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)