William B. Lushbaugh

Genetic Characterization of Trichomonas vaginalis Isolates by Use of Multilocus Sequence Typing

ABSTRACT In this study, we introduce a multilocus sequence typing (MLST) scheme, comprised of seven single-copy housekeeping genes, to genetically characterize Trichomonas vaginalis. Sixty-eight historical and recent isolates of T. vaginalis were sampled from the American Type Culture Collection and female patients at area health care facilities, respectively, to assess the usefulness of this typing method. Forty-three polymorphic nucleotide sites, 51 different alleles, and 60 sequence types were distinguished among the 68 isolates, revealing a diverse T. vaginalis population. Moreover, this discriminatory MLST scheme retains the ability to identify epidemiologically linked isolates such as those collected from sexual partners. Population genetic and phylogenetic analyses determined that T. vaginalis population structure is strongly influenced by recombination and is composed of two separate populations that may be nonclonal. MLST is useful for investigating the epidemiology, genetic diversity, and population structure of T. vaginalis.

Trichomonas vaginalis: Characterization of its glutamate dehydrogenase

An NADP-linked glutamate dehydrogenase (EC 1.4.1.4) was found in the soluble fraction of Trichomonas vaginalis. Its molecular weight was about 230,000 (gel filtration). The enzyme, partially purified by diafiltration and hydroxyapatite column chromatography, was heat stable (1 hr at 57 C). It catalyzed both the amination of alpha-ketoglutarate (mean Km 0.6 mM) and the deamination of glutamate (mean Km 1.2 mM) The optimum pH of the amination reaction was 6.7, and that of the deamination reaction was 8. Glutamate was a competitive inhibitor of the amination reaction (mean Ki 5.6 mM) and alpha-ketoglutarate a partially competitive inhibitor of the deamination reaction (mean Ki 0.45 mM). Both guanosine and inosine diphosphates (1 mM) increased the Km alpha-ketoglutarate fivefold (mean Kis 0.3 and 0.4 mM, respectively). Guanosine diphosphate reduced the Km glutamate 40%. Adenosine di- and triphosphate (1 mM) were ineffective. Because the amination reaction displayed substrate inhibition, guanosine and inosine diphosphates were potent natural inhibitors, and ammonia released by deamination reactions would tend to raise pH (amination operative at acid pH), we hypothesize that the deamination reaction may predominate in the living organism.

Trichomonas vaginalis: analysis of a heat-inducible member of the cytosolic heat-shock-protein 70 multigene family.

Abstract A 2253-nucleotide (nt) transcript for a Trichomonas vaginalis heat-shock protein 70, TVCHSP70, has been isolated that encodes for a protein of 659 amino acids with a predicted molecular weight of 71.3 kDa. TVCHSP70 has a short (10-nt) 5′ untranslated region (UTR), and the 263-nt 3′ UTR is the longest reported for a Trichomonas peptide. Amino-acid sequence analysis and phylogenetic comparison identifies TVCHSP70 as a member of the heat-inducible cytoplasmic HSP70 gene family. Southern-blot data indicate that T. vaginalis contains at least four members of the cytoplasmic HSP70 gene family. Members of the TVCHSP70 family are expressed as 2.3-kb transcripts at low levels during 37 °C culture, and their expression is significantly up-regulated at 43 °C. Slot-blot analysis of seven T. vaginalis clinical isolates demonstrated a 3- to 44-fold up-regulation of TVCHSP70 under conditions of heat shock (43 °C) or oxidative stress (500 μm H2O2) as compared with controls (37 °C).

Three aspecific ATPase in Trichomonas vaginalis

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

Molecular Characterization of a Sarcoplasmic‐Endoplasmic Reticulum Ca+2 ATPase Gene from Trichomonas vaginalis

ABSTRACT. DNA fragments homologous to P‐type cation translocating ATPase genes were identified in Trichomonas vaginalis by polymerase chain reaction (PCR) amplification. The genomic locus corresponding to one PCR fragment, TVCA1, contains a 3,055 base‐pair open reading frame encoding a 108,162 dalton protein composed of 981 amino acids. TVCA1 lacks introns, is present in a single copy, and is expressed as a 3.1 kb transcript with short 5’and 3’untranslated regions. Separate primer extension experiments map the 5’end of the TVCA1 transcript to 12 and 16 nucleotide bases (nt) upstream of the methionine initiation codon. The message polyadenylation site is located 62 nt downstream of the protein termination codon at a CA dinucleotide. The TVCA1 protein sequence shares 57‐58% similarity with rabbit, schistosome, trypanosome and malarial sarcoplasmic‐endoplasmic reticulum calcium (SERCA) pumps, and significantly lower similarity with plasma membrane calcium pumps and cation translocating ATPases of other ion specificities. Structural and functional domains identified in P‐type ATPases as well as 61/68 residues specifically implicated in SERCA pump activity are conserved in TVCA1. However, TVCA1 lacks binding sites for phospholamban regulation, thapsigargin inhibition and the calmodulin dependent protein kinase site phosphorylation present in other SERCA pumps.

Dirofilaria immitis: comparison of cytosolic and mitochondrial glutamate dehydrogenases.

Two membrane-bound glutamate dehydrogenases were found in adult Dirofilaria immitis, an NAD-linked enzyme (EC 1.4.1.2) in the cytosol (C-GDH) and an enzyme equally reactive with NAD or NADP (EC 1.4.1.3) in the mitochondria (M-GDH). The cytosolic enzyme had a pH optimum of 7.8-8.0 and exhibited 30% more activity at 25 C than at 37 C (pH 8.0). The mitochondrial enzyme had a pH optimum at 8.4 and exhibited 27% more activity at 37 C than at 25 C (pH 8.4); it was also more sensitive to heat denaturation. Gel filtration of worm subfractions separated four peaks of C-GDH activity with molecular weights of approximately 610, 285, 180, and less than 100 thousand, and a single major peak of M-GDH activity with a molecular weight of about 335,000. When assayed at pH 8, 37 C, and 200 microM NADH, the Km for the substrate, alpha-ketoglutarate, was equivalent for the two enzymes, but the Km for ADP (activator) was five times greater for M-GDH. When the two enzymes were assayed at pH 8.0, 37 C, and 100 microM NADH, 1 mM ADP approximately doubled and 1 mM ATP halved the velocity observed for each enzyme with no effector present. Under these assay conditions AMP, IDP, GDP, and GTP had opposite effects on the reaction velocities for the two enzymes. When the assay conditions were changed, the effects of added purine nucleotides varied, even directionally. Addition of up to 5 mM glutamate (product) had no significant effect on C-GDH kinetics, nor on the substrate Km of M-GDH.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic Relatedness of Trichomonas vaginalis Reference and Clinical Isolates

We have determined the metronidazole susceptibility status of 20 Trichomonas vaginalis isolates from American Type Culture Collection (ATCC) and assessed the level of genetic relatedness in these isolates using 32 additional T. vaginalis clinical isolates for comparison. These ATCC isolates are commonly used as reference strains in T. vaginalis research and this information provides a rational basis for selection of reference strains for use in comparative studies of T. vaginalis phenotypic and clinical characteristics.

Trichomonas vaginalis: characterization of a family of P-type ATPase genes

P-type ATPases are ion-transporting pumps that enable organisms to control cellular functions and survive changing environmental conditions by regulating internal ion concentrations. Eight P-type ATPases were identified in the amitochondriate protist Trichomonas vaginalis using polymerase chain reaction (PCR) amplification with oligonucleotide primers that recognize conserved motifs present in all P-type ATPases, the ATP phosphorylation site (DKTGTLT) and the ATP binding site (TGDGVND). Phylogenetic analysis and the presence of conserved motifs in predicted peptide sequences identify the Trichomonas ATPases as a sarcoplasmic-endoplasmic reticulum calcium pump (TVCA1); three additional Ca(2+) transporting pumps (TVCA2-4), three phospholipid translocases (TVAPLT1-3), and one P-type ATPase of unknown transport specificity (TVPATP8). Southern blot analyses indicate that the P-type ATPase genes are not linked and are present in single copy, except TVCA2 and TVCA4 which contain additional copies or closely related homologues within the genome. Transcripts of 3.1 kb for TVCA1, 3.0 kb for TVCA2, 2.9 kb for TVCA3, 4.0 kb for TVAPLT1, 4.2 kb for TVAPLT2, 3.9 kb for TVAPLT3, and 3.1 kb for TVPATP8 were detected by Northern blot analysis. No TVCA4 transcript was observed, however, RT-PCR amplification of a transcript product indicates that TVCA4 is expressed. RNA expression of the Trichomonas ATPases, except TVCA3, was constitutive over a range of environmental conditions. TVCA1, TVAPLT3 and TVPATP8 had the highest levels of RNA expression while TVAPLT1 and TVAPLT2 expression was the lowest.