Subcellular localization of the enzymes of the arginine dihydrolase pathway in Trichomonas vaginalis and Tritrichomonas foetus.

The enzymes of the arginine dihydrolase pathway were demonstrated in Tritrichomonas foetus and their subcellular localization determined for both T. foetus and Trichomonas vaginalis. Ornithine carbamyltransferase (anabolic and catabolic activities), ornithine decarboxylase and carbamate kinase activity were localized predominately (56-80%) in the non sedimentable fraction of both species. A large proportion (35-40%) of the arginine deiminase was, however, recovered in the large granular fraction, and this distribution was unchanged by increasing the ionic strength of the buffer. Upon density gradient centrifugation the particles containing arginine deiminase activity had an isopycnic density of 1.09 g/ml in percoll, and separated from hydrogenosomes (1.18 g/ml) and lysosomes (1.12 g/ml). Arginine deiminase was also the only enzyme of the dihydrolase pathway which demonstrated latency upon treatment of the 1.09 g/ml fraction with non-ionic detergents. The results demonstrate the presence of the arginine dihydrolase pathway in T. foetus and indicate that at least a portion of the arginine deiminase in trichomonads is membrane associated.

Polypeptides of hydrogenosome-enriched fractions from rumen ciliate protozoa and trichomonads: immunological studies.

The evolution of hydrogenosomes, energy-generating organelles of rumen ciliate protozoa and the flagellate trichomonads has been the subject of much speculation. Polypeptides of the hydrogenosome-enriched fractions from the rumen ciliates, Dasytricha ruminantium, Isostricha spp., Polyplastron multivesiculatum and Eudiplodinium maggii were separated by SDS-PAGE and compared to analogous polypeptide preparations from Tritrichomonas foetus. Immunoblotting with antisera specific to the hydrogenosomes of T. foetus identified common immunoreactive polypeptides present at estimated molecular masses of 28, 35, 38, 44, 48, 58, 100 and 120 kDa. That at 120 kDa corresponds to a single subunit of the purified pyruvate:ferredoxin oxidoreductase from the hydrogenosome of Trichomonas vaginalis.

Identification of the lipid moiety and further characterization of the novel lipophosphoglycan-like glycoconjugates of Trichomonas vaginalis and Trichomonas foetus.

The lipid moiety of the lipophosphoglycan (LPG)-like glycoconjugates of Trichomonas vaginalis and Trichomonas foetus, parasites of the urogenital tract of human and cattle, respectively, has been isolated and characterized by a combination of enzymatic and chemical degradation, chromatography, and mass spectrometry. The carbohydrate composition of the glycan inositol lipid core is also reported. The glycan inositol core of trichomonad glycoconjugates is unique in having more than one GlcN and is significantly larger than any other glycan core reported so far. T. vaginalis glycoconjugate binds strongly to the lectin RCA-I, which suggest that the macromolecule possesses terminal beta 1,4-linked galactosyl residues. The binding of T. foetus glycoconjugate to the lectin UEA-I suggests the presence of terminal alpha 1,2-linked fucose. Acid hydrolysis of deaminated and reduced LPG products yields a [3H]anhydromannitol-containing product, indicating the presence of unacetylated glucosamine in the trichomonad LPGs. Reductive radiomethylation has been applied to label free amino groups in the hexosamine or other free amine-containing residues of the trichomonad glycoconjugates. Treatment of the LPGs with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis liberates a ceramide substituent. Treatment of LPGs with nitrous acid releases a phospholipid moiety containing myo-inositol and ceramide, implying that the LPGs are anchored in the membrane via an inositol-phosphate-ceramide. Structural characterization of the ceramide by gas-liquid chromatography (GC) and GC-mass spectrometry indicated the presence of the major long-chain base sphinganine (d 18: 0 dihydrosphingosine) and a C 16:0 N-acyl group. Lipophosphoglycans from both parasites contain ceramide as their only lipid moiety. These results suggest that T. vaginalis and T. foetus anchor their LPG-like glycoconjugates on the cell surface via inositol-phosphoceramide and also the glycan inositol core of the macromolecule appears to be unique in nature.

The effects of oxygen on fermentation in Giardia lamblia.

Detailed study of the effects of oxygen on the carbohydrate metabolism of Giardia lamblia revealed that low concentrations of oxygen (< 0.25 microM) produced profound alterations in the carbon balance of this organism. Although this concentration of oxygen could not be detected by mass spectrometry, a marked stimulation of ethanol production was observed. Associated with this was an inhibition of alanine production and oxidation of the intracellular NAD(P)H pool. Higher concentrations of oxygen inhibited ethanol production and further reduced levels of alanine. These results suggest that this stimulation is due to changes in carbon flux. Analysis of cell and medium hydrolysates after the growth of trophozoites in [U-14C]glucose suggests that G. lamblia does not synthesise detectable levels of labelled amino acids, except alanine and to a lesser extent valine, from this sugar. Trophozoites of G. lamblia have both glutamate dehydrogenase and alanine aminotransferase activity. As glutamate is taken up from the medium, it is suggested that glutamate dehydrogenase and alanine aminotransferase cooperate to convert pyruvate to alanine, with the concomitant oxidation of NAD(P)H.

Galactosamine-synthesizing enzymes are induced when Giardia encyst.

Galactosamine, a Giardia filamentous cyst wall specific-sugar, is below the limits of detection in non-encysting trophozoites. Radiolabeling studies suggest that Giardia synthesize galactosamine primarily from endogenous glucose rather than salvage it from the environment. Enzymes responsible for galactosamine synthesis from glucose are induced during encystment and have been characterized in crude homogenates and in supernatant (soluble) fractions. These enzymes (specific activity; time after encystment is induced for maximal activity; x-fold increase) include glucosamine 6-phosphate isomerase (in the deaminating direction, 167 mU mg protein-1; 20 h; x 182-fold; in the aminating direction, 258 mU mg protein-1; 20 h; x 13-fold), glucosamine 6-phosphate N-acetylase (11 mU mg protein-1; 20 h; x 20-fold), phosphoacetylglucosamine mutase (160 mU mg protein-1; 20 h; x 12-fold), UDP-N-acetylglucosamine pyrophosphorylase (22 mU mg protein-1; 48 h; x 8-fold), and UDP-N-acetylglucosamine 4'-epimerase (13 mU mg protein-1; 48 h; x 4000-fold). This represents the first report of these enzymes and of an inducible carbohydrate-synthesizing pathway in any protozoan.

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.

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)

Tritrichomonas foetus: purification and characterization of hydrogenosomal ATP:AMP phosphotransferase (adenylate kinase).

The hydrogenosomal enzyme ATP:AMP phosphotransferase (adenylate kinase) (EC 2.7.4.3) was purified to apparent homogeneity from the bovine parasite Tritrichomonas foetus. A fraction enriched for hydrogenosomes was obtained from cell homogenates which had been subjected to differential and isopycnic centrifugation. Adenylate kinase was solubilized in 50 mM Tris-HCl, pH 7.3, containing 0.8% Triton X-100, and purified by sequential Affi-Gel blue affinity chromatography and high-performance liquid chromatography gel filtration. The purified enzyme, a monomer of Mr 29,000, exhibited Km values of 100, 195, and 83 microM for ADP, ATP, and AMP, respectively. Substituting other mono-, di-, and trinucleotides for AMP, ADP, and ATP gave less than half the maximal activity. Full enzyme activity requires Mg2+, but Mn2+ and Co2+ yield half maximal activity. The enzyme has a broad optimal pH range between pH 6 and 9. The enzyme was competitively inhibited by P1,P5-di(adenosine-5')pentaphosphate, a specific adenylate kinase inhibitor: the Ki was 150 nM. The enzyme was also inhibited with 5,5'-dithiobis(2-nitrobenzoic acid), and this inhibition could be reversed by the addition of 2 mM dithiothreitol. T. foetus adenylate kinase has similar catalytic and physical properties to that of the biologically closely related human parasite Trichomonas vaginalis.

Biochemistry and metabolism of Giardia.

Giardia lamblia, an aerotolerant anaerobe, respires in the presence of oxygen by a flavin, iron-sulfur protein-mediated electron transport system. Glucose appears to be the only sugar catabolized by the Embden-Meyerhof-Parnas and hexose monophosphate pathways, and energy is produced by substrate level phosphorylation. Substrates are incompletely oxidized to CO2, ethanol and acetate by nonsedimentable enzymes. The lack of incorporation of inosine, hypoxanthine, xanthine, formate or glycine into nucleotides indicates an absence of de novo purine synthesis. Only adenine, adenosine, guanine and guanosine are salvaged, and no interconversion of these purines was detected. Salvage of these purines and their nucleosides is accomplished by adenine phosphoribosyltransferase, adenosine hydrolase, guanosine phosphoribosyltransferase and guanine hydrolase. The absence of de novo pyrimidine synthesis was confirmed by the lack of incorporation of bicarbonate, orotate and aspartate into nucleotides, and by the lack of detectable levels of the enzymes of de novo pyrimidine synthesis. Salvage appears to be accomplished by the action of uracil phosphoribosyltransferase, uridine hydrolase, uridine phosphotransferase, cytidine deaminase, cytidine hydrolase, cytosine phosphoribosyltransferase and thymidine phosphotransferase. Nucleotides of uracil may be converted to nucleotides of cytosine by cytidine triphosphate synthetase, but thymidylate synthetase and dihydrofolate reductase activities were not detected. Uptake of pyrmidine nucleosides, and perhaps pyrimidines, appears to be accomplished by carrier-mediated transport, and the common site for uptake of uridine and cytidine is distinct from the site for thymidine. Thymine does not appear to be incorporated into nucleotide pools. Giardia trophozoites appear to rely on preformed lipids rather than synthesizing them de novo.(ABSTRACT TRUNCATED AT 250 WORDS)

Giardia lamblia: characterization of proteinase activity in trophozoites.

The proteinase activity of Giardia lamblia trophozoites, Portland 1 strain, was characterized with respect to substrate specificities and inhibitor sensitivities. Proteinase activity with urea-denatured hemoglobin (UDH), alpha-N-benzoyl-DL-arginine-2-naphthylamide (BANA), and alpha-N-benzoyl-argininamide (BAA) as substrates exhibited pH optima of 5.8, 3.8, and 5.0, respectively. For BANA, the apparent Km was 0.20 mM and the Vmax was 2.56 microM. For BAA, the apparent Km was 4.0 mM and the Vmax was 8.69 microM. Dithiothreitol (DTT, 5 mM) enhanced proteinase activity threefold for UDH, fourfold for BAA, and fivefold for BANA. Iodoacetamide, L-tosylamide-2-phenylethyl chloromethyl ketone (TPCK), and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), each at 1 mM, inhibited proteinase activity by greater than 90% with BANA and BAA. Iodoacetamide inhibited proteinase activity by 35% with UDH; TPCK and TLCK inhibited activity greater than 70% with UDH. Activity on BAA was inhibited by 91% with Zn2+ and activity on UDH was inhibited by 30% with Cu2+. Virtually complete inhibition of proteinase activity on BANA and BAA was obtained with leupeptin and chymostatin at 1 microgram/ml. Pepstatin A, chelators, and other heavy metals had no apparent effect on proteinase activity. Two polypeptide bands (ca. 105 and 40 kDa) indicative of proteinase activity were visualized by sodium dodecyl sulfate-gelatin polyacrylamide gel electrophoresis. The 105 kDa band was visible over the pH range of 4 to 7, but with greater intensity from pH 5 to 7. The 40 kDa band, while present at pH 5, was most intense at pH 6 and 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Respiration in the cysts and trophozoites of Giardia muris.

Cysts and trophozoites of the parasitic protozoon Giardia muris both showed respiratory activity but respiration in cysts was only 10 to 20% that of trophozoites. The O2 dependence of respiration in cysts and trophozoites showed O2 maxima above which respiration decreased. The O2 concentration at which the respiration rate was greatest was higher for cysts than trophozoites. The effects of various inhibitors on cyst and trophozoite respiration suggested that flavoproteins and quinones play some role in respiration. The substrate specificities and the effects of inhibitors on G. muris trophozoites were similar to those observed for Giardia lamblia. Metronidazole, the drug most commonly used in the treatment of giardiasis completely inhibited respiration and motility in trophozoites; however, it had no effect on either respiration or viability in cysts. Menadione, a redox cycling naphthoquinone, stimulated then completely inhibited respiration in cysts and trophozoites; a complete loss of cyst viability or trophozoite motility was also observed. The effects of menadione on G. muris may indicate that redox cycling compounds have potential as chemotherapeutic agents for the treatment of giardiasis.

Giardia cyst wall-specific carbohydrate: evidence for the presence of galactosamine.

Gas chromatographic (GC), mass spectrometric (MS), lectin binding and enzymatic analyses of the carbohydrates from Giardia cyst walls, intact cysts and trophozoites were performed to investigate the carbohydrate composition of Giardia cyst walls and to test the hypothesis that the Giardia cyst wall is composed largely of chitin. Galactosamine, verified by MS, was present in Giardia cyst walls and intact cysts (ca. 47 nmol 10(-6) cysts). Since not even trace amounts of it were detected in trophozoites by either GC or lectin binding, galactosamine is hypothesized to be a cyst wall-specific amino hexose. Based on the putative binding affinity of Phaseolus limensis lectin, galactosamine may be present in cyst walls as N-acetylgalactosamine. Neither glucosamine nor sialic acid were detected in as much as 11 mg dry weight of cysts, cyst walls, or trophozoites. Glucose, the most abundant carbohydrate, and ribose were detected in Giardia cysts and trophozoites. Galactose (ca. 10 nmol 10(-6) cysts) was detected in cysts but not in trophozoites. The lack of detectable levels of (1) glucosamine in cyst wall hydrolysates, (2) cyst staining by Calcofluor M2R, (3) endogenous chitinase activity and (4) N-acetylglucosamine when cysts served as a substrate for exogenous chitinase suggests that the Giardia cyst wall is not composed largely of chitin as previously reported. beta-N-Acetylgalactosaminidase, EC 3.2.1.32, activity was detected in cysts and trophozoites and represents the first carbohydrate splitting hydrolase detected in Giardia.

Giardia lamblia: localization of hydrolase activities in lysosome-like organelles of trophozoites.

Homogenates of Giardia lamblia trophozoites exhibited the following hydrolase activities: acid phosphatase (EC 3.1.3.2), proteinase (EC 3.1.4) with urea-denatured hemoglobin and N-benzoyl-DL-arginine-2-naphthylamide as substrates, deoxyribonuclease (EC 3.1.4.5), and ribonuclease (EC 2.7.7.16). beta-N-Acetylglucosaminidase (EC 3.2.1.30), beta-galactosidase (EC 3.2.1.23), beta-glucuronidase (EC 3.2.1.31), alpha-D-glucosidase (EC 3.2.1.20), beta-D-glucosidase (EC 3.2.1.21), and beta-D-xylosidase (EC 3.2.1.37) activities were below the level of detection. Differential and isopycnic centrifugation of homogenates demonstrated that giardial hydrolases were localized in a single-particle population sedimenting at 7200g for 30 min. The particles had a buoyant density in sucrose of 1.15 and exhibited latency. Latency was completely destroyed by Triton X-100 or 15 cycles of freezing and thawing. After centrifugation of Triton- or freeze-thaw-treated particle fractions, the hydrolase activities, though no longer latent, were still sedimentable suggesting tight binding to the organelle membrane. Latency was destroyed simultaneously for all hydrolases, in direct proportion to the amount of Triton added to a particle preparation or to the number of times a particle preparation was subjected to freezing and thawing. These results support the suggestion that the hydrolases of G. lamblia trophozoites are localized in a single-particle population of lysosome-like organelles.

Giardia lamblia: uptake of pyrimidine nucleosides.

The aerotolerant, anaerobic parasite Giardia lamblia, which depends solely upon salvage pathways for its pyrimidine requirements, was found to transport uridine, cytidine, and thymidine by a carrier mediated mechanism. Support for this conclusion comes from the facts that uptake of radiolabeled uridine, cytidine, and thymidine exhibited saturation kinetics, and uptake of these same radiolabeled nucleosides was inhibited by unlabeled homologs, certain pyrimidine analogs, iodoacetate, and N-ethylmaleimide. Uridine and cytidine (perhaps uracil and cytosine also) are postulated to be transported at a common site which is distinct from the site for thymidine transport. Thymidine does appear to bind nonproductively to the uridine/cytidine transport site, but the reverse of this does not appear to occur.

Extraction and partial characterization of surfactant-soluble antigens from adult female Dirofilaria immitis.

Antigens were solubilized from aqueous-insoluble material of adult female Dirofilaria immitis with Triton X-100 and sodium dodecyl sulfate (SDS) and partially characterized. Concentrations of 2.1-8.3 mg Triton X-100/mg total protein and 1.06-4.26 mg SDS/mg total protein were used to solubilize antigens. Triton X-100 was more efficient in solubilizing protein than SDS; Triton X-100 and SDS released 75% and 26% of the available protein, respectively. Protein mixtures solubilized with different levels of Triton X-100 appeared to be identical in immunodiffusion. Crossed immunoelectrophoresis revealed six antigen systems. Triton X-100 solubilized antigens did not react in immunodiffusion with sera from 7 D. immitis-infected, microfilaremic dogs, but did react with sera from 4 of 5 dogs with occult D. immitis infections.