Jaroslav Kulda

Critical taxonomic revision of Parabasalids with description of one new genus and three new species.

We propose a new classification of Parabasalia which is congruent with both ultrastructural and molecular-phylogenetic studies. We identify six main parabasalid lineages and give them the rank of class: Hypotrichomonadea, Trichomonadea, Tritrichomonadea, Cristamonadea, Trichonymphea, and Spirotrichonymphea. Trichomonadea is characterized by a single mastigont and by the absence of both a comb-like structure and an infrakinetosomal body. Most representatives also possess a lamelliform undulating membrane. Trichomonadea is divided into two monophyletic orders, Trichomonadida (family Trichomonadidae; with a B-type costa) and Honigbergiellida (families Honigbergiellidae, Hexamastigidae and Tricercomitidae; without a costa). The class Tritrichomonadea, with a single order Tritrichomonadida, is ancestrally characterized by a single mastigont with four flagella, and both a comb-like structure and an infrakinetosomal body. The morphologically most complex representatives (family Tritrichomonadidae) possess in addition a rail-type undulating membrane, an A-type costa, and a suprakinetosomal body. These last three characters are absent in families Monocercomonadidae and Simplicimonadidae. The remaining tritrichomonadids, Dientamoebidae, have undergone reductive evolution. Cristamonads (Cristamonadea) are morphologically derived from tritrichomonads. Because we are unable to determine morphologically homogenous monophyletic lineages within cristamonads, we classify all cristamonads into a single family, Lophomonadidae. Hypotrichomonadea, comprising the genera Trichomitus and Hypotrichomonas, resembles Tritrichomonadea by an A-type costa, and by the presence of a comb-like structure in the mastigont. However, they do not possess an infrakinetosomal body, and are not specifically related to Tritrichomonadea in molecular-phylogenetic analyses. Moreover, unlike Tritrichomonadea, Hypotrichomonadea possesses a lamelliform undulating membrane. The remaining parabasalids are of complex morphology and belong to the classes Trichonymphea and Spirotrichonymphea. A new parabasalid genus, Simplicimonas (Tritrichomonadea), and three new species, Tetratrichomonas undula, Hexamastix coercens and Simplicimonas similis, are described.

Cattle pathogen tritrichomonas foetus (Riedmüller, 1928) and pig commensal Tritrichomonas suis (Gruby & Delafond, 1843) belong to the same species.

Abstract A number of reports suggest that the sexually transmitted pathogen of cattle, Tritrichomonas foetus, and a gastrointestinal commensal of pigs, Tritrichomonas suis, are very similar and may be co-specific. A conclusive review of the taxonomic and nomenclatural status of these species has not been presented so far. Toward this end, we reexamined and compared porcine and bovine trichomonads with regard to their morphology, pathogenic potential, and DNA polymorphism. Using light and electron microscopy, no distinguishing features between T. foetus and T. suis strains were found in size, general morphology, and karyomastigont structure. Both bovine and porcine trichomonads showed pathogenic potential in the subcutaneous mouse assays and did not separate into distinct groups according to strain virulence. Three DNA fingerprinting methods (i.e. RFLP, RAPD, and PCR-based analysis of variable-length DNA repeats) that produce species-specific DNA fragment patterns did not distinguish between the bovine and porcine strains. Sequencing of a variable 502-bp DNA fragment as well as comparison of 16S rRNA gene sequences did not reveal species-specific differences between the cattle and porcine strains. Therefore, we conclude that T. foetus and T. suis belong to the same species. To prevent confusion that may arise from T. foetus–T. suis synonymy, we propose to suppress the older name suis and maintain its accustomed junior synonym foetus as a nomen protectum for both cattle and porcine trichomonads. The case has been submitted to the International Commision on Zoological Nomenclature for ruling under its plenary power.

Mechanisms of in vitro development of resistance to metronidazole in Trichomonas vaginalis.

Development of resistance against metronidazole and mechanisms responsible for this process were studied in a sexually transmitted pathogen of humans, Trichomonas vaginalis. Monitoring of changes in metabolism and protein expression that accompanied increasing resistance of strains derived from a common drug-susceptible parent (TV 10-02) showed the multistep character of the process. The aerobic type of resistance known to occur in isolates from patients non-responsive to treatment appeared at the earliest stage, followed by development of the anaerobic type of resistance which was accompanied by gradual loss of hydrogenosomal proteins associated with drug-activating pathways [pyruvate:ferredoxin oxidoreductase (PFOR), hydrogenase, ferredoxin]. Unexpectedly, the loss of PFOR did not result in acquisition of full anaerobic resistance, thus indicating an alternative source of electrons required for the drug activation. These data suggest involvement of the oxidative decarboxylation of malate in hydrogenosomes, catalysed by NAD(+)-dependent malic enzyme and subsequent transfer of reduced equivalents to the drug via NADH:ferredoxin oxidoreductase and ferredoxin. Accordingly, all components of this pathway were eliminated before the resistance was fully developed. Resistant Trichomonas vaginalis compensated the impaired function of hydrogenosomes by enhanced conversion of pyruvate to lactate in the cytosol. Further analysis of the two key enzymes involved in metronidazole activation by Northern blotting and assay for nascent mRNA showed that the insufficient expression of the PFOR protein results from decreased gene transcription, while down-regulation of malic enzyme is controlled at the mRNA level.

Cell Division of Giardia intestinalis: Flagellar Developmental Cycle Involves Transformation and Exchange of Flagella between Mastigonts of a Diplomonad Cell

ABSTRACT Giardia intestinalis is a binucleated diplomonad possessing four pairs of flagella of distinct location and function. Its pathogenic potential depends on the integrity of a complex microtubular cytoskeleton that undergoes a profound but poorly understood reorganization during cell division. We examined the cell division of G. intestinalis with the aid of light and electron microscopy and immunofluorescence methods and present here new observations on the reorganization of the flagellar apparatus in the dividing Giardia. Our results demonstrated the presence of a flagellar maturation process during which the flagella migrate, assume different position, and transform to different flagellar types in progeny until their maturation is completed. For each newly assembled flagellum it takes three cell cycles to become mature. The mature flagellum of Giardia is the caudal one that possesses a privileged basal body at which the microtubules of the adhesive disk nucleate. In contrast to generally accepted assumption that each of the two diplomonad mastigonts develops separately, we found that they are developmentally linked, exchanging their cytoskeletal components at the early phase of mitosis. The presence of the flagellar maturation process in a metamonad protist Giardia suggests that the basal body or centriole maturation is a universal phenomenon that may represent one of the core processes in a eukaryotic cell.

In Vitro Induced Anaerobic Resistance to Metronidazole In Trichomonas Vaginalis

ABSTRACT. Resitance to metronidazole detectable under anaerobic conditions was induced in two Trichomonas vaginalis strains (TV 10‐02 and MRP‐2) by cultivation at gradually increasing pressure of the drug (1‐100 μ/ml) for 12 to 21 months. the resistant derivatives reproduced in anaerobic trypticase‐yeast‐extract‐maltose medium at 100 μ/ml metronidazole and showed very high values of minimal lethal concentration for metronidazole in anaerobic in vitro assays (556‐1,600 μ/ml at 48‐h exposure to the drug). Stepwise selection was necessary to develop the resistance in either strain. Attempts to induce resistance by prolonged maintenance of trichomonads with constant, low or moderate drug concentrations (3‐10 μ/ml) were unsuccessful. Freshly developed resistance to high concentrations of metronidazole was unstable in absence of drug pressure as well as after cryopreservation. Development of stable resistance required further cultivation at 100 μ/ml metronidazole. Unstable substrains did not revert to original susceptibility. They retained a moderate level of resistance, being able to grow at 10 μ/ml metronidazole. the strains with fully developed resistance had no activity of the hydrogenosomal enzymes pyruvate: ferredoxin oxidoreductase and hydrogenase and ceased uptake of [14C]‐metronidazole. These findings indicate that the pyruvate oxidizing pathway responsible for metronidazole activation was inactivated and metabolism of the drug stopped.

Iron-induced changes in pyruvate metabolism of Tritrichomonas foetus and involvement of iron in expression of hydrogenosomal proteins

The main function of the hydrogenosome, a typical organelle of trichomonads, is to convert malate or pyruvate to H(2), CO(2) and acetate by a pathway associated with ATP synthesis. This pathway relies on activity of iron-sulfur proteins such as pyruvate:ferredoxin oxidoreductase (PFOR), hydrogenase and ferredoxin. To examine the effect of iron availability on proper hydrogenosomal function, the metabolic activity of the hydrogenosome and expression of hydrogenosomal enzymes were compared in Tritrichomonas foetus maintained under iron-rich (150 microM iron nitrilotriacetate) or iron-restricted (180 microM 2,2-dipyridyl) conditions in vitro. The activities of PFOR and hydrogenase, and also production of acetate and H(2), were markedly decreased or absent in iron-restricted trichomonads. Moreover, a decrease in activity of the hydrogenosomal malic enzyme, which is a non-Fe-S protein, was also observed. Impaired function of hydrogenosomes under iron-restricted conditions was compensated for by activation of the cytosolic pathway, mediating conversion of pyruvate to ethanol via acetaldehyde. This metabolic switch was fully reversible. Production of hydrogen by iron-restricted trichomonads was restored to the level of organisms grown under iron-rich conditions within 3 h after addition of 150 microM iron nitrilotriacetate. Protein analysis of purified hydrogenosomes from iron-restricted cells showed decreased levels of proteins corresponding to PFOR, malic enzyme and ferredoxin. Accordingly, these cells displayed decreased steady-state level and synthesis of mRNAs encoding PFOR and hydrogenosomal malic enzyme. These data demonstrate that iron is essential for function of the hydrogenosome, show its involvement in the expression of hydrogenosomal proteins and indicate the presence of iron-dependent control of gene transcription in Tt. foetus.

Cattle Pathogen Tritrichomonas foetus () and Pig Commensal Tritrichomonas suis () Belong to the Same SpeciesRiedmüller, 1928Gruby & Delafond, 1843

Abstract A number of reports suggest that the sexually transmitted pathogen of cattle, Tritrichomonas foetus, and a gastrointestinal commensal of pigs, Tritrichomonas suis, are very similar and may be co-specific. A conclusive review of the taxonomic and nomenclatural status of these species has not been presented so far. Toward this end, we reexamined and compared porcine and bovine trichomonads with regard to their morphology, pathogenic potential, and DNA polymorphism. Using light and electron microscopy, no distinguishing features between T. foetus and T. suis strains were found in size, general morphology, and karyomastigont structure. Both bovine and porcine trichomonads showed pathogenic potential in the subcutaneous mouse assays and did not separate into distinct groups according to strain virulence. Three DNA fingerprinting methods (i.e. RFLP, RAPD, and PCR-based analysis of variable-length DNA repeats) that produce species-specific DNA fragment patterns did not distinguish between the bovine and porcine strains. Sequencing of a variable 502-bp DNA fragment as well as comparison of 16S rRNA gene sequences did not reveal species-specific differences between the cattle and porcine strains. Therefore, we conclude that T. foetus and T. suis belong to the same species. To prevent confusion that may arise from T. foetus–T. suis synonymy, we propose to suppress the older name suis and maintain its accustomed junior synonym foetus as a nomen protectum for both cattle and porcine trichomonads. The case has been submitted to the International Commision on Zoological Nomenclature for ruling under its plenary power.

Metabolic differences between metronidazole resistant and susceptible strains of Tritrichomonas foetus

Tritrichomonas foetus mutants resistant to metronidazole lack the hydrogenosomal enzymes pyruvate: ferredoxin oxidoreductase and hydrogenase. Hydrogenosomes of these organisms did not oxidize pyruvate or produce ATP in its presence. Elimination of hydrogenosomal metabolism of pyruvate was compensated by an increased rate of glycolysis. The resistant mutants excreted no organic acids and H2 as metabolic end products. Glycolysis of the resistant T. foetus KV1-1MR-100 can be summarized as 1 mol glucose----2 mol ethanol + 2 mol CO2. The parent strain KV1, excreting H2, CO2 and acidic end products, converted about 10% of glucose to ethanol. Both strains produced ethanol from pyruvate through the action of two cytoplasmic enzymes: pyruvate decarboxylase and alcohol dehydrogenase. The specific activity of the former enzyme, catalyzing nonoxidative decarboxylation of pyruvate to acetaldehyde, was nearly seven times higher in the resistant than in the parent strain. Alcohol dehydrogenase reducing acetaldehyde to ethanol was specific to NADPH; it catalyzed the reverse reaction only slowly, and displayed similar activities in both resistant and sensitive trichomonads. Development of anaerobic metronidazole resistance in T. foetus depended on the loss of pyruvate:ferredoxin oxidoreductase as well as on the ability to increase alcoholic fermentation.

Characterization of Trichomonad Species and Strains by PCR Fingerprinting

ABSTRACT. The random amplified polymorphic DNA (RAPD) technique was used for phylogenetic analysis of trichomonads, for intraspecies genealogical study of Trichomonas vaginalis strains, and for assessment of intrastrain polymorphism in Trichomonas vaginalis. The phylogenetic tree for 12 trichomonad species showed certain discrepancies with current models of trichomonad evolution. However, it shows that RAPD traits retain phylogenetically relevant information. The results of intraspecies analyses of 18 Trichomonas vaginalis strains suggested some concordance between the genetic relationship of strains and their geographic origin. They also suggested a concordance between the strain genetic relationships and the resistance to metronidazole. A concordance was also found with respect to the severity of disease observed in donor patients but not with the results of laboratory virulence assays. No concordance was found between genetic relationship of strains and strain infection with a dsRNA Trichomonas vaginalis virus (TVV). The latter suggests that TVV might be transmitted horizontally among Trichomonas vaginalis populations. The identity of RAPD patterns of clones isolated from in vitro cultures and those of the cultures reisolated independently from the same patient within a period of six weeks suggests that individual Trichomonas vaginalis strains are not polymorphic and that the RAPD patterns are stable. Therefore, the RAPD technique seems useful for addressing various clinically relevant issues.

Molecular Phylogeny of Parabasalids Based on Small Subunit rRNA Sequences, with Emphasis on the Trichomonadinae Subfamily

Abstract We determined small subunit ribosomal DNA sequences from three parabasalid species, Trichomitus batrachorum strain R105, Tetratrichomonas gallinarum, and Pentatrichomonas hominis belonging to the Trichomonadinae subfamily. Unrooted molecular phylogenetic trees inferred by distance, parsimony, and likelihood methods reveal four discrete clades among the parabasalids. TheTrichomonadinae form a robust monophyletic group. Within this subfamily T. gallinarum is closely related to Trichomonas species as supported by morphological data, with P. hominis and Pseudotrypanosoma giganteum occupying basal positions. Our analysis does not place T. batrachorum within the Trichomonadinae. Trichomitus batrachorum (strains R105 and BUB) and Hypotrichomonas acosta form a well-separated cluster, suggesting the genus Trichomitus is polyphyletic. The emergence of T. batrachorum precedes the Trichomonadinae-Tritrichomonadinae dichotomy, emphasizing its pivotal evolutionary position among the Trichomonadidae. A third cluster unites the Devescovinidae and the Calonymphidae. The fourth clade contains the three hypermastigid sequences from the genus Trichonympha, which exhibit the earliest emergence among the parabasalids. The addition of these three new parabasalid species did not however resolve ambiguities regarding the relative branching order of the parabasalid clades. The phylogenetic positions of Tritrichomonas fœtus, Monocercomonas sp., Dientamoeba fragilis, and the unidentified Reticulitermes flavipes gut symbiont 1 remain unclear.