Membrane associated proteins of two Trichomonas gallinae clones vary with the virulence.

Oropharyngeal avian trichomonosis is mainly caused by Trichomonas gallinae, a protozoan parasite that affects the upper digestive tract of birds. Lesions of the disease are characterized by severe inflammation which may result in fatality by starvation. Two genotypes of T. gallinae were found to be widely distributed in different bird species all over the world. Differences in the host distribution and association with lesions of both genotypes have been reported. However, so far no distinct virulence factors of this parasite have been described and studies might suffer from possible co-infections of different genotypes. Therefore, in this paper, we analyzed the virulence capacity of seven clones of the parasite, established by micromanipulation, representing the two most frequent genotypes. Clones of both genotypes caused the maximum score of virulence at day 3 post-inoculation in LMH cells, although significant higher cytopathogenic score was found in ITS-OBT-Tg-1 genotype clones at days 1 and 2, as compared to clones with ITS-OBT-Tg-2. By using one representative clone of each genotype, a comparative proteomic analysis of the membrane proteins enriched fraction has been carried out by a label free approach (Data available via ProteomeXchange: PXD013115). The analysis resulted in 302 proteins of varying abundance. In the clone with the highest initial virulence, proteins related to cell adhesion, such as an immuno-dominant variable surface antigen, a GP63-like protein, an armadillo/beta-catenin-like repeat protein were found more abundant. Additionally, Ras superfamily proteins and calmodulins were more abundant, which might be related to an increased activity in the cytoskeleton re-organization. On the contrary, in the clone with the lowest initial virulence, larger numbers of the identified proteins were related to the carbohydrate metabolism. The results of the present work deliver substantial differences between both clones that could be related to feeding processes and morphological changes, similarly to the closely related pathogen Trichomonas vaginalis.

[CHARACTERISTICS OF PARASITIC PROTOZOA METABOLISM AND POSSIBILITIES OF ANTIPROTOZOA DRUG DEVELOPMENT (REVIEW)].

One of the most poorly studied areas of protozoology is metabolic processes of parasitic protozoa. Study of the biochemistry of parasites required for the development of effective chemotherapy of protozoal diseases. Some amitochondrial parasites of humans, such as Giardia intestinalis, Entamoeba histolytica, Trichomonas sp., living in an environment with low oxygen content, have specialized cellular organelles-hydrogenosomes (like mitochondria provide cells with simple energy). The study of the functioning of these organelles allows us to consider them as targets for the development of аntiprotozoal drugs. The target for chemotherapy in the treatment of trypanosomiasis can be processes related to the characteristics of the glycolytic pathway or a decrease in the level of energy substrate, such as glucose. This leads to a rapid decrease in ATP levels in the cell of the parasite, an overall loss of mobility and disappearance of trypanosomes from the bloodstream of the infected host. Also, glucose transporters located in the membrane of the parasite can be targets for drugs.

The drug ornidazole inhibits photosynthesis in a different mechanism described for protozoa and anaerobic bacteria.

Ornidazole of the 5-nitroimidazole drug family is used to treat protozoan and anaerobic bacterial infections via a mechanism that involves preactivation by reduction of the nitro group, and production of toxic derivatives and radicals. Metronidazole, another drug family member, has been suggested to affect photosynthesis by draining electrons from the electron carrier ferredoxin, thus inhibiting NADP+ reduction and stimulating radical and peroxide production. Here we show, however, that ornidazole inhibits photosynthesis via a different mechanism. While having a minute effect on the photosynthetic electron transport and oxygen photoreduction, ornidazole hinders the activity of two Calvin cycle enzymes, triose-phosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Modeling of ornidazole's interaction with ferredoxin of the protozoan Trichomonas suggests efficient electron tunneling from the iron-sulfur cluster to the nitro group of the drug. A similar docking site of ornidazole at the plant-type ferredoxin does not exist, and the best simulated alternative does not support such efficient tunneling. Notably, TPI was inhibited by ornidazole in the dark or when electron transport was blocked by dichloromethyl diphenylurea, indicating that this inhibition was unrelated to the electron transport machinery. Although TPI and GAPDH isoenzymes are involved in glycolysis and gluconeogenesis, ornidazole's effect on respiration of photoautotrophs is moderate, thus raising its value as an efficient inhibitor of photosynthesis. The scarcity of Calvin cycle inhibitors capable of penetrating cell membranes emphasizes on the value of ornidazole for studying the regulation of this cycle.

RAPD analysis and sequencing of ITS1/5.8S rRNA/ITS2 and Fe-hydrogenase as tools for genetic classification of potentially pathogenic isolates of Trichomonas gallinae.

Trichomonas gallinae is a worldwide parasite that causes oropharyngeal avian trichomonosis. During eight years, 60 axenic isolates were obtained from different bird species and characterized by three molecular methods: RAPD analysis and PCR-sequencing of ITS1/5.8S rRNA/ITS2 fragment and Fe-hydrogenase gene. We have found two genotypes of ITS1/5.8S rRNA/ITS2 widely distributed among bird populations, a new variant and also two sequences with mixed pattern. Genotype ITS-OBT-Tg-1 was associated with the presence of gross lesions in birds. We have found eight genotypes of the Fe-hydrogenase (A1, A2, C2, C2.1, C4, C5, C6 and C7), three of them are new reports (C5, C6 and C7), and also three sequences with mixed pattern. Subtype A1 of the Fe-hydrogenase was also related with the presence of lesions. RAPD analyses included most of the strains isolated from animals with lesions in one of the sub-clusters. Potentially pathogenic isolates of T. gallinae obtained in this study fulfill the following criteria with one exception: isolated from lesions+ITS-OBT-Tg-1 genotype+FeHyd A1+RAPD sub-cluster I2.

Glycogen accumulation and degradation by the trichomonads Trichomonas vaginalis and Trichomonas tenax.

Several species of trichomonad have been shown to accumulate significant quantities of glycogen during growth, suggesting an important role for this compound in cell physiology. We provide the first analysis of the changes in glycogen content and glycogen phosphorylase activity that occur during in vitro growth of two trichomonad species: Trichomonas vaginalis and Trichomonas tenax. Both species accumulated glycogen following inoculation into fresh medium and utilized this compound during logarithmic growth. Glycogen phosphorylase activity also varied during growth in a species-specific manner. The expression of phosphorylase genes in T. vaginalis remained constant during growth and thus transcriptional control did not explain the observed fluctuations in phosphorylase activity. After cloning, expression, and purification, two recombinant glycogen phosphorylases from T. vaginalis and one recombinant glycogen phosphorylase from T. tenax had robust activity and, in contrast to many other eukaryotic glycogen phosphorylases, did not appear to be regulated by reversible protein phosphorylation. Furthermore, allosteric regulation, if present, was not mediated by compounds known to impact the activity of better characterized phosphorylases.

Identification of putative potassium channel homologues in pathogenic protozoa.

K(+) channels play a vital homeostatic role in cells and abnormal activity of these channels can dramatically alter cell function and survival, suggesting that they might be attractive drug targets in pathogenic organisms. Pathogenic protozoa lead to diseases such as malaria, leishmaniasis, trypanosomiasis and dysentery that are responsible for millions of deaths each year worldwide. The genomes of many protozoan parasites have recently been sequenced, allowing rational design of targeted therapies. We analyzed the genomes of pathogenic protozoa and show the existence within them of genes encoding putative homologues of K(+) channels. These protozoan K(+) channel homologues represent novel targets for anti-parasitic drugs. Differences in the sequences and diversity of human and parasite proteins may allow pathogen-specific targeting of these K(+) channel homologues.

Iron-saturated lactoferrin and pathogenic protozoa: could this protein be an iron source for their parasitic style of life?

Iron is an essential nutrient for the survival of pathogens inside a host. As a general strategy against microbes, mammals have evolved complex iron-withholding systems for efficiently decreasing the iron accessible to invaders. Pathogens that inhabit the respiratory, intestinal and genitourinary tracts encounter an iron-deficient environment on the mucosal surface, where ferric iron is chelated by lactoferrin, an extracellular glycoprotein of the innate immune system. However, parasitic protozoa have developed several mechanisms to obtain iron from host holo-lactoferrin. Tritrichomonas fetus, Trichomonas vaginalis, Toxoplasma gondii and Entamoeba histolytica express lactoferrin-binding proteins and use holo-lactoferrin as an iron source for growth in vitro; in some species, these binding proteins are immunogenic and, therefore, may serve as potential vaccine targets. Another mechanism to acquire lactoferrin iron has been reported in Leishmania spp. promastigotes, which use a surface reductase to recognize and reduce ferric iron to the accessible ferrous form. Cysteine proteases that cleave lactoferrin have been reported in E. histolytica. This review summarizes the available information on how parasites uptake and use the iron from lactoferrin to survive in hostile host environments.

Electron microscopy and cytochemistry analysis of the endocytic pathway of pathogenic protozoa.

Endocytosis is essential for eukaryotic cell survival and has been well characterized in mammal and yeast cells. Among protozoa it is also important for evading from host immune defenses and to support intense proliferation characteristic of some life cycle stages. Here we focused on the contribution of morphological and cytochemical studies to the understanding of endocytosis in Trichomonas, Giardia, Entamoeba, Plasmodium, and trypanosomatids, mainly Trypanosoma cruzi, and also Trypanosoma brucei and Leishmania.

The hydrogenosome as a drug target.

Hydrogenosomes are spherical or slightly elongated organelles found in non-mitochondrial organisms. In Trichomonas hydrogenosomes measure between 200 to 500 nm, but under drug treatment they can reach 2 microm. Like mitochondria hydrogenosomes: (1) are surrounded by two closely apposed membranes and present a granular matrix: (2) divide in three different ways: segmentation, partition and the heart form; (3) they may divide at any phase of the cell cycle; (4) produce ATP; (5) participate in the metabolism of pyruvate formed during glycolysis; (6) are the site of molecular hydrogen formation; (7) present a relationship with the endoplasmic reticulum; (8) incorporate calcium; (9) import proteins post-translationally; (10) present cardiolipin. However, there are differences, such as: (1) absence of genetic material, at least in trichomonas; (2) lack a respiratory chain and cytochromes; (3) absence of the F(0)-F(1) ATPase; (4) absence of the tricarboxylic acid cycle; (5) lack of oxidative phosphorylation; (6) presence of peripheral vesicles. Hydrogenosomes are considered an excellent drug target since their metabolic pathway is distinct from those found in mitochondria and thus medicines directed to these organelles will probably not affect the host-cell. The main drug used against trichomonads is metronidazole, although other drugs such as beta-Lapachone, colchicine, Taxol, nocodazole, griseofulvin, cytochalasins, hydroxyurea, among others, have been used in trichomonad studies, showing: (1) flagella internalization forming pseudocyst; (2) dysfunctional hydrogenosomes; (3) hydrogenosomes with abnormal sizes and shapes and with an electron dense deposit called nucleoid; (4) intense autophagy in which hydrogenosomes are removed and further digested in lysosomes.

Energy metabolism among eukaryotic anaerobes in light of Proterozoic ocean chemistry.

Recent years have witnessed major upheavals in views about early eukaryotic evolution. One very significant finding was that mitochondria, including hydrogenosomes and the newly discovered mitosomes, are just as ubiquitous and defining among eukaryotes as the nucleus itself. A second important advance concerns the readjustment, still in progress, about phylogenetic relationships among eukaryotic groups and the roughly six new eukaryotic supergroups that are currently at the focus of much attention. From the standpoint of energy metabolism (the biochemical means through which eukaryotes gain their ATP, thereby enabling any and all evolution of other traits), understanding of mitochondria among eukaryotic anaerobes has improved. The mainstream formulations of endosymbiotic theory did not predict the ubiquity of mitochondria among anaerobic eukaryotes, while an alternative hypothesis that specifically addressed the evolutionary origin of energy metabolism among eukaryotic anaerobes did. Those developments in biology have been paralleled by a similar upheaval in the Earth sciences regarding views about the prevalence of oxygen in the oceans during the Proterozoic (the time from ca 2.5 to 0.6 Ga ago). The new model of Proterozoic ocean chemistry indicates that the oceans were anoxic and sulphidic during most of the Proterozoic. Its proponents suggest the underlying geochemical mechanism to entail the weathering of continental sulphides by atmospheric oxygen to sulphate, which was carried into the oceans as sulphate, fueling marine sulphate reducers (anaerobic, hydrogen sulphide-producing prokaryotes) on a global scale. Taken together, these two mutually compatible developments in biology and geology underscore the evolutionary significance of oxygen-independent ATP-generating pathways in mitochondria, including those of various metazoan groups, as a watermark of the environments within which eukaryotes arose and diversified into their major lineages.

Ancestral roles of eukaryotic frataxin: mitochondrial frataxin function and heterologous expression of hydrogenosomal Trichomonas homologues in trypanosomes.

Frataxin is a small conserved mitochondrial protein; in humans, mutations affecting frataxin expression or function result in Friedreich's ataxia. Much of the current understanding of frataxin function comes from informative studies with yeast models, but considerable debates remain with regard to the primary functions of this ubiquitous protein. We exploit the tractable reverse genetics of Trypanosoma brucei in order to specifically consider the importance of frataxin in an early branching lineage. Using inducible RNAi, we show that frataxin is essential in T. brucei and that its loss results in reduced activity of the marker Fe-S cluster-containing enzyme aconitase in both the mitochondrion and cytosol. Activities of mitochondrial succinate dehydrogenase and fumarase also decreased, but the concentration of reactive oxygen species increased. Trypanosomes lacking frataxin also exhibited a low mitochondrial membrane potential and reduced oxygen consumption. Crucially, however, iron did not accumulate in frataxin-depleted mitochondria, and as T. brucei frataxin does not form large complexes, it suggests that it plays no role in iron storage. Interestingly, RNAi phenotypes were ameliorated by expression of frataxin homologues from hydrogenosomes of another divergent protist Trichomonas vaginalis. Collectively, the data suggest trypanosome frataxin functions primarily only in Fe-S cluster biogenesis and protection from reactive oxygen species.

The evolution of N-glycan-dependent endoplasmic reticulum quality control factors for glycoprotein folding and degradation.

Asn-linked glycans (N-glycans) play important roles in the quality control (QC) of glycoprotein folding in the endoplasmic reticulum (ER) lumen and in ER-associated degradation (ERAD) of proteins by cytosolic proteasomes. A UDP-Glc:glycoprotein glucosyltransferase glucosylates N-glycans of misfolded proteins, which are then bound and refolded by calreticulin and/or calnexin in association with a protein disulfide isomerase. Alternatively, an alpha-1,2-mannosidase (Mns1) and mannosidase-like proteins (ER degradation-enhancing alpha-mannosidase-like proteins 1, 2, and 3) are part of a process that results in the dislocation of misfolded glycoproteins into the cytosol, where proteins are degraded in the proteasome. Recently we found that numerous protists and fungi contain 0-11 sugars in their N-glycan precursors versus 14 sugars in those of animals, plants, fungi, and Dictyostelium. Our goal here was to determine what effect N-glycan precursor diversity has on N-glycan-dependent QC systems of glycoprotein folding and ERAD. N-glycan-dependent QC of folding (UDP-Glc:glycoprotein glucosyltransferase, calreticulin, and/or calnexin) was present and active in some but not all protists containing at least five mannose residues in their N-glycans and was absent in protists lacking Man. In contrast, N-glycan-dependent ERAD appeared to be absent from the majority of protists. However, Trypanosoma and Trichomonas genomes predicted ER degradation-enhancing alpha-mannosidase-like protein and Mns1 orthologs, respectively, each of which had alpha-mannosidase activity in vitro. Phylogenetic analyses suggested that the diversity of N-glycan-dependent QC of glycoprotein folding (and possibly that of ERAD) was best explained by secondary loss. We conclude that N-glycan precursor length has profound effects on N-glycan-dependent QC of glycoprotein folding and ERAD.

A divergent ADP/ATP carrier in the hydrogenosomes of Trichomonas gallinae argues for an independent origin of these organelles.

The evolution of mitochondrial ADP and ATP exchanging proteins (AACs) highlights a key event in the evolution of the eukaryotic cell, as ATP exporting carriers were indispensable in establishing the role of mitochondria as ATP-generating cellular organelles. Hydrogenosomes, i.e. ATP- and hydrogen-generating organelles of certain anaerobic unicellular eukaryotes, are believed to have evolved from the same ancestral endosymbiont that gave rise to present day mitochondria. Notably, the hydrogenosomes of the parasitic anaerobic flagellate Trichomonas seemed to be deficient in mitochondrial-type AACs. Instead, HMP 31, a different member of the mitochondrial carrier family (MCF) with a hitherto unknown function, is abundant in the hydrogenosomal membranes of Trichomonas vaginalis. Here we show that the homologous HMP 31 of closely related Trichomonas gallinae specifically transports ADP and ATP with high efficiency, as do genuine mitochondrial AACs. However, phylogenetic analysis and its resistance against bongkrekic acid (BKA, an efficient inhibitor of mitochondrial-type AACs) identify HMP 31 as a member of the mitochondrial carrier family that is distinct from all mitochondrial and hydrogenosomal AACs studied so far. Thus, our data support the hypothesis that the various hydrogenosomes evolved repeatedly and independently.

Cytokinesis arrest and nuclear fission in low density populations of trichomonad protozoan.

Cell growth of anaerobic protozoan Tritrichomonas foetus was analyzed. This protozoan usually proliferates in extremely high density, but protozoan parasites were dispersed uniformly in F-bouillon medium and cell division stopped temporarily. However, nuclear fission continued and giant polynucleated cells formed. Later, cell division resumed and cells returned to normal form. In conditioned medium, cytokinesis of the dispersed parasites did not stop. Results indicated that T. foetus cells secreted an extracellular factor that influenced cytokinesis.

Trichomonas gallinae: a possible contact-dependent mechanism in the hemolytic activity.

The in vitro hemolytic activity of Trichomonas gallinae was investigated. The parasite was tested against human erythrocytes of groups A, B, AB, and O, and against erythrocytes of six adult animals of different species (rabbit, rat, chicken, horse, bovine, and sheep). Results showed that T. gallinae lysed all human erythrocytes groups, as well as rabbit, rat, chicken, horse, bovine and sheep erythrocytes. No hemolysin released by the parasites could be identified. Hemolysis did not occur with trichomonad culture supernatants, with sonicated extracts of T. gallinae, or with killed organisms. The scanning electron microscopy (SEM) showed that the erythrocytes adhered to the parasite surface and were phagocytosed. These observations suggest that the contact between T. gallinae and erythrocytes may be an important mechanism in the injury caused to the erythrocytes. The hemolytic activity of T. gallinae may be an efficient means of obtaining nutrients for the parasite and allow the investigation of the mechanism used by T. gallinae to damage cellular membranes.

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.

Posttranslational modifications of trichomonad tubulins; identification of multiple glutamylation sites.

The alpha- and beta-tubulins present in cytoskeletons of Tritrichomonas mobilensis are extensively glutamylated. Automated sequencing and mass spectrometry of the carboxyterminal peptides identifies 4 glutamylation sites in alpha- and 2 sites in beta-tubulin. They are marked by asterisks in the terminal sequences GDE*E*E*E*DDG (alpha) and EGE*E*DEEAEA (beta). This is the first report that tubulin glutamylation can occur at multiple sites. Although T. mobilensis has four flagellae the tubulins lack polyglycylation. Thus glycylation is not necessary for formation or function of axonemal microtubules. Alpha-tubulin is completely acetylated at lysine 40 and shows no tyrosine cycle. Peptide sequences establish two distinct beta-tubulins.