Hydrogenosome behavior during the cell cycle in Tritrichomonas foetus

The hydrogenosome is an unusual organelle found in several trichomonad species and other protists living in oxygen poor or anoxic environments. The hydrogenosome behavior in the protist Tritrichomonas foetus, parasite of the urogenital tract of cattle, is reported here. The hydrogenosomes were followed by light and transmission electron microscopy during the whole cell cycle. Videomicroscopy, immunofluorescence microscopy, and immunocytochemistry were also used. It is shown that the hydrogenosomes divide at any phase of the cell cycle and that the organellar division is not synchronized. During the interphase the hydrogenosomes are distributed mainly along the axostyle and costa, and at the beginning of mitosis migrate to around the nucleus. Three forms of hydrogenosome division were seen: (1). segmentation, where elongated hydrogenosomes are further separated by external membranous profiles; (2). partition, where rounded hydrogenosomes, in a bulky form, are further separated by a membranous internal septum and, (3). a new dividing form: heart-shaped hydrogenosomes, which gradually present a membrane invagination leading to the organelle division. The hydrogenosomes divide at any phase of the cell cycle. A necklace of intramembranous particles delimiting the outer hydrogenosomal membrane in the region of organelle division was observed by freeze-etching. Similarities between hydrogenosomes and mitochondria behavior during the cell cycle are discussed.     

Hydrogenosome autophagy: an ultrastructural and cytochemical study.

The process of autophagy was studied in Tritrichomonas foetus under serum deprivation, drug treatment (hydroxyurea, zinc sulfate), and also in normal conditions using routine electron microscopy, freeze-fracture, freeze-substitution, and enzyme cytochemistry. We also used gold particles conjugated with bovine albumin to better characterize the participation of lysosomes in the process of hydrogenosome degradation. Apparently normal hydrogenosomes and also giant, abnormal hydrogenosomes presenting internal membranes were seen in the autophagic process. The first event observed was the rough endoplasmic reticulum surrounding and enclosing the hydrogenosome, forming an isolation membrane. The hydrogenosomes were first sequestered from the remaining cytoplasm and then degraded within lysosomes. The autophagic vacuoles were limited by double or multiple concentric membranes and many contained recognizable hydrogenosomes, probably in the preliminary steps of degradation. Lysosomes seemed to fuse with autophagic vacuoles forming a degradative structure bound by a single membrane and containing hydrogenosomes in various stages of degeneration. Hydrogenosomes appeared partially degraded, forming hydrogenosomal remnants. It was observed that there is a removal of hydrogenosomes in normal cells and in cases of cell toxicity.     

Morphogenesis of the hydrogenosome: An ultrastructural study

Summary— The morphogenesis of hydrogenosomes in several trichomonad species (Tritrichomonas foetus, Trichomonas vaginalis, Tritrichomonas suis, Trichomonas gallinae, Tritrichomonas augusta and Monocercomonas sp) was investigated by transmission electron microscopy of thin sections and freeze-fracture replicas of whole cells or the isolated organelle. Close proximity, and even continuity, between endoplasmic reticulum and hydrogenosomes was observed. Structures were seen connecting hydrogenosomes to each other and to cytoplasmic structures. Morphological evidence is presented showing that in all the trichomonads here studied, hydrogenosomes, like mitochondria, may divide by two distinct processes: segmentation and partition. In the segmentation process, the hydrogenosome grows, becoming enlongated with the appearance of a constriction in the central portion. Microfibrillar structures appear to help the furrowing process, ending with a total fission of the organelle. In the partition process, the division begins by an invagination of the inner hydrogenosome membrane, forming a transversal septum, separating the organelle matrix into two compartments. We suggest that myelin-like structures seen either in close contact with or in the vicinity of the hydrogenosomes may be a source of membrane lipids for hydrogenosome growth.     

Hydrogenosomes in a mixed isolate of Isotricha prostoma and Isotricha intestinalis from ovine rumen contents

1. Both Isotricha intestinalis and I. prostoma possess microbody-like organelles, with a highly granular appearance. 2. These organelles, which are sedimentable at 10(5) g-min, bear no morphological similarity to mitochondria, but are enzymatically similar to organelles possessed by certain other anaerobic protozoa and termed hydrogenosomes. 3. The hydrogenosomes isolated from a preparation of mixed isotrichs bear a closer similarity to those isolated from the other rumen holotrich. Dasytricha ruminantium, than those recently identified in a mixed entodiniomorph preparation, or the trichomonads, in that the enzyme malate dehydrogenase (decarboxylating) is non-sedimentable and phosphoacetyl transferase together with acetate kinase are involved in the transformation of acetyl CoA to acetate. 4. The results enable a scheme of acetate, CO2 and H2 formation from carbohydrates to be proposed and extends the number of protozoa known to possess this organelle.     

Ultrastructural characterization of the isolated hydrogenosome in Tritrichomonas foetus

In the present study we show new aspects of the hydrogenosome ultrastructure as well alterations induced by the fractionation technique. The morphology of freshly isolated hydrogenosomes as well those found in whole cells of Tritrichomonas foetus were examined in thin-sections, in replicas of fast-freezing, and conventional freeze-fracture, freeze-etching, and by high resolution scanning electron microscopy (field emission in-lens scanning electron microscopy). The true surface as well the concave and convex fracture faces of the inner and outer membranes are shown. We showed that after fractionation procedures the hydrogenosome ultrastructure can be changed, since isolated hydrogenosomes present patchwork-like structures, rosettes and the inner hydrogenosomal membrane is displaced. The peripheral vesicle is seen as a distinct compartment, since its content and morphological appearance is quite different from the rest of the organelle. The peripheral vesicle shows a smooth surface but presenting pores with 20 nm in diameter with a density of 7/micron 2 when observed after freeze-etching. We report the existence of characteristic intramembrane particles distribution and density on hydrogenosome membranes of isolated and whole T. foetus, suggesting that this organelle can have its morphology changed as consequence of technical modifications or as expression of its metabolic state.     

Trichomonads, hydrogenosomes and drug resistance

Trichomonas vaginalis and Tritrichomonas foetus are sexually transmitted pathogens of the genito-urinary tract of humans and cattle, respectively. These organisms are amitochondrial anaerobes possessing hydrogenosomes, double membrane-bound organelles involved in catabolic processes extending glycolysis. The oxidative decarboxylation of pyruvate in hydrogenosomes is coupled to ATP synthesis and linked to ferredoxin-mediated electron transport. This pathway is responsible for metabolic activation of 5-nitroimidazole drugs, such as metronidazole, used in chemotherapy of trichomoniasis. Prolonged cultivation of trichomonads under sublethal pressure of metronidazole results in development of drug resistance. In both pathogenic species the resistance develops in a multistep process involving a sequence of stages that differ in drug susceptibility and metabolic activities. Aerobic resistance, similar to that occurring in clinical isolates of T. vaginalis from treatment-refractory patients, appears as the earliest stage. The terminal stage is characterised by stable anaerobic resistance at which the parasites show very high levels of minimal lethal concentration for metronidazole under anaerobic conditions (approximately 1000 microg ml(-1)). The key event in the development of resistance is progressive decrease and eventual loss of the pyruvate:ferredoxin oxidoreductase so that the drug-activating process is averted. In T. vaginalis at least, the development of resistance is also accompanied by decreased expression of ferredoxin. The pyruvate:ferredoxin oxidoreductase deficiency completely precludes metronidazole activation in T. foetus, while T. vaginalis possesses an additional drug-activating system which must be eliminated before the full resistance is acquired. This alternative pathway involves the hydrogenosomal malic enzyme and NAD:ferredoxin oxidoreductase. Metronidazole-resistant trichomonads compensate for the hydrogenosomal deficiency by an increased rate of glycolysis and by changes in their cytosolic pathways. Trichomonas vaginalis enhances lactate fermentation while T. foetus activates pyruvate conversion to ethanol. Drug-resistant T. foetus also increases activity of the cytosolic NADP-dependent malic enzyme, to enhance the pyruvate producing bypass and provide NADPH required by alcohol dehydrogenase. Production of succinate by this species is abolished. Metabolic changes accompanying in-vitro development of metronidazole resistance demonstrate the versatility of trichomonad metabolism and provide an interesting example of how unicellular eukaryotes can adjust their metabolism in response to the pressure of an unfavorable environment.     

Cardiolipin in hydrogenosomes: evidence of symbiotic origin

Hydrogenosomes are found in organisms that lack typical mitochondria. Cardiolipin is a phospholipid located exclusively in bacterial membranes and the inner membrane of mitochondria. Here we show, by cell fractionation, thin-layer chromatography, high-pressure liquid chromatography, and matrix-assisted laser desorption ionization-time of flight mass spectrometry that hydrogenosomes of Tritrichomonas foetus, a cattle vaginal parasite, contain cardiolipin, which is strong evidence for its endosymbiotic origin.     

Tritrichomonas foetus: cytochemical visualization of the endoplasmic reticulum-Golgi complex and lipids

The zinc iodide-osmium tetroxide technique was used to analyze the distribution of the endoplasmic reticulum-Golgi complex system of Tritrichomonas foetus. Interconnections between the cisternae of the endoplasmic reticulum as well as between cisternae of the Golgi complex were observed. The nuclear pores, as well as fenestrations in the Golgi complex, were evident. Three to four profiles of the endoplasmic reticulum were seen in the proximal marginal lamellae, related to the attachment of the recurrent flagellum to the protozoan body. No reaction product was seen in the costae, microtubules, glycogen particles, or hydrogenosomes. Imidazole-buffered osmium tetroxide solution was used to visualize lipids. Electron-dense materials were seen either free in the cytoplasm or within membrane-bounded cytoplasmic vesicles. A high contrast of some membranes, mainly of those which enclosed the hydrogenosomes, was observed in unstained sections.     

The [FeFe] hydrogenase of Nyctotherus ovalis has a chimeric origin

Background

The hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis show how mitochondria can evolve into hydrogenosomes because they possess a mitochondrial genome and parts of an electron-transport chain on the one hand, and a hydrogenase on the other hand. The hydrogenase permits direct reoxidation of NADH because it consists of a [FeFe] hydrogenase module that is fused to two modules, which are homologous to the 24 kDa and the 51 kDa subunits of a mitochondrial complex I.

Results

The [FeFe] hydrogenase belongs to a clade of hydrogenases that are different from well-known eukaryotic hydrogenases. The 24 kDa and the 51 kDa modules are most closely related to homologous modules that function in bacterial [NiFe] hydrogenases. Paralogous, mitochondrial 24 kDa and 51 kDa modules function in the mitochondrial complex I in N. ovalis. The different hydrogenase modules have been fused to form a polyprotein that is targeted into the hydrogenosome.

Conclusion

The hydrogenase and their associated modules have most likely been acquired by independent lateral gene transfer from different sources. This scenario for a concerted lateral gene transfer is in agreement with the evolution of the hydrogenosome from a genuine ciliate mitochondrion by evolutionary tinkering.     

The fine structure of the axostyle and its associations with organelles in Trichomonads

The fine structure of the axostyle in the protists Tritrichomonas foetus and Monocercomonas sp is described using transmission electron microscopy after quick-freezing techniques and immunocytochemistry. The axostyle microtubules presents a lateral projection formed by two protofilaments in addition to the 13 protofilaments normally found in microtubules. The axostyle is associated with other cell structures such as hydrogenosomes, endoplasmic reticulum, sigmoid filaments and glycogen particles. The microtubules of the pelta-axostylar system are connected to each other by bridges regularly spaced with an interval of 9 nm. Labeling of the axostyle was observed after cell incubation with monoclonal antibodies recognizing alpha-tubulin and acetylated-tubulin.     

The nature of anaerobic fungi and their polysaccharide degrading enzymes

Conclusion The discovery of anaerobic fungi has added a new member to the indigenous microorganisms that inhabit the rumen ecosystem. Anaerobic fungi do not appear essential for the survival of ruminants due to their presence in very low numbers, and sometimes absence, in ruminants fed low fiber diets, but their presence may likely be very important in the digestion of fibrous diets. The anaerobic fungi have adapted well to the rumen environment. They are able to ferment a large array of soluble carbohydrates and can synthesize cellular components in an anaerobic environment. The fungi posses hydrogenosomes for the removal of reducing equivalents in the form of molecular hydrogen and the removal of trace oxygen is a accomplished via removal by NADH oxidase. Their positive synergistic interaction with methanogenic bacteria eludes to their highly evolved role in the rumen environment. The fungi also produce resistant sporangia that allows for transfer of species to a new host in an oxygen environment. The anaerobic fungi posses a highly active array of polysaccharide degrading enzymes that may provide an advantage in the highly competitive rumen ecosystem. The production of specific enzymes that hydrolyze the lignocellulosic fraction of plant walls is unique in rumen microorganisms and allows for their attachment and growth on fibrous plant particles that are not available to the rumen bacteria.     

The ultrastructure and possible relationships of four obligate anaerobic chytridiomycete fungi from the rumen of sheep

Zoospores and vegetative growth phases of three cellulolytic rumen chytridiiomycetes, Piromonas, Sphaeromonas and NF1 have been examined by electron microscopy and compared with published and new data on Neocallimastix. The four genera have some 16 distinctive ultrastructural features in common, which collectively may be used to define the group. Some of the common features may individually be sufficient to distinguish these obligate anaerobes from facultative and aerobic chytridiomycetes. These features are the presence of hydrogenosomes at all stages of the life cycle, the presence in rhizoids and sporangia of characteristic crystals coated with hexagonal arrays of particles, and in zoospores the presence of distinct surface layers on the motility organelles and cell body respectively, the organization of the ribosomes into helical and globular arrays and the structures associated with the kinetosomes.     

The anaerobic chytridiomycete fungus Piromyces sp. E2 produces ethanol via pyruvate:formate lyase and an alcohol dehydrogenase E

Anaerobic chytridiomycete fungi possess hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end-products of a prokaryotic-type mixed-acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of pyruvate, which is in marked contrast to the hydrogenosomal metabolism of the anaerobic parabasalian flagellates Trichomonas vaginalis and Tritrichomonas foetus, because these organisms decarboxylate pyruvate with the aid of pyruvate:ferredoxin oxidoreductase (PFO). Here, we show that the chytrids Piromyces sp. E2 and Neocallimastix sp. L2 also possess an alcohol dehydrogenase E (ADHE) that makes them unique among hydrogenosome-bearing anaerobes. We demonstrate that Piromyces sp. E2 routes the final steps of its carbohydrate catabolism via PFL and ADHE: in axenic culture under standard conditions and in the presence of 0.3% fructose, 35% of the carbohydrates were degraded in the cytosol to the end-products ethanol, formate, lactate and succinate, whereas 65% were degraded via the hydrogenosomes to acetate and formate. These observations require a refinement of the previously published metabolic schemes. In particular, the importance of the hydrogenase in this type of hydrogenosome has to be revisited.     

Adenosine triphosphatase activity of Tritrichomonas foetus.

Homogenates of Tritrichomonas foetus exhibited a Mg2+-dependent adenosine triphosphatase (ATPase) activity, with a pH optimum in Tris buffers of 8.2 to 8.3. The activity was not sensitive to oxygen. At high concentrations, quercetin and 4-chloro-7-nitrobenzofurazan inhibited ATPase activity in the cytoplasmic extract by 20 and 70%, respectively, whereas oligomycin, venturicidin, triethyltin, leucinostatin, dibutylchloromethyltin chloride, spegazzinine, efrapeptin, citreoviridin and sodium azide had no effect and N,N'-dicyclohexylcarbodi-imide stimulated the activity somewhat. The activity was localized in a population of small cytoplasmic particles which also contained an acid phosphatase. There was no indication of an association of ATPase with hydrogenosomes. The ATPase activity (or activities) in this aerotolerant anaerobe is different from the ATPases characteristic of mitochondria or of anaerobic bacteria.     

Biochemical Cytology of Trichomonad Flagellates. II. Subcellular Distribution of Oxidoreductases and Hydrolases in Monocercomonas sp.*

A primitive trichomonad, Monocercomonas sp. (strain NS-1:PRR) from Natrix sipedon, was grown axenically in Diamond's medium. Activity of NADH oxidase, malate dehydrogenase, malate dehydrogenase (decarboxylating) and of the anaerobic enzymes, pyruvate synthase and hydrogenase as well as of several hydrolases was demonstrated in homogenates. The subcellular distribution of these activities was studied by means of analytical differential and isopycnic centrifugation of homogenates prepared in 0.25 M sucrose. NADH oxidase and malate dehydrogenase are in the nonsedimentable part of the cytoplasm. Malate dehydrogenase (decarboxylating), pyruvate synthase, and hydrogenase are associated with a large particle which equilibrates at density 1.22. In its properties, this particle corresponds to the microbody-like hydrogenosomes of Tritrichomonas foetus. The 5 hydrolases studied are associated with at least 2 different particle populations: a large particle population equilibrating at densities from 1.10 to 1.16 is the exclusive location of 3 enzymes (β-galactosidase, protease and β-N-acetylglucosaminidase), 2 of which have a pH optimum close to neutrality. These particles contain part of the acid phosphatase and β-glucuronidase. Another part of these 2 enzymes is associated with a separate population of smaller granules with equilibrium densities of 1.16 to 1.18. The 2 types of hydrolase-carrying particles are also biochemically very similar to their counterparts in T. foetus.     

Water exchange between the pregnant ewe,the foetus and its amniotic and allantoic fluids

Water exchange between the ewe and its foetus was measured in two Merino ewes maintained with continuous feeding under thermoneutral conditions from about day 90 of gestation to term. Catheters were established in the maternal pulmonary artery (MPA), the foetal dorsal aorta (FDA) and the amniotic and allantoic sacs. Doses of tritiated water were given into either the MPA or the FDA on five occasions, three for one ewe and two for the other, at least 6 days apart and samples were taken from all catheters for 31 h following the dose. An open, eight-compartment restricted model was developed which simultaneously fitted the tracer data and the ewes water balance, determined by measurement and calculation. The ewes delivered live lambs at term. Water was exchanged between ewe and foetus at 16–43 l h^–1 whereas net flow to the foetus averaged 82 ml day^–1. Turnover times were 20–39 min in the ewes body water, 2–7 min in foetal body water, 10–58 min in amniotic water and 3–22 min in allantoic water; the whole-body half-times were 4.5–5.7 days. The data suggest that intramembranous exchange was the major contributor to water exchange in amniotic and allantoic fluids. The mean residence time of water in the rumen (39–52 min) was shorter than in non-pregnant sheep, suggesting that blood flow to the rumen increased during mid to late gestation.     

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.     

The missing link between hydrogenosomes and mitochondria

Mitochondria typically respire oxygen and possess a small DNA genome. But among various groups of oxygen-shunning eukaryotes, typical mitochondria are often lacking, organelles called hydrogenosomes being found instead. Like mitochondria, hydrogenosomes are surrounded by a double-membrane, produce ATP and sometimes even have cristae. In contrast to mitochondria, hydrogenosomes produce molecular hydrogen through fermentations, lack cytochromes and usually lack DNA. Hydrogenosomes do not fit into the conceptual mold cast by the classical endosymbiont hypothesis about the nature of mitochondria. Accordingly, ideas about their evolutionary origins have focussed on the differences between the two organelles instead of their commonalities. Are hydrogenosomes fundamentally different from mitochondria, the result of a different endosymbiosis? Or are our concepts about the mitochondrial archetype simply too narrow? A new report has uncovered DNA in the hydrogenosomes of anaerobic ciliates. The sequences show that these hydrogenosomes are, without a doubt, mitochondria in the evolutionary sense, even though they differ from typical mitochondria in various biochemical properties. The new findings are a benchmark for our understanding of hydrogenosome origins.     

Investigation of the Secretory Pathway in Trichomonas vaginalis Argues against a Moonlighting Function of Hydrogenosomal Enzymes

The enzymes pyruvate ferredoxin oxidoreductase (PFO), malic enzyme (ME), and the α‐ and β‐subunits of succinyl‐CoA synthetase (SCS) catalyze key steps of energy metabolism in Trichomonas vaginalis hydrogenosomes. These proteins have also been characterized as the adhesins AP120 (PFO), AP65 (ME), AP33, and AP51 (α‐ and β‐SCS), which are localized on the cell surface and mediate the T. vaginalis cytoadherence. However, the mechanisms that facilitate the targeting of these proteins to the cell surface via the secretory pathway and/or to hydrogenosomes are not known. Here we adapted an in vivo biotinylation system to perform highly sensitive tracing of protein trafficking in T. vaginalis. We showed that α‐ and β‐SCS are biotinylated in the cytosol and imported exclusively into the hydrogenosomes. Neither α‐ nor β‐SCS is biotinylated in the endoplasmic reticulum and delivered to the cell surface via the secretory pathway. In contrast, two surface proteins, tetratricopeptide domain‐containing membrane‐associated protein and tetraspanin family surface protein, as well as soluble‐secreted β‐amylase‐1 are biotinylated in the endoplasmic reticulum and delivered through the secretory pathway to their final destinations. Taken together, these results demonstrate that the α‐ and β‐SCS subunits are targeted only to the hydrogenosomes, which argues against their putative moonlighting function.     

Nitrite inhibits hydrogen production and kills the cattle parasite Tritrichomonas foetus

AIMS: To investigate the effects of NaNO2 on the microaerophilic flagellated protozoan, Tritrichomonas foetus KV1, an economically important cattle parasite that inhabits the vagina and can spread rapidly through herds of animals by sexual transmission and leads to abortion of foetal calves. METHODS AND RESULTS: Growth of the parasite was inhibited by 50% in the presence of 4 mm NaNO2; immediate killing occurred at 10 mm. Mass spectrometric monitoring of gases showed that H2 and CO2 evolution were inhibited by NaNO2, and electron paramagnetic resonance spectrometry revealed a signal similar to that of a thiolate-iron-NO complex. Growth with sublethal concentrations of NaNO2 yielded organisms that produced ethanol rather than H2. CONCLUSIONS: NaNO2 probably inactivates FeS protein(s) of hydrogenosomes so as to inhibit the conversion of pyruvate (derived from maltose in the growth medium) to H2 and acetate. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of NaNO2 as a topical antitrichomonal agent in veterinary practice is a possibility. At present, slaughter of infected animals is the favoured method of control.