Absence of a conventional spindle mitotic checkpoint in the binucleated single-celled parasite Giardia intestinalis.

The spindle assembly checkpoint (SAC) joins the machinery of chromosome-to-spindle microtubule attachment with that of the cell cycle to prevent missegregation of chromosomes during mitosis. Although a functioning SAC has been verified in a limited number of organisms, it is regarded as an evolutionarily conserved safeguard mechanism. In this report, we focus on the existence of the SAC in a single-celled parasitic eukaryote, Giardia intestinalis. Giardia belongs to Excavata, a large and diverse supergroup of unicellular eukaryotes in which SAC control has been nearly unexplored. We show that Giardia cells with absent or defective mitotic spindles due to the inhibitory effects of microtubule poisons do not arrest in mitosis; instead, they divide without any delay, enter the subsequent cell cycle and even reduplicate DNA before dying. We identified a limited repertoire of kinetochore and SAC components in the Giardia genome, indicating that this parasite is ill equipped to halt mitosis before the onset of anaphase via SAC control of chromosome-spindle microtubule attachment. Finally, based on overexpression, we show that Giardia Mad2, a core SAC protein in other eukaryotes, localizes along intracytoplasmic portions of caudal flagellar axonemes, but never within nuclei, even in mitotic cells with blocked spindles, where the SAC should be active. These findings are consistent with the absence of a conventional SAC, known from yeast and metazoans, in the parasitic protist Giardia.

How nuclei of Giardia pass through cell differentiation: semi-open mitosis followed by nuclear interconnection.

Differentiation into infectious cysts (encystation) and multiplication of pathogenic trophozoites after hatching from the cyst (excystation) are fundamental processes in the life cycle of the human intestinal parasite Giardia intestinalis. During encystation, a bi-nucleated trophozoite transforms to a dormant tetra-nucleated cyst enveloped by a protective cyst wall. Nuclear division during encystation is not followed by cytokinesis. In contrast to the well-studied mechanism of cyst wall formation, information on nuclei behavior is incomplete and basic cytological data are lacking. Here we present evidence that (1) the nuclei divide by semi-open mitosis during early encystment; (2) the daughter nuclei coming from different parent nuclei are always arranged in pairs; (3) in both pairs, the nuclei are interconnected via bridges formed by fusion of their nuclear envelopes; (4) each interconnected nuclear pair is associated with one basal body tetrad of the undivided diplomonad mastigont; and (5) the interconnection between nuclei persists through the cyst stage being a characteristic feature of encysted Giardia. Based on the presented results, a model of nuclei behavior during Giardia differentiation is proposed.

Propylene glycol and contact-lens solutions containing this diol induce pseudocyst formation in acanthamoebae.

Propylene glycol used as an ophthalmic demulcent in certain contact-lens care systems has been included recently among factors responsible for increasing Acanthamoeba keratitis. In this study, we provide evidence that propylene glycol as well as examined contact-lens solutions containing it induce rapid differentiation of acanthamoebae into pseudocysts. The partial resistance of the pseudocysts and their reversibility to viable trophozoites even after 24-h exposure to the contact-lens solutions indicate a potential risk of infection to contact-lens users.

Stress-induced pseudocyst formation--a newly identified mechanism of protection against organic solvents in acanthamoebae of the T4 genotype.

Differentiation into highly resistant double-walled cysts is a major mechanism allowing amphizoic acanthamoebae to survive under long-lasting, unfavourable environmental conditions. We found that relatively low concentrations of methanol, acetone or DMSO stimulate promptAcanthamoebadifferentiation into a rounded cyst-like stage with a single envelope. To address whether this rapid response differs from the encystment, time-dependent changes in cell surface characteristics and cyst-specific gene expression were monitored in encystating cells and cells differentiating under methanol treatment using microscopic, lectin-binding, PCR and resistance studies. In contrast to the encystment: (1) a single-layered amorphous mannose/glucose coat was the only envelope assembled on the surface of the solvent-treated cells, (2) the cyst-specific protein (CSP21) was not expressed, (3) the coat did not protect cells against acidic pH and (4) in solvent-free encystment medium, the coated cells did not assemble the double-layered wall, thus indicating that these cells were not immature cysts. These findings lead us to specify a terminal stage of rapidAcanthamoebadifferentiation elicited by acute organic solvent stress as "pseudocyst", and to suggest that encystation and pseudocyst formation are distinct stress responses. Moreover, the possibility exists that pseudocysts might form in response to certain contact lens solutions thus increasing resistance of acanthamoebae to disinfecting agents.

Design and validation of an oligonucleotide probe for the detection of protozoa from the order Trichomonadida using chromogenic in situ hybridization.

Infections with protozoal parasites of the order Trichomonadida are often observed in veterinary medicine. Based on the trichomonad species involved these infections are either asymptomatic or can lead to sometimes serious disease. To further study protozoal agents of the order Trichomonadida the establishment of a method to detect trichomonads directly in the tissue, allowing parasite-lesion correlation, is necessary. Here we describe the design and evaluation of an oligonucleotide probe for chromogenic in situ hybridization, theoretically allowing detection of all hitherto known members of the order Trichomonadida. The probe was designed on a region of the 18S ribosomal RNA gene homologue for all representatives of the order Trichomonadida available in the GenBank. Functionality of the probe was proven using protozoal cultures containing different trichomonads (Monocercomonas colubrorum, Hypotrichomonas acosta, Pentatrichomonas hominis, Trichomitus batrachorum, Trichomonas gallinae, Tetratrichomonas gallinarum, Tritrichomonas foetus, and Tritrichomonas augusta). Furthermore, three different tissue sections containing either T. gallinae, T. foetus or Histomonas meleagridis were tested positive. Additionally, to rule out cross-reactivity of the probe a large number of different pathogenic protozoal agents, fungi, bacteria and viruses were tested and gave negative results. The probe presented here can be considered an important tool for diagnosis of all to date described relevant protozoal parasites of the order Trichomonadida in tissue samples.

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.

Molecular phylogeny of diplomonads and enteromonads based on SSU rRNA, alpha-tubulin and HSP90 genes: implications for the evolutionary history of the double karyomastigont of diplomonads.

BACKGROUND: Fornicata is a relatively recently established group of protists that includes the diplokaryotic diplomonads (which have two similar nuclei per cell), and the monokaryotic enteromonads, retortamonads and Carpediemonas, with the more typical one nucleus per cell. The monophyly of the group was confirmed by molecular phylogenetic studies, but neither the internal phylogeny nor its position on the eukaryotic tree has been clearly resolved. RESULTS: Here we have introduced data for three genes (SSU rRNA, alpha-tubulin and HSP90) with a wide taxonomic sampling of Fornicata, including ten isolates of enteromonads, representing the genera Trimitus and Enteromonas, and a new undescribed enteromonad genus. The diplomonad sequences formed two main clades in individual gene and combined gene analyses, with Giardia (and Octomitus) on one side of the basal divergence and Spironucleus, Hexamita and Trepomonas on the other. Contrary to earlier evolutionary scenarios, none of the studied enteromonads appeared basal to diplokaryotic diplomonads. Instead, the enteromonad isolates were all robustly situated within the second of the two diplomonad clades. Furthermore, our analyses suggested that enteromonads do not constitute a monophyletic group, and enteromonad monophyly was statistically rejected in 'approximately unbiased' tests of the combined gene data. CONCLUSION: We suggest that all higher taxa intended to unite multiple enteromonad genera be abandoned, that Trimitus and Enteromonas be considered as part of Hexamitinae, and that the term 'enteromonads' be used in a strictly utilitarian sense. Our result suggests either that the diplokaryotic condition characteristic of diplomonads arose several times independently, or that the monokaryotic cell of enteromonads originated several times independently by secondary reduction from the diplokaryotic state. Both scenarios are evolutionarily complex. More comparative data on the similarity of the genomes of the two nuclei of diplomonads will be necessary to resolve which evolutionary scenario is more probable.

Morphological and molecular diversity of the monocercomonadid genera Monocercomonas, Hexamastix, and Honigbergiella gen. nov.

The family Monocercomonadidae (Parabasala, Trichomonadida) is characterized by the absence of a costa and in most species also of an undulating membrane; both of which are typical structures of trichomonadids. We have examined 25 isolates of Monocercomonadidae species by sequencing of the SSU rDNA and the ITS region and by light and transmission electron microscopy. The isolates formed three distinct phylogenetically unrelated clades: (1) Monocercomonas colubrorum, (2) Monocercomonas ruminantium together with a strain ATCC 50321 designated as Pseudotrichomonas keilini, and (3) Hexamastix. Twenty isolates of Monocercomonas colubrorum split into three clades with no host-specificity. The morphological differences among clades were insufficient to classify them as a separate species. Non-monophyly of the cattle commensal Monocercomonas ruminantium with the type species Monocercomonas colubrorum and absence of Pseudotrichomonas characters in the free-living strain ATCC 50321 led to their reclassification into a new genus (Honigbergiella gen. nov.). The close relationship of these strains indicates a recent switch between a free-living habit and endobiosis. Two strains of Hexamastix represented different species -Hexamastix kirbyi Honigberg 1955 and Hexamastix mitis sp. nov. Polyphyly of the Monocercomonadidae confirmed that the absence of a costa and an undulating membrane are not taxonomically significant characters and were probably secondarily lost in some or all clades. Our observations, however, indicated that other characters - infrakinetosomal body, comb-like structure, marginal lamella, and the type of axostyle - are fully consistent with the position of Monocercomonadidae species in the parabasalian tree and are, therefore, reasonable taxonomic characters.

Cell division of Giardia intestinalis: assembly and disassembly of the adhesive disc, and the cytokinesis.

Trophozoites of Giardia are equipped with a special organelle of attachment, essential for parasite survival and pathogenicity, the ventral disc. Although its basic structure is well established, its reorganization and assembly during cell replication is poorly understood. We addressed some of these problems with aid of conventional, confocal and electron microscopy. We found that dividing Giardia alternates attached and free swimming phases in accordance with functional competence of the parent or newly assembled discs. The division started in attached cells by detachment of the disc microtubules from basal bodies. Shortening and eventual loss of the giardin microribbons, and unfolding of the microtubular layer resulting in collapse of the disc chamber and parasite detachment underlined gradual disassembly of the parent disc skeleton. Two daughter discs assembled on the dorsal side of the attached cell, with their ventral sides exposed on the parent cell surface and their microtubular skeletons growing in counter-clockwise direction. A depression between the assembling discs marked the cleavage plane. The splitting continued during the free-swimming phase with ventral-ventral axial symmetry in a plane of the daughter discs. Finally, the daughter cells with fully developed discs but still connected tail to tail by a cytoplasmic bridge, attached to a substrate and terminated the division by a process resembling adhesion-dependent cytokinesis. The mode of assembly of the daughter discs and plane of the division is compatible with maintenance of the left-right asymmetry of the Giardia cytoskeleton in progeny, which cannot be satisfactorily explained by alternative models proposed so far.

Cell division of Giardia intestinalis: flagellar developmental cycle involves transformation and exchange of flagella between mastigonts of a diplomonad cell.

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.

New evolutionary lineages, unexpected diversity, and host specificity in the parabasalid genus Tetratrichomonas.

We studied morphological and molecular polymorphism of 53 Tetratrichomonas isolates obtained from amphibian, reptilian, mammalian hosts, and from a slug with the aid of protargol staining and analyses of ITS1-5.8S rRNA-ITS2, SSU rRNA, and alpha-tubulin gene sequences. The phylogenetic tree based on the concatenate of all sequences showed the monophyly of the genus Tetratrichomonas with respect to the genus Trichomonas. Our data suggest that two parabasalid genera, Pentatrichomonoides and Trichomonoides, may belong to the genus Tetratrichomonas. Tetratrichomonas isolates were divided into 16 robust host-specific and monophyletic groups that probably represent separate, mostly new, species. As only five Tetratrichomonas species were described from the examined host taxa so far, our study uncovered considerable species diversity within the genus. The wide host range, high level of species-specific host specificity, and newly revealed biodiversity make the genus Tetratrichomonas a valuable model for studying evolution of parasites.

Affiliation of Cochlosoma to trichomonads confirmed by phylogenetic analysis of the small-subunit rRNA gene and a new family concept of the order Trichomonadida.

The protozoan genus Cochlosoma includes parasitic intestinal flagellates of birds and mammals of uncertain taxonomic classification. The presence of an adhesive disc, superficially similar to that of Giardia, led to a proposal that Cochlosoma should be classified as diplomonads. Careful morphological and ultrastructural observations, however, revealed conspicuous homologies to trichomonads. We addressed the question of classification and phylogenetic affiliation of Cochlosoma using the methods of molecular phylogenetics. Analyses based on the 16S rRNA gene sequence of the species Cochlosoma anatis very robustly placed Cochlosoma in the clade of the parabasalid subfamilies Trichomonadinae, Trichomitopsiinae and Pentatrichomonoidinae of the order Trichomonadida (bootstraps >94 %). The data did not provide robust support for any particular position of Cochlosoma within this clade because the sequence suffered from mutational saturation and produced a long branch. The most probable sister taxon of Cochlosoma is the genus Pentatrichomonas, because their relationship was supported specifically by the slowest-mutating, least-saturated positions as determined using the method slow-fast. Classification of the order Trichomonadida was revised to accommodate knowledge about its phylogeny - the family Cochlosomatidae and subfamilies Trichomitopsiinae and Pentatrichomonoidinae were abandoned, Trichomonadidae was amended and new families Tritrichomonadidae (formerly a subfamily) and Trichomitidae were proposed.

Alternative pathway of metronidazole activation in Trichomonas vaginalis hydrogenosomes.

Metronidazole and related 5-nitroimidazoles are the only available drugs in the treatment of human urogenital trichomoniasis caused by the protozoan parasite Trichomonas vaginalis. The drugs are activated to cytotoxic anion radicals by their reduction within the hydrogenosomes. It has been established that electrons required for metronidazole activation are released from pyruvate by the activity of pyruvate:ferredoxin oxidoreductase and transferred to the drug by a low-redox-potential carrier, ferredoxin. Here we describe a novel pathway involved in the drug activation within the hydrogenosome. The source of electrons is malate, another major hydrogenosomal substrate, which is oxidatively decarboxylated to pyruvate and CO2 by NAD-dependent malic enzyme. The electrons released during this reaction are transferred from NADH to ferredoxin by NADH dehydrogenase homologous to the catalytic module of mitochondrial complex I, which uses ferredoxin as electron acceptor. Trichomonads acquire high-level metronidazole resistance only after both pyruvate- and malate-dependent pathways of metronidazole activation are eliminated from the hydrogenosomes.

Inference of the phylogenetic position of oxymonads based on nine genes: support for metamonada and excavata.

Circumscribing major eukaryote groups and resolving higher order relationships between them are among the most challenging tasks facing molecular evolutionists. Recently, evidence suggesting a new supergroup (the Excavata) comprising a wide array of flagellates has been collected. This group consists of diplomonads, retortamonads, Carpediemonas, heteroloboseans, Trimastix, jakobids, and Malawimonas, all of which possess a particular type of ventral feeding groove that is proposed to be homologous. Euglenozoans, parabasalids, and oxymonads have also been associated with Excavata as their relationships to one or more core excavate taxa were demonstrated. However, the main barrier to the general acceptance of Excavata is that its existence is founded primarily on cytoskeletal similarities, without consistent support from molecular phylogenetics. In gene trees, Excavata are typically not recovered together. In this paper, we present an analysis of the phylogenetic position of oxymonads (genus Monocercomonoides) based on concatenation of eight protein sequences (alpha-tubulin, beta-tubulin, gamma-tubulin, EF-1alpha, EF-2, cytosolic (cyt) HSP70, HSP90, and ubiquitin) and 18S rRNA. We demonstrate that the genes are in conflict regarding the position of oxymonads. Concatenation of alpha- and beta-tubulin placed oxymonads in the plant-chromist part of the tree, while the concatenation of other genes recovered a well-supported group of Metamonada (oxymonads, diplomonads, and parabasalids) that branched weakly with euglenozoans--connecting all four excavates included in the analyses and thus providing conditional support for the existence of Excavata.

The phylogenetic position of enteromonads: a challenge for the present models of diplomonad evolution.

Unikaryotic enteromonads and diplokaryotic diplomonads have been regarded as closely related protozoan groups. It has been proposed that diplomonads originated within enteromonads in a single event of karyomastigont duplication. This paper presents the first study to address these questions using molecular phylogenetics. The sequences of the small-subunit rRNA genes for three isolates of enteromonads were determined and a tree constructed with available diplomonad, retortamonad and Carpediemonas sequences. The diplomonad sequences formed two main groups, with the genus Giardia on one side and the genera Spironucleus, Hexamita and Trepomonas on the other. The three enteromonad sequences formed a clade robustly situated within the diplomonads, a position inconsistent with the original evolutionary proposal. The topology of the tree indicates either that the diplokaryotic cell of diplomonads arose several times independently, or that the monokaryotic cell of enteromonads originated by secondary reduction from the diplokaryotic state.

Cryptic species within the Tetratrichomonas gallinarum species complex revealed by molecular polymorphism.

Tetratrichomonas gallinarum is a widespread intestinal parasite of galliform and anseriform birds. The pathogenicity of this species is controversial, presenting an unsettled problem as yet. We analysed the polymorphism and genetic relationship among 29 isolates of T. gallinarum obtained from eight bird species and five T. gallinarum-like isolates from the oral cavity and lower respiratory tract of human patients. Two methods were used for the analyses: RAPD and sequencing of 16S rRNA, 5.8S rRNA, ITS1 and ITS2 genes, both producing consistent and well-supported results. The isolates were divided into five groups, A-E, with eleven subgroups. The distance between groups E, D and the cluster A-B-C considerably exceeded usual intraspecific polymorphism seen in trichomonads. Moreover, the largest subgroup, A2 (containing 18 isolates), was divided into three branches according to the host specificity. All isolates from humans were placed into avian subgroups A2 and B2. We conclude that our isolates represent, at least, three morphospecies or rather complexes of several cryptic species. Since certain species of the T. gallinarum complex can differ in their biological characteristics and some of them can infect humans, the problem of T. gallinarum pathogenicity should be re-examined with regard to specific genetic groups and zoonotic potential of some of these lineages should be considered.

Critical analysis of the topology and rooting of the parabasalian 16S rRNA tree.

The morphological classification of the protozoan phylum Parabasala is not in absolute agreement with the 16S rRNA phylogeny. However, there are strong indications that tree-construction artifacts play a considerable role in the shaping of the 16S rRNA tree. We have performed rigorous analyses designed to minimize such artifacts using the slow-fast and taxa-exclusion methods. The analyses, which included new sequences from the genera Monocercomonas and Hexamastix, in most respects confirmed the previously suggested tree topology and polyphyly of Hypermastigida and Monocercomonadidae but detected one artificial cluster of long branches (Trichonymphidae, Pseudotrichonymphidae, Hexamastix, and Tricercomitus). They also indicated that the rooting of the phylum on the trichonymphid branch is probably wrong and that reliable rooting on the basis of current data is likely impossible. We discuss the tree topology in the view of anagenesis of cytoskeletal and motility organelles and suggest that a robust taxonomic revision requires extensive analysis of other gene sequences.

Pyruvate decarboxylase, the target for omeprazole in metronidazole-resistant and iron-restricted Tritrichomonas foetus.

The substituted benzimidazole omeprazole, used for the treatment of human peptic ulcer disease, inhibits the growth of the metronidazole-resistant bovine pathogen Tritrichomonas foetus in vitro (MIC at which the growth of parasite cultures is inhibited by 50%, 22 microg/ml [63 microM]). The antitrichomonad activity appears to be due to the inhibition of pyruvate decarboxylase (PDC), which is the key enzyme responsible for ethanol production and which is strongly upregulated in metronidazole-resistant trichomonads. PDC was purified to homogeneity from the cytosol of metronidazole-resistant strain. The tetrameric enzyme of 60-kDa subunits is inhibited by omeprazole (50% inhibitory concentration, 16 microg/ml). Metronidazole-susceptible T. foetus, which expresses very little PDC, is only slightly affected. Omeprazole has the same inhibitory effect on T. foetus cells grown under iron-limited conditions. Similarly to metronidazole-resistant cells, T. foetus cells grown under iron-limited conditions have nonfunctional hydrogenosomal metabolism and rely on cytosolic PDC-mediated ethanol fermentation.

Use of random amplified polymorphic DNA (RAPD) analysis for the identification of Giardia intestinalis subtypes and phylogenetic tree construction.

A comparison of random amplified polymorphic DNA (RAPD) was used to investigate genetic polymorphisms among 25 isolates of Giardia intestinalis and to assess the utility of RAPD for subtype detection and genealogical analysis. Using data obtained for six human and 19 animal-derived isolates in polymerase chain reactions using 13 different primers, phylogenetic trees were constructed and bootstrap values computed by the program FreeTree. Three major clades were distinguished, corresponding to previously defined genetic assemblages A, B, and E. The purported specificity of assemblage E genotypes for artiodactyl hosts was supported. Assemblages A and B showed wide host spectra, including human and animal hosts. No correlation was found between the genotype of analyzed isolates and the presence or absence of the double-stranded RNA Giardiavirus. The results indicate that RAPD data provide reliable genetic information that can be used for both "fingerprinting" and genealogical purposes.