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PubMed1987

Hydrogenosomal ATP:AMP phosphotransferase of Trichomonas vaginalis.

1. ATP:AMP phosphotransferase (adenylate kinase) is present in Trichomonas vaginalis, primarily with hydrogenosomal localization. 2. Adenylate kinase has been purified from hydrogenosome-enriched fractions by solubilization with Triton X-100 and KCl followed by affinity chromatography and gel filtration. 3. The enzyme has a Mr = 28,000, a broad pH optimum of pH 7-9, requirement for Mg2+ and specificity for adenine and deoxyadenine nucleotides. 4. The activity is competetively inhibited by P1,P5-di(adenosine-5') pentaphosphate (Ki 200 nM) and reversibly inactivated by 5,5'-dithiobis-(2-nitrobenzoate). 5. Catalytic properties of this enzyme are similar to those of enzymes from other organisms. Other properties indicate its uniqueness, however, since its molecular mass and Ki for P1,P5-di(adenosine-5'-)-pentaphosphate bring it closer to the mitochrondrial isoenzyme, while it shares a requirement for reduced thiol groups with the cytosolic isoenzyme.

Adenylate Kinase↗
PubMed1991

Three aspecific ATPases in Trichomonas vaginalis.

1. Three aspecific ATPases were found in the sedimentible fractions of Trichomonas vaginalis. 2. One, with a pH optimum of 5.5, was equally activated by Ca2+ or Mg2+, moderately stable, preferred nucleotide diphosphates as substrates, and was inhibited by vanadate, oligomycin, nitrate and Na+. 3. A second, with a pH optimum of 7.5, was activated by Mg2+, preferred guanosine diphosphate as substrate, and was the least stable and most subject to inhibitors (vanadate, oligomycin, NEM, NBD-Cl, azide and Cl-). 4. The third, pH optimum 8.0, was activated by Ca2+, was latent and the most stable, reacted equally well with nucleotide tri- or diphosphates, and was the least susceptible to inhibitors (vanadate and NEM). 5. All exhibited proton-translocating ability.

Adenosine Triphosphatases↗
PubMedDate not supplied

Neonatal Trichomonas vaginalis infection.

Clinical infection with Trichomonas vaginalis (T. vaginalis) in the neonate is an unusual occurrence. We present a case of T. vaginalis in a 2-week-old girl with vaginal discharge. Our patient had complete resolution of symptoms after treatment with metronidazole. We found few references in the medical literature on the treatment of this condition. This report discusses the pathogenesis and treatment of T. vaginalis in the neonate.

Female↗
PubMed2006

Midpregnancy genitourinary tract infection with Chlamydia trachomatis: association with subsequent preterm delivery in women with bacterial vaginosis and Trichomonas vaginalis.

OBJECTIVE: The objective of the study was to estimate whether midpregnancy genitourinary tract infection with Chlamydia trachomatis is associated with an increased risk of subsequent preterm delivery. STUDY DESIGN: Infection with C. trachomatis was determined using a ligase chain reaction assay (performed in batch after delivery) of voided urine samples collected at the randomization visit (16(0/7) to 23(6/7) weeks' gestation) and the follow-up visit (24(0/7) to 29(6/7) weeks) among 2470 gravide women with bacterial vaginosis or Trichomonas vaginalis infection enrolled in 2 multicenter randomized antibiotic treatment trials (metronidazole versus. placebo). RESULTS: The overall prevalence of genitourinary tract C. trachomatis infection at both visits was 10%. Preterm delivery less than 37 weeks' or less than 35 weeks' gestational age was not associated with the presence or absence of C. trachomatis infection at either the randomization (less than 37 weeks: 14% versus 13%, P=.58; less than 35 weeks: 6.4% versus 5.5%, P=.55) or the follow-up visit (less than 37 weeks: 13% versus 11%, P=.33; less than 35 weeks: 4.4% versus 3.7, P=.62). Treatment with an antibiotic effective against chlamydia infection was not associated with a statistically significant difference in preterm delivery. CONCLUSION: In this secondary analysis, midtrimester chlamydia infection was not associated with an increased risk of preterm birth. Treatment of chlamydia was not associated with a decreased frequency of preterm birth.

Anti-Bacterial Agents↗
PubMed2005

HeLa cell nucleus, a source of thymidine for Trichomonas vaginalis growing in vitro.

Trichomonas vaginalis is a parasitic protist incapable of de novo purine and pyrimidine biosynthesis. The lack of these de novo syntheses of nucleotides is supplemented with purine and pyrimidine salvage pathways. Likewise, T. vaginalis is incapable of converting its ribonucleotides to deoxyribonucleotides. Therefore, the parasite must rely on the salvage of exogenous deoxyribonucleosides for DNA synthesis. It has been demonstrated that the parasite can incorporate external adenine and guanine in vitro, but no in vivo nucleotide source has been identified so far. Accordingly, we set out to determine if the parasite could incorporate 3H-thymidine from the nuclei of a cervical-derived cell line into its own DNA. By light and electron microscopy we found that the parasite was able to interact directly, both with mechanically isolated HeLa cell nuclei and with the nuclei released after the disruption of HeLa cell monolayers by the parasite. This study shows that T. vaginalis was capable of incorporating 3H-thymidine from labeled HeLa cells into its own DNA suggesting that the nuclei of this cervical cell line could be an in vivo source of nucleotides for T. vaginalis.

Animals↗
PubMed2006

Location of the cell-binding domain of CP65, a 65kDa cysteine proteinase involved in Trichomonas vaginalis cytotoxicity.

The cysteine proteinase (CP) of 65kDa, CP65, binds to the surface of HeLa cells and is involved in Trichomonas vaginalis cellular damage. To identify and locate the CP65 cellular-binding domain, we enriched the CP65 protein band by ammonium sulfate fractionation and ion-exchange chromatography and the N-terminal sequence was obtained. A 618bp gene fragment was obtained by PCR using genomic DNA as template and primers derived from the N-terminal sequence of CP65 and the Asn papain-catalytic conserved region. This gene fragment encodes for 206 amino acid (aa) residues corresponding to the N-terminal region of a mature CP with 67-76% identity to the reported trichomonad cathepsin-L-like CPs. This gene fragment was expressed in a bacterial system for antibody production and functional analysis. Antibodies against the native trichomonad CP65 recognized the recombinant protein, referred to as rCP65, confirming its relationship with the CP65 gene. The rCP65 protein was bound to the surface of HeLa cells and competed with the native CP65 for binding. Antibodies to the rCP65 (alpha-rCP65) reacted with the trichomonad CP65 located on the parasite surface, and inhibited trichomonal cytotoxicity in a concentration-dependent manner. These data strongly suggest that this gene fragment encodes for the putative cell-binding domain (CBD) of CP65 located at its N-terminal region.

Amino Acid Sequence↗
PubMed2005

N-acetyl-beta-D-hexosaminidase from Trichomonas vaginalis: substrate specificity and activity of inhibitors.

Among chitinolytic activities previously described in Trichomonas vaginalis, N-acetyl-beta-D-hexosaminidase (NAHase) was the enzyme system expressing the highest level of specific activity. We report here some biochemical characteristics of NAHase purified from T. vaginalis. We found at first that the use of 4-methylumbellifferyl-substrate was responsible for a substrate affinity for the enzyme, about 1000-fold higher than those when using p-nitrophenyl-substrates (PNP). Whereas the optimum pH was 7.0 using PNP-substrate, it was at 4.5 using 4-methylumbelliferyl-substrate. Four different substrates were compared for their action on T. vaginalis NAHase and we have found that N-acetyl-beta-D-glucosaminide substrate was the most specific. DTT had no effect on enzyme activity suggesting that thiol group are not involved at the catalytic site. The use of previously described inhibitors showed a positive correlation between trichomonacidal activity and NAHase inhibition.

Animals↗
PubMed2003

Characterization of an ecto-5'-nucleotidase (EC 3.1.3.5) activity in intact cells of Trichomonas vaginalis.

The enzymatic properties of an ecto-5'-nucleotidase were described in Trichomonas vaginalis. The enzyme hydrolyzes nucleoside monophosphates in optimum pH values of 7.5 and 6.5 for the 30236 strain and for the 30238 strain, respectively. Mg(2+) and Ca(2+) were activators of AMP hydrolysis in both strains. The apparent K(m) (Michaelis constant) values for Mg(2+)-AMP were 111.4+/-28.1 microM (mean+/-SD, n=3) for 30236 strain and 420.2+/-35.7 microM (mean+/-SD, n=3) for 30238 strain. The ecto-5'-nucleotidase activity was insensitive to levamisole and tetramisole, inhibitors of alkaline phosphatases, whereas alpha,beta-methylene-ADP inhibited the enzymatic activity of both strains. Our results showed that the AMP hydrolysis presents differences in some kinetic parameters between the two strains investigated. An analysis of the enzymatic chain involved in the ATP hydrolysis to adenosine will contribute to understanding the biochemical aspects of the parasite and the mechanisms related to host-parasite interactions.

5'-Nucleotidase↗
PubMed2005

Trichomonas vaginalis: identification of a triacylglycerol acylhydrolase.

This work describes the identification of a triacylglycerol lipase named TVLip directly onto blood-LB-agar plates by hemolytic screening of a Trichomonas vaginalis cDNA expression library. Sharing significant similarity in the primary sequence with other lipases, the theoretical 3D structure of the TVLip was resolved. The structure reveals the predictive conserved characteristics of other lipases from EC3.1.1.3 group, although presenting one amino acid change in the catalytic triad Ser-His-Asp. Finally, analysis of Northern blot indicates that the expression of the TVLip gene is up-regulated by iron.

Amino Acid Sequence↗
PubMed2004

Malic enzymes of Trichomonas vaginalis: two enzyme families, two distinct origins.

The cytosolic malic enzyme of the amitochondriate protist Trichomonas vaginalis was purified to homogeneity and characterized. The corresponding gene was sequenced and compared with its hydrogenosomal homologue from the same organism. The enzymes were found to differ in coenzyme specificity, molecular mass and physiological role. The cytosolic malic enzyme is a dimer consisting of two 42-kDa subunits with strict specificity for nicotinamide adenine dinucleotide phosphate (NADP(+)), and has a presumed function of pyruvate and NADPH production. The hydrogenosomal malic enzyme is a tetramer of 60-kDa subunits that preferentially utilizes nicotinamide adenine dinucleotide (NAD(+)) to NADP(+). The hydrogenosomal enzyme supplies the hydrogenosome with pyruvate for further catabolic processes linked with substrate-level phosphorylation. Phylogenetic analysis of malic enzymes showed the existence of two distinct families of these enzymes in nature, which differ in subunit size. The trichomonad cytosolic malic enzyme belongs to the small subunit-type family that occurs almost exclusively in prokaryotes. In contrast, the hydrogenosomal malic enzyme displays a close relationship with the large subunit-type family of the enzyme, which is found in mitochondria, plastids and the cytosol of eukaryotes. The eubacterial origin of trichomonad cytosolic malic enzyme suggests an occurrence of horizontal gene transfer from a eubacterium to the ancestor of T. vaginalis. The presence of both prokaryotic and eukaryotic type of malic enzyme in different compartments of a single eukaryotic cell appears to be unique in nature.

Amino Acid Sequence↗
PubMed2004

Two novel asparaginyl endopeptidase-like cysteine proteinases from the protist Trichomonas vaginalis: their evolutionary relationship within the clan CD cysteine proteinases.

Cysteine proteinases (CPs) are important virulence factors of the protozoan parasite Trichomonas vaginalis. A total of six genes coding for cathepsin L-like CPs belonging to clan CA have been identified in T. vaginalis. At least 23 distinct spots with proteolytic activity have been detected by two-dimensional (2-D) substrate gel electrophoresis from in vitro grown parasites; however, only few of them have been characterized. In this work, we detected six spots with proteolytic activity and molecular weights between 25 and 35 kDa. The six proteinases correspond to two distinct CP families: the papain-like family, represented by four spots with pIs between 4.5 and 5.5; and the legumain-like family represented by two spots with pI 6.3 and 6.5. Next, we obtained two cDNAs encoding for legumain-like CPs from T. vaginalis, which were named Tvlegu-1 and Tvlegu-2. The size of these cDNA clones were 1225 and 1364 bp, which encoded for 388 and 415 amino acids, respectively. Their putative translation products have molecular masses of 42.8 and 47.2 kDa, corresponding to inactive legumain-like CP precursors. The two sequences share approximately 40% identity at the amino acid level. These protein products can be classified within a branch of the legumain-like family in clan CD cysteine proteinases due to their sensitivity to specific proteinases inhibitors, their DNA sequences, and phylogenetic reconstruction. However, they do not correspond either to the typical asparaginyl endopeptidase or the glycosylphosphatidylinositol (GPI): protein transamidase subfamilies. These results suggest that the TVLEGU-1 and TVLEGU-2 peptidases are likely to be part of a new subfamily within the legumain-like family of clan CD cysteine proteinases. Furthermore, they could be one of the missing links between prokaryotic and eukaryotic CPs in clan CD enzymes.

Amino Acid Sequence↗
PubMed2006

The activity of protease inhibitors against Giardia duodenalis and metronidazole-resistant Trichomonas vaginalis.

Antiretroviral protease inhibitors were assessed in vitro for their activity against Giardia duodenalis and Trichomonas vaginalis. Kaletra (a co-formulation of ritonavir and lopinavir) was the most effective overall, with 50% effective drug concentrations (EC(50)) of 1.1-2.7 microM (ritonavir concentration) against G. duodenalis and 6.8-8 microM against metronidazole-sensitive and clinically metronidazole-resistant T. vaginalis. Minimal inhibitory concentrations were 2-2.5 microM and 10-50 microM for G. duodenalis and T. vaginalis, respectively. Within the range of human plasma concentrations for ritonavir, only G. duodenalis was inhibited. Lopinavir alone was less inhibitory than ritonavir but was associated with a blockage in cytokinesis of G. duodenalis trophozoites. Saquinavir was not effective. These findings are significant considering the association between human immunodeficiency virus and T. vaginalis, and between G. duodenalis and homosexual behaviour.

Animals↗
PubMed2006

Genotyping Trichomonas vaginalis.

A genotyping method has been developed to distinguish each Trichomonas vaginalis isolate and has provided the first genome mapping studies of this protist with an estimated 180Mb genome. The technique was developed using high molecular weight DNA prepared from five laboratory isolates from Australia and USA and 20 clinical isolates from South Africa. Inhibition of the notorious T. vaginalis endogenous nucleases by addition of potent inhibitors was essential to the success of this study. Chromosomal DNA larger than 2.2Mb was macrorestricted to a minimum segment size of approximately 50kb, separated by pulsed field gel electrophoresis and hybridised with a variety of gene probes. Each isolate generated a unique pattern that was distinguished by each of the probes. Four single copy gene probes (fd, hmp35, ibp39 and pfoD) were identified but probes which identified several bands (pfoB and alpha-scs) per isolate were most informative for genotyping. The pyruvate:ferredoxin oxidoreductase B gene probe identified two to seven copies of pfoB (or its closely related homologue pfoA) per genome in different isolates and is an obvious candidate probe to identify epidemiological linkage between infections by this genotyping method. Cleavage of the genomes into smaller fragments failed to distinguish isolates from diverse locations indicating the proximal regions of genes are conserved.

Animals↗
PubMed2004

Heme-iron increases levels of AP65-mediated adherence by Trichomonas vaginalis.

Trichomonas vaginalis is a protozoan responsible for the number one, non-viral sexually transmitted disease. Surface proteins (AP65, AP51, AP33 and AP23) mediate adherence to vaginal epithelial cells (VECs). Iron increases growth of trichomonads and synthesis and surface placement of adhesins. We observed by immunofluorescence using monoclonal antibody (mAb) 12G4 the placement of AP65 on surfaces of trichomonads supplemented with hemoglobin or hemin as a source of iron. We, therefore, tested the hypothesis that heme-bound iron is an alternative source of iron important to trichomonal growth and regulation of expression of the adhesin genes. Here we show that the inhibition of parasite growth by the iron chelator 2,2-dipyridal is rescued by hemoglobin or hemin, but not protoporphyrin IX. Importantly, trichomonads grown in iron-limiting medium supplemented with free iron, hemoglobin and hemin had elevated levels of ap65 transcript that were 12.6-, 12.3- and 9.2-fold higher, respectively, than low-iron organisms, as determined by RT-PCR. Similarly, the amounts of AP65 were 8.9-, 11.2-, and 4.8-fold higher in parasites grown in free iron, hemoglobin and hemin, respectively, than organisms in low-iron medium. The heme-iron-regulated AP65 increased adherence of parasites to immortalized VECs. Not surprisingly, parasites pretreated with anti-AP65 serum IgG had decreased adherence compared to organisms incubated with prebleed serum IgG. These data illustrate that heme-bound iron is a source of iron similar to lactoferrin. This work extends our findings about the multiple sources of iron for regulating virulence genes of T. vaginalis.

2,2'-Dipyridyl↗
PubMed2006

Trichomonas vaginalis initiator binding protein (IBP39) and RNA polymerase II large subunit carboxy terminal domain interaction.

The core promoter that directs RNA polymerase to the start of transcription in the protist Trichomonas vaginalis is an initiator (Inr) element recognized by the Inr Binding Protein, IBP39. This nuclear protein is composed of two domains: a 14.5 kDa amino (N-terminal) and a 25 kDa carboxy terminal domain (C-domain). Here we describe the identification of an IBP39-interacting protein by screening a T. vaginalis expression library using a two-hybrid system with the IBP39 C-domain as bait. The CTD of the large subunit of RNAP II was found to specifically interact with the C-domain. The specificity and nature of the interaction between the CTD of RNAP II and the C-domain of IBP39 was validated by three independent biochemical methods: co-immunoprecipitation with epitope-tagged proteins, affinity chromatography and enzyme linked ligand sorbent (ELLSA) assays. Binding was shown to involve hydrophobic bonds and to have a disassociation constant (K(d)) of 690 nM (+/-55). These results confirm and extend our previous binding studies using a peptide composed of the last nine amino acids of RNAP II CTD [Schumacher MA, Lau AOT, Johnson PJ. Structural basis of core promoter recognition in a primitive eukaryote. Cell 2003;115:413-24] that predicted an interaction between the CTD and IBP39. These data further demonstrate that IBP39 minimally possesses two functional domains: a N-terminal DNA binding domain (that recognizes the Inr) [Liston DR, Johnson PJ. Analysis of a ubiquitous promoter element in a primitive eukaryote: early evolution of the initiator element. Mol Cell Biol 1999;19:2380-8] and a C-terminal protein binding domain that recognizes the RNAP II CTD, an interaction that may be critical for recruiting RNAP II for initiation of transcription.

Animals↗
PubMed2003

Scanning electron microscopy study of Trichomonas gallinae.

A scanning electron microscopy (SEM) study of Trichomonas gallinae (Rivolta, 1878), provided more information about the morphology of this flagellated protozoan. SEM showed the morphological features of the trophozoites; the emergence of the anterior flagella, the structure of the undulating membrane, the position and shape of the pelta, axostyle and posterior flagellum. Of special interest were the pseudocyst forms.

Animals↗
PubMed2001

Differences between coding and non-coding regions in the Trichomonas vaginalis genome: an actin gene as a locus model(1).

The sequence of a cloned genomic fragment of Trichomonas vaginalis containing a complete actin gene was determined. An uninterrupted open reading frame of 1128 nucleotides was found that codes for an actin gene. Two overlapped consensus promoter sequences for T. vaginalis were found 12 nucleotides upstream the actin initiation codon. In addition to actin, two incomplete open reading frames were found at the 5' and 3' ends of the clone. These two sequences are expressed and showed similarity to adenylate cyclase genes and a yeast hypothetical protein. The overall sequence showed a higher G+C content and a lower frequency of repeated sequences in the coding regions when compared with the non-coding regions. A similar unequal nucleotide distribution was found in various T. vaginalis genes retrieved from data bases.

Actins↗
PubMed2002

Trichomonas vaginalis: microtubule cytoskeleton distribution using fluorescent taxoid.

Trichomonas vaginalis is a flagellated parasitic protist of the human urogenital tract. The parasite has a poorly known cytoskeleton formed by an axostyle and a pelta, which are formed by stable structures such as microtubules, essential for the maintenance of cell shape and organization. FLUTAX-2 is an active fluorescent derivative of Taxol, binds to alphabeta-tubulin dimer polymerized. In this paper we present the analysis of microtubule distribution in living trophozoites of T. vaginalis using FLUTAX-2.

Animals↗