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Nucleoside uptake by Trichomonas vaginalis.

A rapid sampling technique was used to follow nucleoside uptake by Trichomonas vaginalis. The results indicated that nucleoside uptake is biphasic with time. Adenosine, guanosine, and uridine uptake is carrier mediated, transported substrate is rapidly metabolised to nucleotides. Two separate carriers appear to exist, one which transports all nucleosides and a second which transports adenosine, guanosine and uridine. Both carriers have more than one binding site for nucleosides. The first carrier has sites for adenosine and pyrimidine nucleosides, and a separate site for purine nucleosides. The second carrier has a site for adenosine and uridine and a separate site for guanosine. Adenosine uptake could not be completely inhibited by nitrobenzylthionucleosides. The rate of nucleoside uptake by T. vaginalis is sufficient to sustain growth.

Adenosine↗

Effect of DL-alpha-difluoromethylornithine on polyamine synthesis and interconversion in Trichomonas vaginalis grown in a semi-defined medium.

Growth of Trichomonas vaginalis in a semi-defined medium was inhibited by 5 mM DL-alpha-difluoromethylornithine (DFMO). Using high pressure liquid chromatography (HPLC) analysis, putrescine and cadaverine levels were found to be 90 and 100% reduced, respectively after 120 h exposure, whilst spermidine and spermine levels were unchanged. Putrescine (40 microM) and cadaverine (6 microM) were detected in the spent media from control cultures. Neither of these diamines was detected in spent media from 72 h DFMO-treated cultures. Changes in intracellular levels of amine precursors were also determined by HPLC. There was a transient increase in ornithine to 39 nmol (mg protein)-1 at 48 h in the DFMO-treated cells while it remained undetectable in control cells throughout the experiment. Arginine and citrulline levels remained high, decreasing to control levels only after 72 h. Only spermine (1 mM) rescued DFMO-treated cells, and this is discussed with respect to the presence of a putative spermine-specific oxidase designated by its sensitivity to aminoguanidine. Aerobic incubation of growing (normal) cells with [14C]spermine resulted in the production of an unknown metabolite (19% of total label), whose content was reduced to 5% under anaerobic conditions. Decarboxylated S-adenosylmethionine remained undetectable in DFMO-treated cells, and the methylation index (ratio of S-adenosylmethionine to S-adenosylhomocysteine) did not change from the control value of 9.3. Ornithine decarboxylase, S-adenosylmethionine synthetase, S-adenosylmethionine:L-homocysteine methyltransferase, and S-adenosylhomocysteine hydrolase enzyme activities were detected. However, S-adenosylmethionine decarboxylase, spermidine synthase or spermine synthase were not detected. These findings are discussed with reference to the arginine dihydrolase pathway whose end products are putrescine and ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗

Presence of a fructose-2,6-bisphosphate-insensitive pyrophosphate: fructose-6-phosphate phosphotransferase in the anaerobic protozoa Tritrichomonas foetus, Trichomonas vaginalis and Isotricha prostoma.

Extracts of the anaerobic protozoa Tritrichomonas foetus, Trichomonas vaginalis and Isotricha prostoma contained a high activity (0.5-1 mumol min-1 (mg protein)-1) of pyrophosphate:fructose-6-phosphate phosphotransferase (PPi-PFK), but no detectable ATP: fructose-6-phosphate phosphotransferase. PPi-PFK from I. prostoma was purified close to homogeneity by adsorption on phospho-Ultrogel and elution with fructose-1,6-bisphosphate, and subsequent anion-exchange chromatography. The enzyme had an Mr of 95,000 as determined by gel filtration and consisted of subunits of Mr 48,000. PPi-PFK from I. prostoma and from T. foetus displayed hyperbolic kinetics with respect to their substrates and were not affected by fructose-2,6-bisphosphate. In sharp contrast with what has been found in other eukaryotes, no evidence could be found for the presence of fructose-2,6-bisphosphate in the two trichomonads, in I. prostoma and in Entamoeba histolytica.

Animals↗

Fatty acid and sterol metabolism of cultured Trichomonas vaginalis and Tritrichomonas foetus.

Trichomonas vaginalis and Tritrichomonas foetus grown in a fetal calf serum-based culture medium, contained as major lipids (i.e., greater than 10% of total) cholesterol, phosphatidylethanolamine and phosphatidylcholine. T. vaginalis also contained sphingomyelin and T. foetus glycophosphosphingolipids. The culture medium contained (greater than 10%) cholesterol, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine. The fatty acyl groups of these major lipids of the trichomonads and the culture medium were similar. Those present in amounts greater than 5% of the total fatty acyl groups for a given lipid were myristic, palmitic, hexadecaenoic, stearic, oleic, linoleic, arachidonic and docosahexaenoic. When the trichomonads were exposed to radiolabeled lipids and lipid precursors, [14C]-labeled acetate and potential acetate precursors (glucose, threonine) were poorly incorporated and failed to label the fatty acyl groups of the trichomonad lipids. [14C]-labeled, C12-C22 saturated and unsaturated fatty acids were incorporated, unaltered, into phosphoglycerides and sphingolipids (sphingomyelin and glycophosphosphingolipids), but not into cholesteryl esters or triacylglycerols. Phosphoglycerides were preferentially labeled with unsaturated fatty acids and sphingolipids with saturated ones. This information inferred that the trichomonads: 1) were unable to biosynthesize fatty acids de novo, 2) took up unesterified fatty acids from the culture medium and used them in phosphoglyceride and sphingolipid biosynthesis and/or turnover, 4) did not use unesterified fatty acids in the biosynthesis or turnover of cholesteryl esters or triacylglycerols. Phosphatidylcholine and phosphatidylethanolamine, with [14C]labeled fatty acyl groups, and sphingomyelin, with 14C-labeled choline, were incorporated by the trichomonads. The phospholipids strongly labeled phosphoglycerides and sphingolipids, but not triacylglycerols, while the radioactivity of sphingomyelin [14C]choline remained associated solely with trichomonad sphingomyelin. Triacylglycerol, with 14C-labeled fatty acyl groups, was also incorporated, and labeled phosphoglycerides and sphingolipids. The results of those experiments suggested that trichomonads: (1) could take up culture medium phospholipids and triacylglycerols; (2) actively deacylated and reacylated phospholipids, but not triacylglycerols; (3) hydrolyzed exogenous triacylglycerols and used their fatty acyl groups for phospholipid acylations. Radiolabeled acetate, mevalonate and squalene were not incorporated into trichomonad cholesterol or cholesteryl esters. [14C]Cholesterol was incorporated unaltered, but was not esterified.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Trichomonas vaginalis requires traces of oxygen and high concentrations of carbon dioxide for optimal growth.

The effects of O2 and CO2 on the growth in culture of Trichomonas vaginalis strain C1-NIH were investigated. Growth under pre-purified N2 in the absence of CO2 supplementation gave a doubling time of 4.4 h; when traces of O2 (less than 0.25 microM) were present, the doubling time was 3.5 h. Organisms grew most rapidly (doubling time 2.3 h) with traces of O2 (less than 0.25 microM) and with the CO2 level controlled at 5 mM. The balance of fermentation products from maltose was greatly influenced by supplied gases. Under strictly anaerobic conditions at 5 mM CO2, equimolar glycerol and lactate accounted for more than 95% of the measured products, whereas lower CO2 increased acetate production. Under microaerobic conditions (O2 less than 0.25 microM) acetate was the major product when CO2 was limited to that evolved endogenously; again 5 mM CO2 favoured glycerol and lactate production. Activities of key enzymes measured in cell-free extracts (pyruvate:ferredoxin oxidoreductase, hydrogenase, glycerol kinase, malate dehydrogenase (decarboxylating) and lactate dehydrogenase) altered with growth conditions commensurately with observed changes in metabolic flux patterns. These results suggest that T. vaginalis is optimally adapted to conditions it experiences in situ in the vagina (traces of O2, high CO2).

Acetates↗

Guanosine kinase from Trichomonas vaginalis.

A novel guanosine kinase was partially purified from the parasitic protozoan Trichomonas vaginalis. Unlike nucleoside kinases from other sources, the preferred substrate for this enzyme was guanosine (Vmax/Km = 120). The enzyme also catalyzed the phosphorylation of inosine (Vmax/Km = 3), uridine (2), adenosine (0.5), cytidine (0.2), and 2'-deoxyguanosine (0.1). The 2'-deoxyribonucleosides of adenine, hypoxanthine, uracil, cytosine and thymine were not phosphorylated. The Km for ATP was 6.6 microM. The enzyme was extremely labile in the absence of ATP. As a result, only a 20-fold purification with 25% recovery of activity was possible. However, this preparation was free of nucleoside phosphorylase, nucleoside phosphotransferase and the distinct uridine kinase. The enzyme had a broad optimum of pH 6.5-8. It had a molecular weight of 15000.

Animals↗

Inorganic pyrophosphatase of Trichomonas vaginalis.

Trichomonas vaginalis homogenates were found to have an acid inorganic pyrophosphatase activity with a specific activity at pH 4.8 of about 7 nmol min-1 (mg protein)-1. This activity was localized predominantly in hydrolase containing particles, showed structure-bound latency and was tightly membrane-bound. The activity showed no magnesium dependence, a Km of about 2 mM inorganic pyrophosphate, a pH optimum of 5.2 and was inhibited by fluoride at millimolar levels. No evidence was obtained for the existence of a cytosolic magnesium-dependent activity but the existence of a low level of magnesium-independent cytosolic activity cannot be excluded. These observations correlate with the importance of cytosolic inorganic pyrophosphate in the carbohydrate catabolism in T. vaginalis.

Acid Phosphatase↗

Phospholipid metabolism of cultured Trichomonas vaginalis and Tritrichomonas foetus.

Trichomonas vaginalis and Tritrichomonas foetus grown in a fetal calf serum-based culture medium were exposed to radiolabeled phospholipids and lipid precursors to determine the extent to which these organisms can incorporate complex lipids and/or de novo synthesize their major membrane phosphoglycerides. Phosphatidylethanolamine and phosphatidylcholine were the dominant phospholipids (40-50% of extractable phospholipids), with acidic lipids, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and O-acylphosphatidylglycerol accounting for the remaining phosphoglycerides. T. vaginalis was rich in sphingomyelin while T. foetus lacks significant amounts of this lipid. Incubation with [32P]orthophosphate resulted in only modest incorporation into extractable phospholipids; the most striking observation being the failure to label choline-containing lipids (phosphatidylcholine, lysophosphatidylcholine and sphingomyelin). Phosphatidylethanolamine was heavily labeled with modest labeling observed in the acidic phosphoglycerides. [U-14C]Glucose failed to label choline-containing lipids in T. foetus but did so in T. vaginalis, with phosphatidylethanolamine again being heavily labeled. Choline, phosphorylcholine, ethanolamine, serine, inositol, glycerol and methionine were incorporated poorly or failed to label the expected phosphoglycerides in either of the trichomonads, demonstrating an impairment in synthesis. Intact phosphoglycerides, labeled in the fatty acyl groups, labeled most phospholipids indicating that turnover of membrane lipids can occur with respect to the acyl component of the phospholipids. Fluorescent probes attached to phosphoglyceride molecules support observations seen with radiolabeled phosphoglycerides. Though trichomonads are able to transacylate phosphoglycerides, it is evident that the trichomonads lack a variety of enzymatic activities necessary for de novo synthesis of complex phosphoglycerides and must rely on environmental sources to supply them.

Animals↗

Pyruvate kinase from Trichomonas vaginalis, an allosteric enzyme stimulated by ribose 5-phosphate and glycerate 3-phosphate.

Trichomonas vaginalis pyruvate kinase was purified over 1750 fold to a specific activity greater than 100 mumol min-1 (mg protein)-1. The enzyme is a tetramer of M(r) 266,000, consisting of subunits of M(r) 53,000 and 56,000 in equivalent amounts. Its activity was dependent on the presence of magnesium but was not stimulated by potassium or ammonium. The enzyme exhibited positive cooperativity towards phosphoenolpyruvate and was inhibited by inorganic phosphate, which increased the sigmoidicity of the saturation curve for phosphoenolpyruvate without affecting maximal activity. It was heterotropically stimulated by ribose 5-phosphate and glycerate 3-phosphate, not previously known to act on eukaryotic pyruvate kinases, but was unaffected by known effectors of most pyruvate kinases, including fructose 1,6-bisphosphate and fructose 2,6-bisphosphate.

Allosteric Regulation↗

Influence of oxygen on the fermentative metabolism of metronidazole-sensitive and resistant strains of Trichomonas vaginalis.

The microaerophilic protozoon Trichomonas vaginals responds to extracellular changes in oxygen concentration: acetate, lactate, ethanol, H2 and CO2 formation, as well as glucose-depletion rates, are affected. All these variables except ethanol production rates, also differed between clinically metronidazole-sensitive (1910) and resistant (IR78 and CDC85) strains. Most interesting were the greatly increased glucose-scavenging rates of resistant isolates and their low specific activities of hydrogenase and H2 formation rates by comparison with the metronidazole-sensitive strain. Results suggest that all three strains of this parasite are well adapted to the O2 levels prevailing in situ (13-56 microM). Thus, vaginal oxygen tensions have more pronounced effects on the balances of fermentation products in the resistant strains, and results indicate that these strains may then use hydrogenosomal pathways to their advantage.

Adaptation, Physiological↗

Lipophosphoglycan-like glycoconjugate of Tritrichomonas foetus and Trichomonas vaginalis.

Lipophosphoglycan-like glycoconjugates were isolated, purified and partially characterized from Tritrichomonas foetus and Trichomonas vaginalis. Cell surface radiolabeling of both trichomonads by the galactose oxidase/NaB[3H]4 technique indicated that the glycoconjugate was located on the cell surface of the parasites. The glycoconjugates were extracted from the delipidated residue fraction with the solvent, water/ethanol/diethylether/pyridine/NH4OH (15:15:5:1:0.017) and were purified to homogeneity by Sepharose CL-4B followed by octyl-Sepharose chromatography and methanol precipitation. The glycoconjugates migrated as broad bands upon SDS-PAGE. The T. foetus glycoconjugate contained large amounts of fucose along with some mannose, galactose, glucosamine and glucose and trace amounts of galactosamine and inositol. The T. vaginalis glycoconjugate appeared to contain large amounts of glucosamine and galactose along with some glucose, mannose and traces of galactosamine and inositol. The surface-labeled glycoconjugates from both parasites was found to be deaminated with nitrous acid and susceptible to phosphatidylinositol-specific phospholipase C, indicating the presence of a phospholipid anchor. Furthermore, these glycoconjugate were found to contain phosphate and were labile to hydrolysis by mild acid, strongly suggesting that the intact molecule is related to Leishmania lipophosphoglycans (LPG). The most striking and the unique features of these glycoconjugate molecules are the presence of large amounts of fucose in T. foetus and glucosamine in T. vaginalis along with the presence of galactosamine in both parasites. These results indicate that these glycoconjugates are new types of LPG-like molecules expressed on the trichomonad cell surface and are structurally distinct from Leishmania LPG.

Animals↗

Analysis of a single-domain P-glycoprotein-like gene in the early-diverging protist Trichomonas vaginalis.

We have characterized a gene encoding an Adenosine triphosphate (ATP) Binding Cassette (ABC) transmembrane protein from Trichomonas vaginalis, an early-diverging protozoan parasite. This gene, Tvpgp1, encodes a 589-amino acid protein with an amino-terminal hydrophobic region, 6 potential membrane-spanning segments and a carboxy-terminal ATP binding site. Tvpgp1 is most similar in sequence to mammalian P-glycoproteins, 170 kDa transport proteins which are frequently overexpressed in multiple drug-resistant (Mdr) tumor cell lines. However, Tvpgp1 is half the size of typical P-glycoproteins which are tandem duplications. These data suggest that the duplication/fusion events which gave rise to the bipartite structure comprised of 2 similar halves which characterize eukaryotic P-glycoproteins may have occurred after the divergence of trichomonads (Parabasalia) from the main line of eukaryotic evolutionary descent. We have examined 7 metronidazole resistant strains of T. vaginalis to determine whether the Tvpgp1 gene is overexpressed or amplified. 2 drug resistant strains show a 2-3-fold overexpression and one shows a 20-fold overexpression of Tvpgp mRNA. The gene is not amplified in any of the drug resistant strains. On the contrary, 4 of the 7 resistant strains lack one of 2 Tvpgp genes found in drug-sensitive strains.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Beta-tubulin genes of Trichomonas vaginalis.

Microtubules, formed by polymerization of alpha and beta-tubulins, are major structural components of the mitotic spindle, cytoskeleton, and flagella, and are also an important target for the antiparasitic benzimidazole drugs. Trichomonas vaginalis, a flagellated protozoan responsible for urogenital tract infections in humans, is highly sensitive to certain benzimidazoles in vitro. As a first step towards defining the roles of microtubules in this organism, the regulation of their expression, and the basis for their benzimidazole sensitivity, we have characterized the genes encoding T. vaginalis beta-tubulin. A combination of genomic DNA cloning using bacteriophage lambda and PCR amplification using conserved beta-tubulin gene primers was employed. Southern blots of DNA from two different T. vaginalis strains suggest there are 6-7 beta-tubulin gene copies. Sequencing identified three distinct genes: btub1, btub2, and btub3. Amplification of cDNA with gene-specific primers indicated that the relative expression of RNA transcripts was btub1 > btub2 >> btub3. The promoter region from btub1 includes a 15-bp repeat also found (with 1-bp difference) upstream of the T. vaginalis ferredoxin gene. Primer extension suggests the 5' leader of the mRNA transcribed from btub1 is only 10 nucleotides long, similar to the lengths found in other anaerobic protozoa. In 152 residues examined by PCR, btub2 and btub3 differed by 1 and 12 amino acids, respectively, from btub1. All three sequences, however, have diverged considerably (20-24%) from beta-tubulins of other protozoa. T. vaginalis beta-tubulins include residues Tyr167 and Phe200, previously implicated in resistance and sensitivity, respectively, to the benzimidazole derivative benomyl.

Amino Acid Sequence↗

Primary structure of the hydrogenosomal adenylate kinase of Trichomonas vaginalis and its phylogenetic relationships.

Hydrogenosomal adenylate kinase of the amitochondriate protist, Trichomonas vaginalis, has been purified and the sequence of its 39 amino-terminal residues established. Based on this sequence and a conserved internal region of the enzyme, a probe was obtained by DNA polymerase chain reaction and used to isolate a genomic DNA clone containing the gene of this enzyme. This gene exists probably as a single copy in T. vaginalis and is not interrupted by introns. The open reading frame obtained codes for a large type adenylate kinase with a mature molecular mass of 24.5 kDa. The T. vaginalis enzyme is homologous with adenylate kinases of other eukaryotes and eubacteria. Strongly conserved parts and residues of the molecule are conserved also in this enzyme. Phylogenetic trees obtained with various methods placed the T. vaginalis adenylate kinase close to the point where the different subfamilies of this enzyme branch from each other, indicating that the T. vaginalis enzyme has no close relationship to any of these subfamilies and that it separated early from other adenylate kinases. The conceptual translation predicts the existence of an amino-terminal nonapeptide absent from the protein purified from hydrogenosomes, similar to the processed amino-terminal extensions of other hydrogenosomal proteins. These extensions have been considered as putative targeting and import signals.

Adenylate Kinase↗

Molecular characterization of the alpha-subunit of Trichomonas vaginalis hydrogenosomal succinyl CoA synthetase.

The anaerobic, parasitic protist, Trichomonas vaginalis, is characterized by the absence of mitochondria and the presence of double membrane bound organelles called hydrogenosomes. Succinyl-coenzyme A synthetase is a hydrogenosomal enzyme which catalyzes the formation of ATP via substrate-level phosphorylation. We have characterized genes encoding the alpha subunit of the hydrogenosomal protein succinyl-coenzyme A synthetase (SCS). The alpha-SCS of T. vaginalis is encoded by a multigene family composed of 3 similar genes that do not appear allelic. These 3 alpha-SCS genes encode a protein with a calculated molecular mass of approximately 32.5 kDa that has > 50% identity (> 70% similarity with alpha-SCSs from Escherichia coli, Thermus flavus, and rat liver mitochondria. Antibodies raised against recombinant T vaginalis alpha-SCS expressed in bacteria were used to isolate alpha-SCS proteins from purified hydrogenosomes. These proteins partition into the soluble fraction of hydrogenosomes treated with sodium carbonate at high pH, consistent with a matrix localization in the organelle. Amino-terminal sequencing of purified alpha-SCS proteins shows that mature proteins lack a short, leader sequence of 9 amino acids. These amino terminal sequences which are cleaved from T. vaginalis alpha-SCSs are similar to each other and to all other leader sequences identifed on hydrogenosomal proteins.

Amino Acid Sequence↗

Molecular characterisation of adenosylhomocysteinase from Trichomonas vaginalis.

The enzyme S-adenosylhomocysteine hydrolase (SAHH) has been identified as a potential target for chemotherapy in protozoan parasites including Trichomonas vaginalis. To investigate this area of trichomonad metabolism in more detail, we have isolated and characterised a gene which encodes this activity from the WAA38 strain of this parasite. The gene was isolated by probing a Bg/II genomic mini-library with a fragment of the gene generated by thermal cycling using degenerate oligonucleotide primers. A 5.9-kb Bg/II clone was isolated and has been partially sequenced to reveal a 1458-bp open reading frame which encodes a 486-residue polypeptide (computed molecular mass of 53.4 kDa). The deduced amino acid sequence showed a high degree of sequence similarity to the hydrolases from other species, but was most similar to the enzyme from photosynthetic organisms. The trichomonal sahh gene also contains two "insertion sequences', one of which appears to be unique to this parasite while the second has previously been found only in photosynthetic organisms and in Plasmodium falciparum. Characterisation of the sahh mRNA from T. vaginalis confirmed that both of these insertion sequences (encoding 9 and 37 amino acid residues, respectively) are expressed in the protein product. The sahh mRNA is similar to those characterised from other protozoa in having a short, 12-bp untranslated 5'-leader sequence but the leader sequence does not conform well with the consensus sequence of the other mRNAs. Finally, Southern blots and sequence differences between genomic and cDNA clones indicate that there are multiple copies of the sahh gene in T. vaginalis.

Adenosylhomocysteinase↗

Aspartate:2-oxoglutarate aminotransferase from Trichomonas vaginalis: comparison with pig heart cytoplasmic enzyme.

The aspartate:2-oxoglutarate aminotransferase from the protozoon Trichomonas vaginalis exists as a mixture of sub-forms of identical Mr and amino acid composition, and of similar catalytic properties. The amino acid composition closely resembles that of aspartate aminotransferase from prokaryotic and vertebrate sources. Some molecular and catalytic properties of the T. vaginalis aspartate aminotransferase are compared with those of the cytoplasmic pig heart enzyme. A major difference is in the ability of the trichomonal enzyme to transaminate aromatic amino acids and 2-oxo acids. A range of inhibitors have been used to compare the active-site regions of the T. vaginalis and cytoplasmic pig heart aspartate aminotransferases.

Amino Acids↗

Probing the active sites of aspartate: 2-oxoglutarate aminotransferases from Trichomonas vaginalis and pig heart cytoplasm using substrate analogues.

1. Series of structural analogues of the substrates and products of the aspartate: 2-oxoglutarate aminotransferase reaction have been tested as reversible inhibitors of the purified aspartate aminotransferases from the protozoon Trichomonas vaginalis and from pig heart cytoplasm. 2. The results highlight differences and similarities between the active site regions of the two enzymes which are relevant to a better understanding of the nature of the enzyme/substrate interactions which influence substrate specificity.

Amino Acids↗