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.
- Klíčová slova
- Albendazole, Giardia, Kinetochore, Mad2, Single-celled eukaryote, Spindle assembly checkpoint,
- MeSH
- aparát dělícího vřeténka fyziologie MeSH
- Giardia lamblia genetika izolace a purifikace fyziologie MeSH
- kinetochory fyziologie MeSH
- kontrolní body M fáze buněčného cyklu fyziologie MeSH
- proteiny buněčného cyklu metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- proteiny buněčného cyklu MeSH
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.
- MeSH
- buněčná diferenciace * MeSH
- buněčné jádro genetika MeSH
- Giardia cytologie genetika růst a vývoj MeSH
- giardiáza parazitologie MeSH
- lidé MeSH
- mitóza * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- Acanthamoeba účinky léků fyziologie ultrastruktura MeSH
- buněčná stěna fyziologie ultrastruktura MeSH
- dimethylsulfoxid farmakologie MeSH
- ethanol farmakologie MeSH
- fyziologická adaptace * MeSH
- fyziologický stres * MeSH
- genotyp MeSH
- methanol farmakologie MeSH
- rozpouštědla MeSH
- stanovení celkové genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- dimethylsulfoxid MeSH
- ethanol MeSH
- methanol MeSH
- rozpouštědla MeSH
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.
- MeSH
- Acanthamoeba klasifikace účinky léků patogenita fyziologie MeSH
- akantamébová keratitida etiologie MeSH
- fluorescenční mikroskopie MeSH
- genotyp MeSH
- lidé MeSH
- propylenglykol škodlivé účinky MeSH
- rizikové faktory MeSH
- roztoky pro kontaktní čočky škodlivé účinky chemie MeSH
- transmisní elektronová mikroskopie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- propylenglykol MeSH
- roztoky pro kontaktní čočky MeSH
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.
- MeSH
- fylogeneze MeSH
- geny rRNA MeSH
- mezerníky ribozomální DNA chemie genetika MeSH
- mikroskopie MeSH
- molekulární sekvence - údaje MeSH
- organely ultrastruktura MeSH
- Parabasalidea klasifikace genetika ultrastruktura MeSH
- protozoální DNA chemie genetika MeSH
- protozoální proteiny genetika MeSH
- ribozomální DNA chemie genetika MeSH
- RNA protozoální genetika MeSH
- RNA ribozomální 18S genetika MeSH
- sekvenční analýza DNA MeSH
- shluková analýza MeSH
- transmisní elektronová mikroskopie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- mezerníky ribozomální DNA MeSH
- protozoální DNA MeSH
- protozoální proteiny MeSH
- ribozomální DNA MeSH
- RNA protozoální MeSH
- RNA ribozomální 18S MeSH
- MeSH
- Eukaryota klasifikace genetika MeSH
- fylogeneze * MeSH
- genetická variace * MeSH
- molekulární sekvence - údaje MeSH
- ribozomální DNA chemie genetika MeSH
- sekvenční analýza DNA MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ribozomální DNA MeSH
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.
- MeSH
- fylogeneze MeSH
- geny rRNA MeSH
- mezerníky ribozomální DNA genetika MeSH
- molekulární sekvence - údaje MeSH
- organely ultrastruktura MeSH
- protozoální DNA chemie genetika MeSH
- ribozomální DNA chemie genetika MeSH
- RNA protozoální genetika MeSH
- RNA ribozomální 18S genetika MeSH
- sekvenční analýza DNA MeSH
- sekvenční homologie nukleových kyselin MeSH
- transmisní elektronová mikroskopie MeSH
- Trichomonadida klasifikace cytologie genetika ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- mezerníky ribozomální DNA MeSH
- protozoální DNA MeSH
- ribozomální DNA MeSH
- RNA protozoální MeSH
- RNA ribozomální 18S MeSH
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.
- MeSH
- buněčné dělení genetika fyziologie MeSH
- cytokineze fyziologie MeSH
- dělení bunečného jádra genetika fyziologie MeSH
- flagella parazitologie fyziologie ultrastruktura MeSH
- fluorescenční mikroskopie metody MeSH
- Giardia lamblia cytologie genetika fyziologie MeSH
- mikroskopie elektronová rastrovací metody MeSH
- mikrotubuly genetika fyziologie ultrastruktura MeSH
- transmisní elektronová mikroskopie metody MeSH
- trofozoiti parazitologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- Bayesova věta MeSH
- fylogeneze MeSH
- genetická variace genetika MeSH
- interakce hostitele a parazita genetika MeSH
- mezerníky ribozomální DNA genetika MeSH
- molekulární evoluce * MeSH
- molekulární sekvence - údaje MeSH
- myši parazitologie MeSH
- prasata parazitologie MeSH
- protozoální DNA chemie genetika MeSH
- RNA ribozomální 5.8S genetika MeSH
- sekvenční analýza DNA MeSH
- Trichomonadida klasifikace genetika MeSH
- zvířata MeSH
- Check Tag
- myši parazitologie MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- mezerníky ribozomální DNA MeSH
- protozoální DNA MeSH
- RNA ribozomální 5.8S MeSH
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.
- MeSH
- buněčné dělení fyziologie MeSH
- flagella fyziologie MeSH
- fluorescenční protilátková technika MeSH
- Giardia lamblia cytologie ultrastruktura MeSH
- konfokální mikroskopie MeSH
- transmisní elektronová mikroskopie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH