Fine structure and isozymic characterization of trichomonadid protozoa.

Tritrichomonas suis and T. foetus are characterized herein at the ultrastructural and biochemical levels. Microcinematography and measurements, scanning and transmission electron microscopy, cytochemistry for carbohydrate detection (Thiéry technique), and isozyme electrophoresis analysis were performed. In all, 11 different strains from 5 species of parasites were studied (T. foetus, T. suis, Trichomonas gallinae, T. vaginalis, and Monocercomonas sp.). A total of 11 enzymes were scored. Fine-structure study using scanning and transmission electron microscopy demonstrated that T. suis and T. foetus are identical morphologically. The high degree of isozymatic similarity noted between T. suis and T. foetus is consistent with the hypothesis that they may be different strains of the same species.

Purification and basic characteristics of sialidase from Tritrichomonas mobilensis.

Sialidase, produced by Tritrichomonas mobilensis, a colonic parasite of squirrel monkeys, was purified by adsorption on human RBCs in ice-cold culture supernatant followed by release in PBS at 37 degrees C. The enzyme was purified by size-exclusion chromatography of RBC-eluted material giving a single peak with sialidase activity of molecular weight approx. 630 kDa, representing a quadrimer of the complex of three subunits. SDS-PAGE under reducing conditions showed three bands of 56, 61, and 66 kDa, identical with the molecular weight of the three subunits of T. mobilensis sialic acid-specific lectin (Babál et al., 1994). The enzyme treatment changed the agglutination of human RBCs by other lectins: created agglutinability with galactose-specific peanut agglutinin, but did not change agglutination with alpha 2,6 sialic acid linkage-specific Sambucus nigra lectin and linkage nonspecific TML. A 4-fold decrease of the agglutination with alpha 2,3 sialic acid linkage-specific Maackia amurensis lectin was observed. Histochemistry of kidney glomeruli after T. mobilensis sialidase treatment showed peanut agglutinin positivity on membranes of podocytes indicating selectivity for alpha 2,3 linked sialic acid. The sialidase did not hydrolyze colominic acid and was inhibited by 2,3-dehydro-2-deoxy-NeuAc. Divalent cations were not required for activity. The enzyme activity was optimal at pH 6.5-7 with RBCs as substrate and could be stored for 1 year at 4 degrees C.

Some properties of sialic-acid binding systems in Tritrichomonas suis and Tritrichomonas foetus.

Hemagglutination of normal and enzyme-treated red blood cells and its inhibition, in vitro adherence to porcine caecal mucus and kinetic properties of neuraminidase were carried out with Tritrichomonas suis and T. foetus. All tested strains adhered extensively to porcine caecal mucus in vitro and agglutinated human (A1, A2, B and O), rabbit, porcine and hen red blood cells. Different inhibitors were efficacious in hemagglutination activity (HA) tests using neuraminidase treated and untreated red blood cells. The Scatchard plot showed an independent type of cooperativity in porcine strain 41, while in bovine strain KVC-1, a positive type of cooperativity was observed.

Hydrogenosomal succinate thiokinase in Tritrichomonas foetus and Trichomonas vaginalis.

Succinate thiokinase displays a diversity of nucleotide specificity and molecular size throughout Nature. Eukaryotes and Gram-positive bacteria possess distinct 'small' (dimeric) thiokinase enzymes which are specific for adenine (ADP) or guanine (GDP) nucleotides, whereas Gram-negative bacteria contain a single 'large' (tetrameric) enzyme which utilizes both nucleotides. Succinate thiokinase activities, both ADP- and GDP-dependent, were shown to be hydrogenosomal in Tritrichomonas foetus and Trichomonas vaginalis. Surprisingly, the 'small' enzyme was found in T. foetus whereas T. vaginalis contained a 'large' enzyme.

Cleavage of proteins of reproductive secretions by extracellular proteinases of Tritrichomonas foetus.

Cleavage of host defense proteins from reproductive secretions was investigated as a potential virulence mechanism for Tritrichomonas foetus extracellular proteinases. Three categories of susceptibility to digestion were found among the defense proteins tested. Cleavage of fibrinogen, fibronectin, and albumin occurred rapidly with more than 50% of these digested within 30 min. Lactoferrin, immunoglobulin G1, and immunoglobulin G2 were more than 50% digested after 4 h. Transferrin, immunoglobulin M, and immunoglobulin A were the most resistant to the Tritrichomonas foetus extracellular proteinases, since 50% or more of the parent molecule remained after 24 h. The responsible proteinases were classified as cysteine (thiol) proteinases because cleavage was inhibited by the cysteine proteinase specific inhibitor, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane and not by the serine proteinase specific inhibitor, phenylmethylsulfonyl fluoride. In addition, alpha 2-macroglobulin, but not alpha 1-antitrypsin, inhibits the action of the proteinases. The ratio of this naturally occurring inhibitor to the quantity of proteinases released may determine whether the above substrates are cleaved in vivo. Since these substrates are implicated in iron acquisition, cell adherence, and acquired immunity, Tritrichomonas foetus proteinases are likely to play a role in host-parasite interactions.

Characteristics of a leucine aminoacyl transfer RNA synthetase from Tritrichomonas augusta.

This study has investigated the characteristics of a leucine aminoacyl transfer RNA synthetase enzyme from Tritrichomonas augusta. Differential centrifugation and DEAE-cellulose column chromatography were used for partial enzyme purification. The column purification increased the synthetase activity 125-fold over the unfractionated cell extract. The conditions for maximum [3H] leucine charging were 37 degrees C for 20 min, with protein at 180 micrograms ml-1 using yeast leucine tRNA as an acceptor. The optimal reaction conditions were 14 mM-Mg acetate, 3 mM-ATP, 3 mM-spermidine and 5.5 mM-putrescine. Acceptor activity with T. augusta transfer RNA was 8-fold higher than with yeast transfer RNA and 25-fold higher than with Escherichia coli transfer RNA. The partially purified enzyme fraction had comparable changing activities for both leucine and valine.

Proteinases in Trichomonas vaginalis and Tritrichomonas mobilensis are not exclusively of cysteine type.

High molecular weight proteinases of Trichomonas vaginalis (with apparent Mr values 142 and greater than 220 kDa) and Tritrichomonas mobilensis (Mr 67, 86, 104 and 120 kDa), optimally active at pH8, were analysed in gelatin-containing polyacrylamide gels. All of these proteinases were resistant to serine-, aspartic- as well as cysteine proteinase inhibitors. Both proteolytic bands in T. vaginalis and two proteinases in T. mobilensis (67 and 104 kDa) were inhibited by EDTA and EGTA suggesting that they belong to the metallo-proteinase class. The 67 kDa proteinase of T. mobilensis was inhibited also by o-phenanthroline. The other two bands of T. mobilensis (86, 120 kDa) were not classified to any proteinase group since they appeared to be resistant to the chelating agents tested in this study.

Proteolytic activity in Tritrichomonas mobilensis.

Cell extracts of an entero-invasive protozoon of squirrel monkeys, Tritrichomonas mobilensis, contained relatively high proteolytic activity, measured on hide powder azure (HPA). Multiple proteinase forms, optimally active at pH 5-7, were detected by electrophoretic analysis in gelatin-containing polyacrylamide gels. Three major proteinase bands of apparent low molecular weights, Mr 18, 23 and 30 kDa, were seen on gels. Inhibition-activation studies suggest that only cysteine proteinases were involved in HPAase and gelatinolytic activities of T. mobilensis cell extracts.

The specificity of trichomonad cysteine proteinases analysed using fluorogenic substrates and specific inhibitors.

The multiple cysteine proteinases of Trichomonas vaginalis and Tritrichomonas foetus, both those retained intracellularly and those released, were separated using gelatin-SDS-PAGE, and their activity towards a range of 15 fluorogenic peptidyl aminomethylcoumarins determined together with their relative sensitivity to inhibitors. Three types of enzyme were apparent in T. vaginalis: (i) an 86-kDa enzyme active only on Z-Arg-Arg-NHMec; (ii) a 54-kDa proteinase which was most active on Z-Phe-Arg-NHMec but also able to hydrolyse N-t-Boc-Val-Leu-Lys-NHMec, Suc-Ala-Phe-Lys-NHMec, H-Pro-Phe-Arg-NHMec and Z-Arg-Arg-NHMec; and (iii) a group of six enzymes which preferentially hydrolysed substrates with bulky residues at the P2 and P3 positions. N-t-Boc-Val-Leu-Lys-NHMec and H-Leu-Val-Tyr-NHMec were the best substrates for the latter group. The 86-kDa proteinase was inactivated by E-64, but only at high concentrations, and was relatively insensitive to the peptidyl diazomethanes. The other proteinases were inhibited by low concentrations of E-64 and by Z-Phe-Ala-CHN2, and to a lesser extent by Z-Phe-Phe-CHN2. Differences between the proteinases of T. foetus were also demonstrated. All of them were active on Z-Arg-Arg-NHMec, but their activity towards other substrates varied. Three predominantly extracellular proteinases (25, 27 and 34 kDa), hydrolysed Z-Arg-Arg-NHMec specifically. Other proteinases (apparent Mr of 20,000 and 32,000) hydrolysed a number of other substrates, with the 32-kDa enzyme having greater activity towards N-t-Boc-Val-Leu-Lys-NHMec and H-Leu-Val-Tyr-NHMec than towards Z-Arg-Arg-NHMec. At a high concentration (270 microM), E-64 inhibited all of the T. foetus enzymes, but lower concentrations were less effective, with the 18-kDa proteinase being particularly insensitive. Z-Phe-Ala-CHN2 and Z-Phe-Phe-CHN2 were relatively poor inhibitors. The results demonstrate that the proteinases of both species are a heterogeneous group with respect to specificity, and have highlighted significant differences between the enzymes of T. vaginalis and T. foetus. The information on the specificities will be useful for assessing the features required in proteinase inhibitors if they are to be of potential value as antitrichomonal agents.

Mycophenolic acid and thiazole adenine dinucleotide inhibition of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: implications on enzyme mechanism.

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) with the conversion of NAD to NADH. An ordered sequential mechanism where IMP is the first substrate bound and XMP is the last product released was proposed for Tritrichomonas foetus IMPDH on the basis of product inhibition studies. Thiazole adenine dinucleotide (TAD) is an uncompetitive inhibitor versus IMP and a noncompetitive inhibitor versus NAD, which suggests that TAD binds to both E-IMP and E-XMP. Mycophenolic acid is also an uncompetitive inhibitor versus IMP and noncompetitive versus NAD. Multiple-inhibitor experiments show that TAD and mycophenolic acid are mutually exclusive with each other and with NADH. Therefore, mycophenolic acid most probably binds to the dinucleotide site of T. foetus IMPDH. The mycophenolic acid binding site was further localized to the nicotinamide subsite within the dinucleotide site: mycophenolic acid was mutually exclusive with tiazofurin, but could form ternary enzyme complexes with ADP or adenosine diphosphate ribose. NAD inhibits the IMPDH reaction at concentrations greater than 3 mM. NAD substrate inhibition is uncompetitive versus IMP, which suggests that NAD inhibits by binding to E-XMP. TAD is mutually exclusive with both NAD and NADH in multiple-inhibitor experiments, which suggests that there is one dinucleotide binding site. The ordered mechanism predicts that multiple-inhibitor experiments with XMP and TAD, mycophenolic acid, or NAD should have an interaction constant (alpha) between 0 and 1. However, alpha was greater than 1 in all cases.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The release of hydrolases from Trichomonas vaginalis and Tritrichomonas foetus.

Trichomonas vaginalis and Tritrichomonas foetus were found to release large amounts of beta-N-acetylglucosaminidase (EC 3.2.1.30), alpha-mannosidase (EC 3.2.1.24), beta-glucosidase (EC 3.2.1.21), acid phosphatase (EC 3.1.3.2) and proteinases during axenic growth in vitro. The enzymes were released continually throughout the growth phase, with the extracellular activity being of the same order as that within the cells. There was differential release of proteinases from Trichomonas vaginalis. The subcellular localization of the hydrolases was determined by differential and isopycnic centrifugation. The intracellular enzymes were shown to be mostly located within particle populations. Centrifugation on Percoll gradients allowed the separation of sub-populations of the particles in T. vaginalis; two distinct sub-populations were apparent with equilibrium densities in 20% (v/v) Percoll of 1.035 and 1.050 g cm-3 respectively. The higher density particles were rich in the hydrolases released most abundantly, suggesting a possible link between enzyme release and these organelles. Distinct subpopulations of hydrolase-containing particles were not detected in Tritrichomonas foetus. The results demonstrate that hydrolytic enzyme release represents a major activity during trichomonad growth.

Free radical intermediates in the reaction of pyruvate:ferredoxin oxidoreductase in Tritrichomonas foetus hydrogenosomes.

Aerobic incubations of the Tritrichomonas foetus hydrogenosomal fraction containing pyruvate, CoA, and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) gave spectra of two radical adducts. One was a carbon-centered radical adduct of DMPO. This radical was centered at C-3 of pyruvate as determined in experiments using [13C]pyruvate. The other radical detected was identified as the CoA radical adduct of DMPO by comparison with an adduct obtained by incubating CoA with DMPO, H2O2 and horseradish peroxidase. Deletion of CoA led to an increased stability of the carbon-centered radical adduct of DMPO, disappearance of the thiyl radical adduct of DMPO, and appearance of a hydroxyl radical adduct of DMPO. Superoxide dismutase suppressed the appearance of the DMPO-hydroxyl radical adduct but did not have any inhibitory effect on the appearance of the other adducts. Catalase had no significant effect on any of the adducts. Addition of pyruvate to these hydrogenosomal preparations stimulated oxygen consumption. Addition of CoA led to a further increase in the rate of O2 uptake but had no effect in the absence of pyruvate. The formation of two substrate free radicals as intermediates in the generation of acetyl-CoA represents a novel mechanism for this enzymatic reaction and indicates that the pyruvate:ferredoxin oxidoreductase from T. foetus differs significantly from the pyridine nucleotide-dependent pyruvate dehydrogenase complex of other eukaryotic cells in its catalytic mechanism.

Purification, characterization, and kinetic analysis of inosine 5'-monophosphate dehydrogenase of Tritrichomonas foetus.

The IMP dehydrogenase of Tritrichomonas foetus, a parasitic protozoan incapable of de novo biosynthesis of purine nucleotides, has been purified about 1000-fold to apparent homogeneity. The purified enzyme demonstrated a 20-fold higher substrate turnover rate than the pure IMP dehydrogenase from sarcoma ascites tumor cells. It has a subunit molecular weight of 58,000, aggregates to a size of 380,000 at low ionic strength, and partly dissociates to a molecular weight of 270,000 in high salt concentrations. Unlike the IMP dehydrogenase of bacteria and mammals, the T. foetus enzyme does not require K+ for activity. The analysis of initial velocity and product inhibition data is consistent with a sequential, ordered bi bi kinetic mechanism for the parasite enzyme-catalyzed reaction, in which IMP binds before NAD+ and NADH is released before XMP. This is in contrast to the partially random mechanism of the bacterial enzyme which involves the formation of an enzyme-K+-(IMP) complex. Mycophenolic acid inhibits T. foetus IMP dehydrogenase uncompetitively versus both IMP and NAD+ with an apparent Ki of 9 microM. This value, which is several hundred-fold higher than that for mammalian IMP dehydrogenase, suggests significantly different binding properties of the mycophenolic acid site in T. foetus IMP dehydrogenase, which might be amenable to specific inhibitor design.

The use of a highly sensitive electrophoretic method to compare the proteinases of trichomonads.

A highly sensitive electrophoretic method involving gelatin-containing polyacrylamide gels has been used to analyse trichomonad proteinases. Multiple forms, optimally active at pH 5-6, were present in all four species examined, but the species could be distinguished from one another by both quantitative and qualitative differences. The intestinal parasites, Trichomitus batrachorum and Pentatrichomonas hominis, had lower specific activities than the urogenital parasites, Trichomonas vaginalis and Tritrichomonas foetus, and, in the case of P. hominis, there were fewer enzyme forms. The high activity proteinases of Tritrichomonas foetus had low apparent molecular weights (less than 25 kDa), while the predominant enzymes of Trichomonas vaginalis were of high apparent molecular weight (68-110 kDa). Distinct differences were also observed between the proteinase patterns of various isolates of T. vaginalis. All of the enzymes were stimulated by dithiothreitol, suggesting that they were cysteine proteinases. This was confirmed for the T. vaginalis and Tritrichomonas foetus proteinases from their inhibition by antipain, leupeptin, TLCK and iodoacetic acid. The method allows the detection of proteinases in samples of Trichomonas vaginalis containing as few as 10(4) cells or as little as 1 microgram protein. It was also possible to detect proteinase activity released into the medium. For both T. vaginalis and Tritrichomonas foetus, the extracellular enzymes present during early log phase were qualitatively different from the intracellular proteinases, although the latter were present in samples of media obtained from later cultures (cell densities greater than 1 X 10(5) parasites ml-1). The results show the potential of this technique for detecting proteinases in trichomonad samples in studies aimed at determining proteinase function in pathogenesis and host-parasite relationships.

Trichomonas species: homocysteine desulphurase and serine sulphydrase activities.

Homocysteine desulphurase (EC 4.4.1.2) and serine sulphydrase (EC 4.2.1.22) activities in various lines of Trichomonas vaginalis, both metronidazole resistant and sensitive, and other trichomonad species were assessed. T. vaginalis contained the highest homocysteine desulphurase and serine sulphydrase activities of all the species. Although the levels of the enzyme activity in T. vaginalis isolates differed, no correlation between the activities and sensitivity to metronidazole was apparent. T. vaginalis homocysteine desulphurase catalysed both the hydrolysis of homocysteine to hydrogen sulphide, ammonia, and 2-oxoacid, and an exchange reaction between homocysteine and 2-mercaptoethanol. Homocysteine desulphurase was detected as a single enzyme band on isoelectric focusing, whereas several isoenzymes of serine sulphydrase were found. There were large differences in serine sulphydrase isoenzyme patterns between T. vaginalis lines and between species. Several isoenzymes were amplified in cells grown with 10(-5) M DL-propargylglycine for 24 hr. T. vaginalis homocysteine desulphurase and serine sulphydrase activities were inhibited by bithionol, hexachlorophene, and dichlorophene. These compounds also inhibited growth in vitro of T. vaginalis at concentrations similar to those that inhibited the enzymes.

Comparative study of ferredoxin-linked and oxygen-metabolizing enzymes of trichomonads.

1. The activities of pyruvate:methyl viologen oxidoreductase (EC 1.2.7.1), hydrogenase (EC 1.18.99.1), NADH:methyl viologen oxidoreductase (EC 1.6.99.3), NADPH:methyl viologen oxidoreductase (EC 1.6.99.1), NADH oxidase (EC 1.6.99.3) and NADPH oxidase (EC 1.6.99.1) were determined for Trichomonas vaginalis, Tritrichomonas foetus and Trichomitus batrachorum. 2. The three trichomonad species were found to differ significantly, especially with respect to NADH oxidase and NADH:methyl viologen oxidoreductase activities. 3. The species differences in ferredoxin-linked and oxygen-metabolising enzymes may be related to the ways in which the trichomonads are adapted for growth in their respective hosts.

Glycerol, a metabolic end product of Trichomonas vaginalis and Tritrichomonas foetus.

Glycerol was demonstrated as an end product of anaerobic glucose metabolism in Trichomonas vaginalis and Tritrichomonas foetus, produced in addition to acetate, H2, CO2, and lactate or succinate. In T. vaginalis strain C-1, glycerol amounted to 16% of the fermentation products and was formed at an average rate of 38 nmol min-1 (mg protein)-1. Corresponding figures for T. foetus strain KV1 were 7% and 4.8 nmol min-1 (mg protein)-1. The amounts of glycerol detected compensated almost exactly for the deficits in fermentation products recognized earlier, thus complete redox balances can now be provided for both organisms. The metronidazole-resistant T. foetus strain KV1-1MR-100 excreted only negligible amounts of glycerol and carried out an ethanol-CO2 fermentation. Aerobiosis hardly affected glycerol formation in T. vaginalis strains C-1 and NYH 286, but almost completely abolished it in T. foetus strain KV1. An NADP-dependent glycerol 3-phosphate dehydrogenase and a Mg2+-dependent glycerol 3-phosphatase were detected in the cytosol of both species. The phosphatase is distinct from the particle-bound nonspecific acid phosphatase. Glycerol kinase activity was not detected in either organism. Enhanced pCO2 did not affect the ratio of fermentation products in T. vaginalis strain C-1, but significantly increased the amount of succinate, and decreased the amounts of acetate, H2, and CO2, formed by T. foetus.

S-Adenosylhomocysteine hydrolase activity in Trichomonas vaginalis and other trichomonads.

S-Adenosylhomocysteine hydrolase has been detected in crude homogenates of Trichomonas vaginalis, Tritrichomonas foetus and Trichomitus batrachorum at activities of 14, 1.2 and 3.3 nmol min-1 mg-1 protein, respectively. The enzyme from T. vaginalis was found to be soluble with pH optimum of 8.0 and apparent Km values for adenosine and homocysteine of 100 and 155 microM, respectively. Ara A was shown to inhibit the T. vaginalis enzyme but only at relatively high concentration (I50 100 microM), whereas sinefungin and 2'-deoxyadenosine had only small inhibitory effects. EDTA (I50 6 mM) and various divalent cations also inhibited the enzyme from T. vaginalis.

Purification and properties of a secondary alcohol dehydrogenase from the parasitic protozoan Tritrichomonas foetus.

A novel secondary alcohol dehydrogenase has been isolated from Tritrichomonas foetus, the protozoan parasite which is responsible for bovine trichomonal abortion. The enzyme has been obtained in apparently homogeneous form after a 120-fold purification from cell homogenates, thus indicating that this activity constitutes an unusually high 1% of the total cytosolic protein. The native Mr = 115,000, determined by polyacrylamide gel electrophoresis. Mobility on sodium dodecyl sulfate gels suggests that the enzyme is composed of 6-8 subunits, identical as to molecular size (Mr = 17,000). The enzyme catalyzes the reversible oxidation of 2-propanol to acetone, using NADP+ (and not NAD+) as the redox-active co-substrate. Other small secondary alcohols, such as 2-butanol, 2- and 3-pentanol, cyclobutanol, and cyclopentanol are substrates, as are the corresponding ketones of these alcohols. Primary alcohols, such as ethanol and 1-propanol, are oxidized at rates less than 5% of that observed for 2-propanol. Product inhibition studies demonstrate an ordered kinetic mechanism, wherein the co-substrate (NADP+/NADPH) binds to the enzyme prior to binding of the substrate (alcohol/ketone).