Comparison of european Trichobilharzia species based on ITS1 and ITS2 sequences.

Schistosomes are parasites of considerable medical and veterinary importance and, therefore, all aspects of their biology have been intensively studied. In contrast, our knowledge of species of the largest genus, Trichobilharzia, is insufficient. Because morphological characterization of Trichobilharzia species provides a limited number of criteria for species determination, molecular data are required. In the present paper, we sequenced internal transcribed spacers ITS1 and ITS2, and 5.8S ribosomal RNA (rRNA) genes of 3 European Trichobilharzia species (T. regenti, T. szidati and T. franki). We showed that ITS1 and ITS2 sequences can be used in species identification. Repetitive elements were found in ITS1 of all 3 Trichobilharzia species; their number and length varied depending on the species. Phylogenetic analysis showed that the visceral T. franki is more related to the nasal T. regenti, than to the visceral T. szidati. The newly designed primer, which is specific for T. regenti, might be used as a tool for diagnosis of this potential pathogen.

Concordance between genetic relatedness and phenotypic similarities of Trichomonas vaginalis strains.

BACKGROUND: Despite the medical importance of trichomoniasis, little is known about the genetic relatedness of Trichomonas vaginalis strains with similar biological characteristics. Furthermore, the distribution of endobionts such as mycoplasmas or Trichomonas vaginalis virus (TVV) in the T. vaginalis metapopulation is poorly characterised. RESULTS: We assayed the relationship between 20 strains of T. vaginalis from 8 countries using the Random Amplified Polymorphic DNA (RAPD) analysis with 27 random primers. The genealogical tree was constructed and its bootstrap values were computed using the program FreeTree. Using the permutation tail probability tests we found that the topology of the tree reflected both the pattern of resistance to metronidazole (the major anti-trichomonal drug) (p < 0.01) and the pattern of infection of strains by mycoplasmas (p < 0.05). However, the tree did not reflect pattern of virulence, geographic origin or infection by TVV. Despite low bootstrap support for many branches, the significant clustering of strains with similar drug susceptibility suggests that the tree approaches the true genealogy of strains. The clustering of mycoplasma positive strains may be an experimental artifact, caused by shared RAPD characters which are dependent on the presence of mycoplasma DNA. CONCLUSIONS: Our results confirmed both the suitability of the RAPD technique for genealogical studies in T. vaginalis and previous conclusions on the relatedness of metronidazol resistant strains. However, our studies indicate that testing analysed strains for the presence of endobionts and assessment of the robustness of tree topologies by bootstrap analysis seem to be obligatory steps in such analyses.

Phylogenetic relationships of class II fumarase genes from trichomonad species.

Class II fumarase sequences were obtained by polymerase chain reaction from five trichomonad species. All residues known to be highly conserved in this enzyme were present. Nuclear run-on assays showed that one of the two genes identified in Tritrichomonas foetus was expressed, whereas no fumarase transcripts were detected in the related species Trichomonas vaginalis. These findings corroborate previous biochemical data. Fumarase genes were also expressed in Monocercomonas sp. and Tetratrichomonas gallinarum but not in Pentatrichomonas hominis, Trichomonas gallinae, Trichomonas tenax, and Trichomitus batrachorum under the culture conditions used. Molecular trees inferred by likelihood methods reveal that trichomonad sequences have no affinity to described class II fumarase genes from other eukaryotes. The absence of functional mitochondria in protists such as trichomonads suggests that they diverged from other eukaryotes prior to the alpha-proteobacterial symbiosis that led to mitochondria. Furthermore, they are basal to other eukaryotes in rRNA analyses. However, support for the early-branching status of trichomonads and other amitochondriate protists based on phylogenetic analyses of multiple data sets has been equivocal. Although the presence of hydrogenosomes suggests that trichomonads once had mitochondria, their class II iron-independent fumarase sequences differ markedly from those of other mitochondriate eukaryotes. All of the class II fumarase genes described from other eukaryotes are of apparent alpha-proteobacterial origin and hence a marker of mitochondrial evolution. In contrast, the class II fumarase from trichomonads emerges among other eubacterial homologs. This is intriguing evidence for an independent acquisition of these genes in trichomonads apart from the mitochondrial endosymbiosis event that gave rise to the form present in other eukaryotes. The ancestral trichomonad class II fumarase may represent a prokaryotic form that was replaced in other eukaryotes after the divergence of trichomonads with the movement of endosymbiont genes into the nucleus. Alternatively, it may have been acquired via a separate endosymbiotic event or lateral gene transfer.

Unusual diversity in alpha-amanitin sensitivity of RNA polymerases in trichomonads.

Previous studies in the parasitic protist Trichomonas vaginalis have revealed that protein coding genes are transcribed by an alpha-amanitin-resistant RNA polymerase (RNAP) II. To investigate whether this unusual property is a general characteristic of trichomonads, we addressed the physiology of RNA synthesis in lysolecithin-permeabilized cells. Unlike in T. vaginalis, RNAP II in Tritrichomonas foetus was highly sensitive to the inhibitor alpha-amanitin. On the other hand, RNAP III, identified by its sensitivity to the specific inhibitor tagetitoxin, was found to be resistant to alpha-amanitin in Tritrichomonas foetus, but showed a typical intermediate sensitivity in T. vaginalis. Extension of this study to an additional seven trichomonad species confirmed this genera specific pattern of alpha-amanitin sensitivity and highlighted an unusual diversity in RNAPs among trichomonads, a closely related group of unicellular eukaryotes.

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.

Correlation between aerobic and anaerobic resistance to metronidazole in trichomonads: application of a new computer program for permutation tests.

An indispensable step of any comparative study is testing the concordance between the distribution of phenetic traits and the evolutionary history of the taxa under study. Here we present a computer program TREEPT which can perform these tasks on the basis of a permutation test. The use of the program was demonstrated on trichomonad drug sensitivity data. The program can also perform permutation tests analogous to the t-test, analysis of variance (ANOVA) and correlation analysis and is available at http://www.natur.cuni.cz/flegr/PROGRAMS/+ ++TREEPT.ZIP and http://www.karlin. mff.cuni.cz/-zaboj/treept.

Transcription of the Trypanosoma brucei spliced leader RNA gene is dependent only on the presence of upstream regulatory elements.

The spliced leader (SL) RNA plays a key role in mRNA maturation in trypanosomatid protozoa by providing the SL sequence, which is joined to the 5' end of every mRNA. As a first step towards a better understanding of the biogenesis and function of the SL RNA, we expressed a tagged SL RNA gene in a cell-free system of procyclic Trypanosoma brucei cells. Transcription initiates at + 1 can be detected as early as 1 min after addition of extract. Transcription of the SL RNA gene in vitro, as well as in permeable cells, is mediated by an alpha-amanitin/tagetitoxin resistant complex, suggesting a promoter that is intermediate between a classical RNA polymerase II and RNA polymerase III promoter. An analysis of the promoter architecture of the SL RNA gene revealed that regulatory elements are located upstream of the coding region and that the SL sequence, in contrast to the nematode SL sequence, is not required for T. brucei SL RNA gene transcription.

Characterization of trichomonad species and strains by PCR fingerprinting.

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.