Energy metabolism of the anaerobic protozoon Giardia lamblia.

Cells of the aerotolerant anaerobe Giardia lamblia respire in the presence of oxygen. Endogenous respiration is stimulated by glucose but not by other carbohydrates and Krebs cycle intermediates. Endogenous and glucose-stimulated respiration are insensitive to cyanide, malonate, and 2,4-dinitrophenol, but are inhibited by atabrin and iodoacetamide. G. lamblia produces ethanol, acetate and CO2 both aerobically and anaerobically either from endogenous reserves or exogenous glucose. Molecular hydrogen is not produced. The following enzyme activities were detected in homogenates: hexokinase, fructose-biphosphate aldolase, pyruvate kinase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malate dehydrogenase (decarboxylating), pyruvate synthase, acetyl-CoA synthetase, alcohol dehydrogenase (NADP+), NADH dehydrogenase, NADPH dehydrogenase, NADPH oxidoreductase and superoxide dismutase. The enzymes of energy and carbohydrate metabolism are nonsedimentable (109 000 x g for 30 min). Activities of lactate dehydrogenase, hydrogenase, phosphate acetyltransferase, acetate kinase, citrate synthase, succinate dehydrogenase, fumarate hydratase and catalase were below the limits of detection. The results suggest the occurrence of glycolysis, energy production by substrate level phosphorylation and a flavin, iron-sulfur protein mediated electron transport system as well as the absence of cytochrome mediated oxidative phosphorylation and functional Krebs cycle.

Pyrimidine metabolism in Giardia lamblia trophozoites.

The pyrimidine metabolism of Giardia lamblia trophozoites (Portland I strain) was studied using whole trophozoites and trophozoite homogenates. Pyrimidines and pyrimidine nucleosides were readily incorporated into nucleic acids. Orotic and aspartic acid incorporations were below the level of detection. Enzymes of the pyrimidine salvage pathway (i.e., thymidine and uridine phosphorylases and thymidine and uridine kinases) were detected in trophozoite homogenates, but the activities of de novo pyrimidine synthesis enzymes (i.e., carbamoyl-phosphate synthase, aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase) were below the level of detection in these same homogenates. The evidence presented supports the conclusion that G. lamblia trophozoites appear incapable of synthesizing pyrimidines de novo but are capable of salvaging preformed pyrimidines and pyrimidine nucleosides from the growth medium and the enzymes of this pyrimidine salvage pathway are not organelle associated.

Respiration of Tritrichomonas foetus: components detected in hydrogenosomes and in intact cells by electron paramagnetic resonance spectrometry.

Electron paramagnetic resonance spectrometry (EPR) techniques revealed a complex set of redox components in intact respiring cells of Tritrichomonas foetus, an aerotolerant anaerobic protozoon which lacks mitochondria. One of the two main systems of terminal respiration of this organism is the hydrogenosomal system, which oxidizes pyruvate and other substrates. EPR analysis of hydrogenosome-enriched fractions, prepared by differential centrifugation, revealed the presence of at least five redox components with midpoint potentials ranging from -305 to -115 mV; most of these are likely to represent iron-sulfur clusters. Signals indicating relatively stable flavin free radicals were also observed. Involvement of these multiple redox components in the electron transport system of T. foetus hydrogenosomes is suggested.

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.

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.

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)

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.

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.

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.

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.

Pyrimidine metabolism in Tritrichomonas foetus.

The pyrimidine metabolism of Tritrichomonas foetus (KV 1) was studied using whole cells and cell homogenates. Pyrimidines and pyrimidine nucleosides were readily incorporated into nucleic acids. Orotate and aspartate were not incorporated into pyrimidine bases. Enzymes of the pyrimidine salvage pathway (i.e., thymidine and uridine phosphorylases and uridine kinase) were detected in trophozoite homogenates, but the activities of de novo pyrimidine synthesis enzymes (i.e., carbamoylphosphate synthase, aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase) were below the level of detection in these same homogenates. The evidence presented supports the proposal that T. foetus is incapable of synthesizing pyrimidines de novo but is capable of salvaging preformed pyrimidines and pyrimidine nucleosides from the growth medium and that enzymes of this parasite's pyrimidine salvage pathway are not organelle-associated.

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.

Activation of Polynuclear Aromatic Hydrocarbons to Mutagens by the Marine Ciliate Parauronema acutum.

The marine ciliate Parauronema acutum converted 2-aminofluorene and 2-acetylaminofluorene to compounds with mutagenic activity in the Ames Salmonella test. The ciliate, however, did not activate benzo (alpha)pyrene or benzanthracene or destroy the mutagenic properties of nitrosoguanidine. Homogenates, when substituted for the liver S-9 fraction in the Salmonella/microsome test, activated 2-aminofluorene and 2-acetylaminofluorene to mutagens. Benzo(alpha)pyrene and benzanthracene were not activated, nor was nitrosoguanidine inactivated. Phenobarbitol did not induce or increase the amount of activating activity. The activation showed no requirement for the reduced nicotinamide adenine dinucleotide phosphate-regenerating system required by liver P-450 cytochromes. Upon differential sedimentation of a cell homogenate, the majority of the activity sedimented with a small-particle fraction with sedimentation properties like those of microsomes from higher eucaryotes. Benzo(alpha)pyrene, although not metabolized, was accumulated by cultures of P. acutum at a linear rate and was not appreciably released (10%) after removal of benzo(alpha)pyrene from the incubation medium. Hence, this ciliate could convert certain polynuclear aromatic hydrocarbons to mutagens and accumulate others.

Antitrichomonad action, mutagenicity, and reduction of metronidazole and other nitroimidazoles.

Twelve 4- and 5-nitroimidazole derivatives, including metronidazole and two of its metabolites, tinidazole, dimetridazole, and nimorazole, were tested for antitrichomonad action on Tritrichomonas foetus (KV(1)) and Trichomonas vaginalis (ATCC 30001) for mutagenicity on a nitroreductase-positive (TA 100) and a nitroreductase-deficient (TA 100-FR(1)) strain of Salmonella typhimurium, as well as for the reducibility of the nitro group by T. foetus homogenates. Compounds with activity <1% of that of metronidazole are regarded as inactive. All antitrichomonad compounds induce mutations and can be reduced. S. typhimurium TA 100 gave mutations under both aerobiosis and anaerobiosis; TA 100-FR(1), however, gave mutations only under anaerobiosis. Certain compounds that are reducible, and the nonreducible derivatives, were inactive. Metronidazole and its inactive 4-nitro analogue were reduced in a four-electron process in ferredoxin- or methyl viologen-mediated reactions with the same velocity. The results underscore the role of the reduction of the nitro group in the antitrichomonad and in the mutagenic activity of nitroimidazoles.

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.

Uptake of metronidazole and its effect on viability in trichomonads and Entamoeba invadens under anaerobic and aerobic conditions.

[(14)C]metronidazole used at the chemotherapeutic concentration of 10 mug/ml is taken up rapidly by the anaerobic protozoa Tritrichomonas foetus, Trichomonas vaginalis, and Entamoeba invadens kept under anaerobic conditions. It can be calculated that within 30 to 60 min the intracellular concentration of the label is 50 to 100 times higher than in the medium. The presence of air markedly suppresses the uptake in the trichomonads and abolishes it in E. invadens. The suppression disappears after anaerobic conditions are established. The rate of uptake in T. foetus is dependent on the concentration of the drug in the range studied (1 to 200 mug/ml). Analysis of double reciprocal plots suggests that the drug enters the cells predominantly or exclusively by diffusion. The major factor driving the uptake is most likely the intracellular biotransformation of the compound. If less than 3 mug of drug per mg of protein is taken up by T. foetus no decrease in viability is observed. Above this level the cytotoxic activity corresponds roughly to the amount accumulated in the cell, irrespective of whether the conditions are anaerobic or aerobic.