 h, and no detectable protein is observed after 72 h. Experiments replacing the CAT gene with the puromycin N-acetyltransferase (PAC) gene in the Tet regulated expression construct have demonstrated the use of this system for testing putative toxic and essential genes. The establishment of regulated gene expression of exogenous genes in T. vaginalis represents a crucial step towards determining the function of proteins in this divergent parasite.

Acetyltransferases↗
PubMed1996

The contribution of the arginine dihydrolase pathway to energy metabolism by Trichomonas vaginalis.

The enzymes of the arginine dihydrolase pathway were measured in Trichomonas vaginalis hydrogenosome-deficient lines MR-5 and MR-100, and the parent strain TV 10-02. The activities and substrate affinities of arginine deiminase, carbamate kinase and ornithine decarboxylase were similar for the hydrogenosome-deficient lines and the parent TV 10-02. The activity of catabolic ornithine carbamyltransferase, however, was found to be 5-7-fold elevated in the hydrogenosome-deficient lines; the apparent K(m) for citrulline was similar for all of the lines. Putrescine biosynthesis by the hydrogenosome-deficient cell lines was found to be significantly higher than the parent. Incubation of strain MR-100 with U-[14C]-arginine resulted in a 5-fold greater amount of 14CO2 liberated compared to the parent strain TV 10-02. Inclusion of the ornithine decarboxylase inhibitor difluoromethylornithine in these incubations reduced the CO2 production of strain TV 10-02 by 42%, but only inhibited the MR-100 strain by 14.5%, indicative that the majority of the CO2 liberated from arginine by this strain is derived from the elevated activity of ornithine carbamyltransferase. Despite the increased flow through the arginine dihydrolase pathway, the energy gain to the parasite is approximately 10% of that from glucose, thus, under the growth conditions used in this study carbohydrate metabolism provides the bulk of the ATP for the parasite.

Animals↗
PubMed1996

Iron-ascorbate cleavable malic enzyme from hydrogenosomes of Trichomonas vaginalis: purification and characterization.

Two isoforms of NAD(P)(+)-dependent malic enzyme (EC 1.1.1.39) were isolated from hydrogenosomes of Trichomonas vaginalis. A positively charged isoform at pH 7 was obtained in a single purification step using cation-exchange chromatography. The second isoform, negatively charged at pH 7.5, was partially purified using a combination of anion-exchange and affinity chromatography. Both isoforms displayed similar physical and kinetic properties. Molecular weight determination of the native enzyme suggested a homotetrameric arrangement of the 60 kDa subunits. The enzyme utilized NAD+ (Km, 6-6.3 microM) preferentially to NADP+ (Km, 125-145 microM). The NAD(+)-dependent activity showed a broad pH optimum with maximum under alkaline conditions (pH 9) likely to be present inside hydrogenosomes. Immunocytochemical studies using a polyclonal rabbit antibody raised against purified T. vaginalis malic enzyme proved hydrogenosomal localization of the enzyme. Subfractionation of hydrogenosomes suggested an association of the malic enzyme with the hydrogenosomal membranes. The 60 kDa malic enzyme subunit was highly sensitive to non-enzymatic cleavage by an iron-ascorbate system resulting in two enzymatically inactive fragments of about 31 kDa. Microsequencing of the fragments revealed that the 60 kDa subunit was cleaved at the metal-binding site between Asp279-Ile280. The enzyme inactivation was inhibited by an excess of manganese. Iron-dependent posttranslational modification might contribute to the regulation of malic enzyme activity in vivo.

Amino Acid Sequence↗
PubMed1998

Evidence for an uncommon alpha-actinin protein in Trichomonas vaginalis.

As part of our ongoing project of identification of actin-binding proteins implicated in the cell transition (flagellate to amoeboid/adherent) of Trichomonas vaginalis, we have characterized an alpha-actinin-related protein in this parasite. The protein (P100) has a molecular mass of 100 kDa and an isoelectric point of 5.5. A monoclonal antibody raised against this protein co-localizes with the actin network. P100 gene transcripts are co-expressed with actin throughout the cell cycle. Analysis of the deduced protein sequence reveals three domains: an N-terminal actin-binding region; a central region rich in alpha-helix; and a C-terminal domain with Ca(2+)-binding capacity. Whereas the N- and C-terminal regions are well-conserved as compared to other alpha-actinins, we observe in the central region an atypical distribution of residues in five repeats. The sequence of the repeats does not show any homology with the rod domain of the other alpha-actinins, except for the first repeat which shows some similarity. The four other repeats of T. vaginalis P100 appear to result from a duplication event which is not detectable in the other sequences.

5' Untranslated Regions↗
PubMed1999

Alternative 2-keto acid oxidoreductase activities in Trichomonas vaginalis.

We have induced high levels of resistance to metronidazole (1 mM or 170 microg ml(-1)) in two different strains of Trichomonas vaginalis (BRIS/92/STDL/F1623 and BRIS/92/STDL/B7708) and have used one strain to identify two alternative T. vaginalis 2-keto acid oxidoreductases (KOR) both of which are distinct from the already characterised pyruvate:ferredoxin oxidoreductase (PFOR). Unlike the characterised PFOR which is severely down-regulated in metronidazole-resistant parasites, both of the alternative KORs are fully active in metronidazole-resistant T. vaginalis. The first, KORI, localized in all membrane fractions but predominantly in the hydrogenosome fraction, is soluble in Triton X-100 and the second, KOR2, is extractable in 1 M acetate from membrane fractions of metronidazole-resistant parasites. PFOR and both KORI and KOR2 use a broad range of 2-keto acids as substrates (pyruvate, alpha-ketobutyrate, alpha-ketomalonate), including the deaminated forms of aromatic amino acids (indolepyruvate and phenylpyruvate). However, unlike PFOR neither KORI or KOR2 was able to use oz-ketoglutarate. Deaminated forms of branched chain amino acids (alpha-ketoisovalerate) were not substrates for T. vaginalis KORs. Since KOR I and KOR2 do not apparently donate electrons to ferredoxin, and are not down-regulated in metronidazole-resistant parasites, we propose that KORI and KOR2 provide metronidazole-resistant parasites with an alternative energy production pathway(s) which circumvents metronidazole activation.

Animals↗
PubMed2000

Competition and protease sensitivity assays provide evidence for the existence of a hydrogenosomal protein import machinery in Trichomonas vaginalis.

Hydrogenosomes are double membrane bounded redox organelles found in a number of amitochondriate protists and fungi. They are involved in carbohydrate metabolism and ATP synthesis and thus resemble mitochondria. Molecular analysis of the hydrogenosomal heat shock proteins Hsp70, Hsp60 and Hsp10 in Trichomonas vaginalis, one of the deepest-branching eukaryotes known to date, has revealed that these group exclusively with mitochondrial heat shock proteins. This finding indicates strongly that a progenitor organelle which gave rise to contemporary mitochondria and hydrogenosomes existed early in eukaryotic life. This hypothesis is further supported by similarities of hydrogenosomal and mitochondrial biogenesis. It was shown that T. vaginalis hydrogenosomal proteins are synthesized on free ribosomes in the cytosol with an N-terminal presequence that carries targeting information and is cleaved upon import into the organelle. Furthermore, as in mitochondrial import, hydrogenosomal protein import requires ATP, an electrochemical transmembrane potential and cytosolic protein factor(s). Here we demonstrate that inhibition of hydrogenosomal protein import occurs (i) in the presence of a synthetic presequence peptide and (ii) after pretreatment of hydrogenosomes with the protease trypsin. Trypsin pretreatment affects two hydrogenosomal membrane proteins of 31 and 70 kDa, respectively. Thus, we present evidence that import is saturable and that proteinaceous hydrogenosomal import receptor(s) exist. These results are a first step towards a characterization of the hydrogenosomal import machinery which should provide further insights into the relationship of hydrogenosomes and mitochondria and the evolution of protein targeting into organelles of endosymbiotic origin.

Animals↗
PubMed1998

Analysis of the uptake of the fluorescent marker 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) by hydrogenosomes in Trichomonas vaginalis.

The fluorescent dye 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) has been widely used as an indicator of cytosolic pH. Here we report that BCECF localizes to hydrogenosomes (hydrogen-generating organelles found in several phylogenetically separate groups of anaerobic protists) in Trichomonas vaginalis, where it was observable by fluorescence microscopy. Its cellular location was confirmed by treatment of BCECF-loaded cells with diaminobenzidine and hydrogen peroxide together with UV illumination. This produced an osmiophilic precipitate in the matrix of hydrogenosomes, observable by electron microscopy. Use of a short (7.5 min) loading period, loading on ice, use of concentrations of BCECF (acetoxymethyl ester) down to 10 nM, and inclusion of the anion channel blockers probenicid or sulfinpyrazone, or the K+/H+ ionophore nigericin in the loading buffer all failed to prevent hydrogenosomal accumulation of BCECF. This uptake was best observed when intact cells were loaded with the ester form of BCECF, but could also be seen using free BCECF following either incubation with ruptured cells or electroporation of intact cells. Hydrogenosomal BCECF loading was also obtained with washed cell lysates, without cytoplasm or metabolic substrates. We tested a range of other fluorogenic dyes designed for cytosolic labeling, and found that the calcium indicator fura-2 (acetoxymethyl ester) and the cell viability marker fluorescein diacetate also labeled hydrogenosomes. The results illustrate a novel use for BCECF as a fluorescent marker for hydrogenosomes (the first such marker), but present a warning against the indiscriminate use of fluorogenic ester dyes to measure properties of the cytosol in hydrogenosome-containing organisms - the dyes may also be indicating the properties of the hydrogenosome.

Animals↗
PubMed1988

Determination of cytopathogenic effects of Trichomonas vaginalis on cultured cells by assaying 51Cr release.

By use of an assay for 51Cr release, it was attempted to determine the cytopathogenic effects of Trichomonas vaginalis on mammalian cultured cells. 51Cr was released from Na2(51)CrO4-labeled HeLa, L-929, FL, Vero and LLC-MK 2 cells. HeLa cells were most sensitive to the damage, followed by L-929, FL, Vero and LLC-MK 2 cells. The effect depended upon the number of parasite and the incubation period. The maximum 51Cr release from the labeled cells was observed at 37 degrees C and pH 6.0. These results attest that this assay is reliable for determining the cytopathogenic effects of T. vaginalis on the target cells.

Animals↗
PubMedDate not supplied

Trichomonas vaginalis has two fibronectin-like iron-regulated genes.

BACKGROUND: Trichomonas vaginalis, a protozoan parasite of the human urogenital tract, interacts with fibronectin (FN), a glycoprotein of the extracellular matrix. We, therefore, attempted to identify genes of this eukaryote encoding FN-binding proteins. METHODS: A cDNA clone, C1, representing an incomplete gene was obtained from an expression library based on its FN-binding ability and was characterized. The full-length 378-bp gene encoding a 14.8-kDa protein of 125 amino acids was obtained. RESULTS: The amino acid sequences revealed homology with the type III-14 repeat of the heparin-binding domain at the carboxyl terminal end of FN. This fibronectin-like protein gene, flp1, was single copy in all the T. vaginalis isolates examined. Levels of flp1 transcript were elevated in cells grown under low-iron conditions. Another low-iron-regulated gene, flp2, with 70 and 67.5% identity to flp1 at the nucleotide and amino acid levels, respectively, was recovered from the trichomonad genome. Both flp1 and flp2 had consensus Inr promoter-like elements immediately adjacent to the start codon. flp2 also contained an additional Inr element followed by an ATG 24-bp within the gene. CONCLUSIONS: Unlike flp2, the flp1 gene had AU-rich destabilizing elements in the 3'-untranslated region (UTR).

Amino Acid Sequence↗
PubMed2003

Effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis.

Here we report the effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) hydrolysis was 5- to 7-fold higher for the fresh clinical strain, when compared with the ATCC (American Type Culture Collection) strain. ATP hydrolysis was activated in the presence of metronidazole in the ATCC strain, whilst it was inhibited 33% by 50 microM tinidazole in a fresh clinical isolate. The treatment of cells in the presence of metronidazole for 2 h inhibited ATP and ADP hydrolysis, whilst treatment with tinidazole inhibited ATP and ADP hydrolysis only in the fresh clinical isolate. The drugs did not change the ecto-5'-nucleotidase activity for both strains. Our results suggest that the modulation of extracellular ATP and ADP levels during treatment with these drugs could be a parasitic defence strategy as a survival mechanism in an adverse environment.

5'-Nucleotidase↗
PubMed2001

Evolutionary relationships of the glucokinase from the amitochondriate protist, Trichomonas vaginalis.

Two genes coding for Trichomonas vaginalis glucokinase were isolated and sequenced. The putative translation products have molecular masses of 41,584 and 41,772 Da, corresponding to 375 and 377 amino acids, respectively. These values agree with data determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the enzyme purified from the organism. The two sequences showed 78% amino acid identity. The sequences and their phylogenetic reconstruction show that they are members of a glucokinase/fructokinase protein family found in eubacteria and also in the eukaryote Giardia lamblia and are only distantly related to typical eukaryotic hexokinases. The results indicate that the evolutionary past of this enzyme, catalyzing the first step of glycolysis in T. vaginalis, is different from that of the enzyme performing this key role in almost all other eukaryotes.

Amino Acid Sequence↗
PubMed2001

Unique phylogenetic relationships of glucokinase and glucosephosphate isomerase of the amitochondriate eukaryotes Giardia intestinalis, Spironucleus barkhanus and Trichomonas vaginalis.

Glucokinase (GK) and glucosephosphate isomerase (GPI), the first two enzymes of the glycolytic pathway of the diplomonads Giardia intestinalis and Spironucleus barkhanus, Type I amitochondriate eukaryotes, were sequenced. GPI of the parabasalid Trichomonas vaginalis was also sequenced. The diplomonad GKs belong to a family of specific GKs present in cyanobacteria, in some proteobacteria and also in T. vaginalis, a Type II amitochondriate protist. These enzymes are not part of the hexokinase family, which is broadly distributed among eukaryotes, including the Type I amitochondriate parasite Entamoeba histolytica. G. intestinalis GK expressed in Escherichia coli was specific for glucose and glucosamine, as are its eubacterial homologs. The sequence of diplomonad and trichomonad GPIs formed a monophyletic group more closely related to cyanobacterial and chloroplast sequences than to cytosolic GPIs of other eukaryotes and prokaryotes. The findings show that certain enzymes of the energy metabolism of these amitochondriate protists originated from sources different than those of other eukaryotes. The observation that the two diplomonads and T. vaginalis share the same unusual GK and GPI is consistent with gene trees that suggest a close relationship between diplomonads and parabasalids. The intriguing relationships of these enzymes to cyanobacterial (and chloroplast) enzymes might reflect horizontal gene transfer between the common ancestor of the diplomonad and parabasalid lineages and the ancestor of cyanobacteria.

Animals↗