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Metalloenzymes such as the superoxide dismutases (SODs) form part of a defense mechanism that helps protect obligate and facultative aerobic organisms from oxygen toxicity and damage. Here, we report the presence in the trypanosomatid genomes of four SOD genes: soda, sodb1, sodb2, and a newly identified sodc. All four genes of Trypanosoma brucei have been cloned (Tbsods), sequenced, and overexpressed in Escherichia coli and shown to encode active dimeric FeSOD isozymes. Homology modeling of the structures of all four enzymes using available X-ray crystal structures of homologs showed that the four TbSOD structures were nearly identical. Subcellular localization using GFP-fusion proteins in procyclic insect trypomastigotes shows that TbSODB1 is mainly cytosolic, with a minor glycosomal component, TbSODB2 is mainly glycosomal with some activity in the cytosol, and TbSODA and TbSODC are both mitochondrial isozymes. Phylogenetic studies of all available trypanosomatid SODs and 106 dimeric FeSODs and closely related cambialistic dimeric SOD sequences suggest that the trypanosomatid SODs have all been acquired by more than one event of horizontal gene transfer, followed by events of gene duplication.

Objective: To investigate the incidence of the association of Trichomonas and Pneumocystis in the lung., Study Design: Sixty-six bronchoalveolar lavage fluid (BALF) samples from immunocompromised patients with pneumocystosis were retrospectively examined microscopically., Results: Trichomonads were found as coinfecting agents in 60% of BALF samples. The frequency and abundance of trichomonads was increased, up to 100%, in cases rich in Pneumocystis., Conclusion: The data suggest that pulmonary Trichomonas infection occurs frequently in the course of Pneumocystis pneumonia. The role of trichomonads in causing alveolar damage during Pneumocystis pneumonia is hypothetical.

Small-subunit (SSU) rRNA gene sequences were obtained by PCR from 12 Blastocystis isolates from humans, rats, and reptiles for which elongation factor 1alpha (EF-1alpha) gene sequences are already available. These new sequences were analyzed by the Bayesian method in a broad phylogeny including, for the first time, all Blastocystis sequences available in the databases. Phylogenetic trees identified seven well-resolved groups plus several discrete lineages that could represent newly defined clades. Comparative analysis of SSU rRNA- and EF-1alpha-based trees obtained by maximum-likelihood methods from a restricted sampling (13 isolates) revealed overall agreement between the two phylogenies. In spite of their morphological similarity, sequence divergence among Blastocystis isolates reflected considerable genetic diversity that could be correlated with the existence of potentially >/=12 different species within the genus. Based on this analysis and previous PCR-based genotype classification data, six of these major groups might consist of Blastocystis isolates from both humans and other animal hosts, confirming the low host specificity of Blastocystis. Our results also strongly suggest the existence of numerous zoonotic isolates with frequent animal-to-human and human-to-animal transmissions and of a large potential reservoir in animals for infections in humans.

The molecular phylogeny of parabasalids has mainly been inferred from small subunit (SSU) rRNA sequences and has conflicted substantially with systematics based on morphological and ultrastructural characters. This raises the important question, how congruent are protein and SSU rRNA trees? New sequences from seven diverse parabasalids (six trichomonads and one hypermastigid) were added to data sets of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase, alpha-tubulin and beta-tubulin and used to construct phylogenetic trees. The GAPDH tree was well resolved and identical in topology to the SSU rRNA tree. This both validates the rRNA tree and suggests that GAPDH should be a valuable tool in further phylogenetic studies of parabasalids. In particular, the GAPDH tree confirmed the polyphyly of Monocercomonadidae and Trichomonadidae and the basal position of Trichonympha agilis among parabasalids. Moreover, GAPDH strengthened the hypothesis of secondary loss of cytoskeletal structures in Monocercomonadidae such as Monocercomonas and Hypotrichomonas. In contrast to GAPDH, the enolase and both tubulin trees are poorly resolved and rather uninformative about parabasalian phylogeny, although two of these trees also identify T. agilis as representing the basal-most lineage of parabasalids. Although all four protein genes show multiple gene duplications (for 3-6 of the seven taxa examined), most duplications appear to be relatively recent (i.e., species-specific) and not a problem for phylogeny reconstruction. Only for enolase are there more ancient duplications that may confound phylogenetic interpretation.

The iron- and manganese-containing superoxide dismutases (Fe/Mn-SOD) share the same chemical function and spatial structure but can be distinguished according to their modes of oligomerization and their metal ion specificity. They appear as homodimers or homotetramers and usually require a specific metal for activity. On the basis of 261 aligned SOD sequences and 12 superimposed x-ray structures, two phenetic trees were constructed, one sequence-based and the other structure-based. Their comparison reveals the imperfect correlation of sequence and structural changes; hyperthermophilicity requires the largest sequence alterations, whereas dimer/tetramer and manganese/iron specificities are induced by the most sizable structural differences within the monomers. A systematic investigation of sequence and structure characteristics conserved in all aligned SOD sequences or in subsets sharing common oligomeric and/or metal specificities was performed. Several residues were identified as guaranteeing the common function and dimeric conformation, others as determining the tetramer formation, and yet others as potentially responsible for metal specificity. Some form cation-pi interactions between an aromatic ring and a fully or partially positively charged group, suggesting that these interactions play a significant role in the structure and function of SOD enzymes. Dimer/tetramer- and iron/manganese-specific fingerprints were derived from the set of conserved residues; they can be used to propose selected residue substitutions in view of the experimental validation of our in silico derived hypotheses.

Some protozoans, such as Trichomonad species, do not possess mitochondria. Most of the time, they harbor another type of membrane-bounded organelle, called hydrogenosome from its capacity to produce H(2). This is the case for the human parasite Trichomonas vaginalis. Some other parasites, such as the protist Giardia lamblia, do not harbor any of these organelles. From this observation arises naturally a naive question: How do cells die when the mitochondrion, the cornerstone of apoptotic process, is absent? Data strongly suggest that the mitochondrion and the hydrogenosome arose from a common ancestral endosymbiont. But hydrogenosomes do not appear to directly substitute for mitochondria in apoptotic functions. Thus, it appears judicious to examine more closely the genome of unicellular cells, which do not harbor mitochondria, and search for new molecules that could participate in the apoptotic process in these microorganisms.

A 41-year-old man was hospitalized, presenting increasing dyspnea and extensive ground-glass opacities on chest X-ray. Infection by human immunodeficiency virus was confirmed. Cytologic examination of bronchoalveolar lavage fluid revealed numerous trichomonads and aggregates of Pneumocystis sp. Treatment was followed by rapid improvement of respiratory symptoms and chest X-ray. The trichomonad species found in the lungs was identified as Trichomonas vaginalis by small-subunit rRNA gene amplification and sequencing. With the exception of rare cases of contamination of newborn babies during delivery, T. vaginalis has never been found in lungs in healthy or immunocompromised adults. In the present case, T. vaginalis is found as coinfecting agent. Our data, like those found in the literature, suggest that trichomonads are overlooked parasites that may be regularly implicated in diverse human pathologies.

The behavior of microtubular structures during division was followed by immunofluorescence in Trichomonas vaginalis using an anti-alpha-tubulin monoclonal antibody together with nuclear staining by DAPI, allowing us to describe successive mitotic stages. In contrast to recent reports, we showed that: (1) the microtubular axostyle-pelta complex depolymerized during division, (2) the flagella were assembled during mitosis, and (3) the flagellar number was restored in each daughter kinetid before cytokinesis. Observation of griseofulvin-treated T. vaginalis cells revealed that the elongation of the mitotic spindle or paradesmosis was not the main motile force separating the daughter kinetids to opposite poles during division, suggesting the existence of other mechanisms and/or molecules involved in this morphogenetic event. Examination of treated cells re-incubated in fresh medium showed the nucleation of microtubules radiating from the perinuclear area, the origin of which is discussed. Finally, we confirm the effectiveness of griseofulvin against T. vaginalis and propose that this antifungal drug could be a promising antitrichomonal agent.

Small subunit rRNA sequences were obtained by polymerase chain reaction from trichomonad symbionts of termites that belong to the polymastigont Calonymphidae, including Snyderella tabogae, Calonympha grassii, and Metacoronympha senta. The yet-unidentified sequence Nk9 previously obtained from the termite Neotermes koshunensis, has also been shown to derive from the Devescovinidae Devescovina sp. by in situ hybridization. These new sequences were analyzed by distance, parsimony, and likelihood methods in a broad phylogeny including all identified parabasalid sequences available in databases. All analyses revealed the emergence of a very well supported Devescovinidae/Calonymphidae group but showed an unexpected dichotomy of the Calonymphidae represented by the "Coronympha" and "Calonympha" groups. It strongly suggests that the polymastigont state observed in the Calonymphidae might be explained by at least two independent evolutionary events. In a second phylogenetic analysis, some yet-unidentified parabasalid sequences likely deriving from the Devescovinidae/Calonymphidae taxa, were added to our data set. This analysis confirmed the polyphyly of the Calonymphidae. A tentative identification is proposed for each of these sequences, and hypotheses on the origin of the Devescovinidae and Calonymphidae are discussed. Tritrichomonas foetus or a close relative might be the best candidate for the ancestor of the Devescovinidae, fairly consistent with morphology-based hypotheses. Regarding the Calonymphidae, the origin of the "Coronympha" group might be found within the Devescovinidae, related to Foaina, whereas the "Calonympha" group may directly descend from Tritrichomonas or related species.

One of hallmarks of apoptosis is the degradation and concomitant compaction of chromatin. It is assumed that caspases and caspase-independent pathways are rate limiting for the development of nuclear apoptosis. The caspase-independent pathway involves apoptosis-inducing factor (AIF) and leads to DNA fragmentation and peripheral chromatin condensation. Both pathways are the result of activation of death signals that the mitochondrion receives, integrates, and responds to with the release of various molecules (e.g., cytochrome c and AIF). In fact, both pathways have in common the final point of the DNA fragmentation and the mitochondrial origin of molecules that initiate the apoptotic events. Here, we examine the question of whether apoptosis or apoptotic-like processes exist in a unicellular organism that lacks mitochondria. We herein show that a form of cell death with some features resembling apoptosis is indeed present in Trichomonas vaginalis. Characterization of morphological aspects implicated in this event together with the preliminary biochemical data provided may lead to new insight about the evolutionary relationships between the different forms of programmed cell death identified so far.

We have isolated and analysed an alpha-tubulin-encoding gene (atub1) in an early-diverging eukaryote, Trichomonas vaginalis. The complete atub1 open reading frame included 1.356 bp encoding a polypeptide of 452 amino-acyl residues. A second alpha-tubulin gene (atub2) was amplified by PCR using primers derived from consensus alpha-tubulin amino acid sequences. Both T. vaginalis alpha-tubulin sequences showed high identity to those described in other parabasalids (94.4%-97.3%), and exhibited a high degree of similarity to sequences from Metazoa (such as pig brain) and diplomonads (such as Giardia). Despite large evolutionary distances previously observed between trichomonads and mammals, the three-dimensional model of the T. vaginalis tubulin dimer was very similar to that of pig brain. Possible correlations between alpha-tubulin sequences and posttranslational modifications (PTMs) were examined. Our observations corroborated previous data obtained in T. vaginalis using specific anti-PTMs antibodies. As described in the related species Tritrichomonas mobilensis, microtubules are likely acetylated, non-tyrosinated, glutamylated, and non-glycylated in T. vaginalis. Evolutionary considerations concerning the time of appearance of these tubulin PTMs are also discussed since trichomonads are potentially one of the earliest diverging eukaryotic lineages.

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.

The phylogenetic position of the trichomonad, Histomonas meleagridis was determined by analysis of small subunit rRNAs. Molecular trees including all identified parabasalid sequences available in data bases were inferred by distance, parsimony, and likelihood methods. All reveal a close relationship between H. meleagridis, and Dientamoeba fragilis. Moreover, small subunit rRNAs of both amoeboid species have a reduced G + C content and increased chain length relative to other parabasalids. Finally, the rRNA genes from H. meleagridis and D. fragilis share a recent common ancestor with Tritrichomonasfoetus, which exhibits a more developed cytoskeleton. This indicates that Histomonas and Dientamoeba secondarily lost most of the typical trichomonad cytoskeletal structures and hence, do not represent primitive morphologies. A global phylogeny of parabasalids revealed significant discrepancies with morphology-based classifications, such as the polyphyly of most of the parabasalid families and classes included in our study.

Small subunit rDNA genes were amplified by polymerase chain reaction using specific primers from mixed-population DNA obtained from the whole hindgut of the termite Calotermes flavicollis. Comparative sequence analysis of the clones revealed two kinds of sequences that were both from parabasalid symbionts. In a molecular tree inferred by distance, parsimony and likelihood methods, and including 27 parabasalid sequences retrieved from the data bases, the sequences of the group II (clones Cf5 and Cf6) were closely related to the Devescovinidae/Calonymphidae species and thus were assigned to the Devescovinidae Foaina. The sequence of the group I (clone Cf1) emerged within the Trichomonadinae and strongly clustered with Tetratrichomonas gallinarum. On the basis of morphological data, the Monocercomonadidae Hexamastix termitis might be the most likely origin of this sequence.

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. The Trichomonadinae 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 faetus, Monocercomonas sp., Dientamoeba fragilis, and the unidentified Reticulitermes flavipes gut symbiont 1 remain unclear.

The Parabasala are a primitive group of protists divided into two classes: the trichomonads and the hypermastigids. Until recently, phylogeny and taxonomy of parabasalids were mainly based on the comparative analysis of morphological characters primarily linked to the development of their cytoskeleton. Recent use of molecular markers, such as small subunit (SSU) rRNA has led to now insights into the systematics of the Parabasala and other groups of prolists. An updated phylogeny based on SSU rRNA is provided and compared to that inferred from ultrastructural data. The SSU rRNA phylogeny contradicts the dogma equating simple characters with pumitive characters. Hypermastigids, possessing a hyperdeveloped cytoskeleton, exhibit the most basal emergence in the parabasalid lineage. Other observations emerge from the SSU rRNA analysis, such as the secondary loss of some cytoskeleton structures in all representatives of the Monocercomonadidae, the existence of secondarily free living taxa (reversibility of parasitism) and the evidence against the co-evolution of the endobiotic parabasalids and their animal hosts. According to phylogenies based on SSU rRNA, all the trichomonad families are not monophyletic groups, putting into question the validity of current taxonomic assignments. The precise branching order of some taxa remains unclear, but this issue can possibly be addressed by the molecular analysis of additional parabasalids. The goal of such additional analyses would be to propose, in a near future, a revision of the taxonomy of this group of protists that takes into account both molecular and morphological data.

A superoxide dismutase (SOD) gene of the parasitic protist Trichomonas vaginalis was cloned, sequenced, expressed in Escherichia coli, and its gene product characterized. It is an iron-containing dimeric protein with a monomeric mass of 22,067 Da. Southern blots analyses suggested the presence of seven iron-containing (FeSOD) gene copies. Hydrophobic cluster analysis revealed some peculiarities in the 2D structure of the FeSOD from T. vaginalis and a strong structural conservation between prokaryotic and eukaryotic FeSODs. Phylogenetic reconstruction of the SOD sequences confirmed the dichotomy between FeSODs and manganese-containing SODs. FeSODs of protists appeared to group together with homologous proteobacterial enzymes suggesting a possible origin of eukaryotic FeSODs through an endosymbiotic event.