Characterization of telomeres and telomerase from the single-celled eukaryote Giardia intestinalis.

The ends of linear chromosomes, telomeres, are most commonly maintained by the enzyme telomerase. Our study presents the characteristics of telomeres and telomerase from the single-celled parasitic eukaryote Giardia intestinalis. Using fluorescence in situ hybridization, we localized telomeres during all stages of the trophozoite cell cycle and demonstrated differences in the observed number of telomeric foci, indicating telomere clustering. The length of Giardia telomeres was determined in different cell lines derived from WB clinical isolate using terminal restriction fragment analysis and ranged from 0.5 to 2.5kb; moreover, a BAL-31 digestion experiment did not reveal any long interstitial telomeric sequences in the genome. Despite the absence of the specific T motif in the telomerase catalytic subunit, the presence of an active telomerase enzyme synthesising telomeric repeats in Giardia was proved by a Telomere repeat amplification protocol assay, and its localization in nuclei was determined by the expression of recombinant GiTERT. Except for the Giardia-type TAGGG telomeric repeat, Giardia telomerase was proved to synthesize in vitro also other repeat variants, TAAGG and TAAGGG. In summary, despite its unusual characteristics, including a structurally divergent but active telomerase, unique terminal sequences and relatively short telomeres, the present data support the view that the chromosomal termini in Giardia are maintained in a conservative manner that is common to other eukaryotes.

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.

Constitutive aneuploidy and genomic instability in the single-celled eukaryote Giardia intestinalis.

Giardia intestinalis is an important single-celled human pathogen. Interestingly, this organism has two equal-sized transcriptionally active nuclei, each considered diploid. By evaluating condensed chromosome numbers and visualizing homologous chromosomes by fluorescent in situ hybridization, we determined that the Giardia cells are constitutively aneuploid. We observed karyotype inter-and intra-population heterogeneity in eight cell lines from two clinical isolates, suggesting constant karyotype evolution during in vitro cultivation. High levels of chromosomal instability and frequent mitotic missegregations observed in four cell lines correlated with a proliferative disadvantage and growth retardation. Other cell lines, although derived from the same clinical isolate, revealed a stable yet aneuploid karyotype. We suggest that both chromatid missegregations and structural rearrangements contribute to shaping the Giardia genome, leading to whole-chromosome aneuploidy, unequal gene distribution, and a genomic divergence of the two nuclei within one cell. Aneuploidy in Giardia is further propagated without p53-mediated cell cycle arrest and might have been a key mechanism in generating the genetic diversity of this human pathogen.

Structural organization of very small chromosomes: study on a single-celled evolutionary distant eukaryote Giardia intestinalis.

During mitotic prophase, chromosomes of the pathogenic unicellular eukaryote Giardia intestinalis condense in each of the cell's two nuclei. In this study, Giardia chromosomes were investigated using light microscopy, high-resolution field emission scanning electron microscopy, and in situ hybridization. For the first time, we describe the overall morphology, condensation stages, and mitotic segregation of these chromosomes. Despite the absence of several genes involved in the cohesion and condensation pathways in the Giardia genome, we observed chromatin organization similar to those found in eukaryotes, i.e., 10-nm nucleosomal fibrils, 30-nm fibrils coiled to chromomeres or in parallel arrangements, and closely aligned sister chromatids. DNA molecules of Giardia terminate with telomeric repeats that we visualized on each of the four chromatid endings of metaphase chromosomes. Giardia chromosomes lack primary and secondary constrictions, thus preventing their classification based on the position of the centromere. The anaphase poleward segregation of sister chromatids is atypical in orientation and tends to generate lagging chromatids between daughter nuclei. In the Giardia genome database, we identified two putative members of the kleisin family thought to be responsible for condensin ring establishment. Thus far, Giardia chromosomes (300 nm to 1.5 µm) are the smallest chromosomes that were analyzed at the ultrastructural level. This study complements the existing molecular and sequencing data on Giardia chromosomes with cytological and ultrastructural information.

The effect of metronidazole on the cell cycle and DNA in metronidazole-susceptible and -resistant Giardia cell lines.

Metronidazole (MTZ) is used as the drug of choice to treat Giardia infections. It is believed that the prodrug is transformed intracellularly into toxic intermediates that interact with cellular components, leading to cell death. The present study aimed to describe the effects of MTZ treatment on DNA and cell cycle progression in MTZ-sensitive and in vitro-derived MTZ-resistant cell lines. Detection of the phosphorylated form of histone H2A in cell nuclei together with electrophoresis of genomic DNA, flow cytometry analysis and incubation of cells with other drugs (albendazole or neomycin) demonstrated that DNA damage in MTZ-treated cells is clearly conditioned by the presence of this drug. The flow cytometry analysis and a BrdU labeling assay showed that the sublethal drug concentration affects the replication phase of the cell cycle. Cells incubated with lethal drug concentration exhibit unchanged DNA profile, only about 50% of cells are positive for γH2A and lose an ability to attach to a surface after few hours of incubation. It is likely that the early reaction of cells to lethal concentration of MTZ is not primarily initiated by the reaction to DNA damage but rather by the immediate interaction of MTZ with biomolecules where activated MTZ is generated. Interestingly, in MTZ-resistant lines incubated in the presence of the drug, about 40% of cells remain permanently positive for γH2A without any effects on the cell cycle progression suggesting that DNA damage caused by MTZ treatment persists in these cells. Accelerated mutagenesis caused by MTZ-induced DNA damage may therefore be a new factor contributing to the development of natural resistance.

Molecular evidence of Pneumocystis jirovecii reinfection in a haemato-oncology patient.

For many years Pneumocystis pneumonia was thought to be caused by the reactivation of a latent infection, but several studies have demonstrated that Pneumocystis jirovecii infection can be acquired de novo. On the basis of our results obtained from a patient with recurrent pneumocystosis, we support the hypothesis that recurrent episodes are caused by reinfection.

Giardia intestinalis: aphidicolin influence on the trophozoite cell cycle.

This study is a thorough examination of the effects of the DNA polymerase inhibitor aphidicolin on the nuclear cycle and cell cycle progression characteristics, as well as their reversibility, in Giardia intestinalis. Giardia trophozoites are arrested in the G1/S-junction after aphidicolin treatment according to their DNA content. However, cell growth continues and trophozoites arrested with aphidicolin resemble cells in the G2 phase and trophozoites in ageing cultures. Extensive treatment with aphidicolin causes side effects and we detected positive signals for phosphorylated histone H2A, which, in mammalian cells, is involved in a signalling pathway triggered as a reaction to double stranded DNA breaks. These results suggest that aphidicolin causes dissociation of the nuclear and cytoplasmic cycles, a phenomenon that has also been described for other inhibitors in mammalian cell lines. Thus, if aphidicolin is used for synchronization of Giardia trophozoites, this fact must be accounted for, and treatment with aphidicolin must be minimal.

SlowFaster, a user-friendly program for slow-fast analysis and its application on phylogeny of Blastocystis.

BACKGROUND: Slow-fast analysis is a simple and effective method to reduce the influence of substitution saturation, one of the causes of phylogenetic noise and long branch attraction (LBA) artifacts. In several steps of increasing stringency, the slow-fast analysis omits the fastest substituting alignment positions from the analysed dataset and thus increases its signal/noise ratio. RESULTS: Our program SlowFaster automates the process of assessing the substitution rate of the alignment positions and the process of producing new alignments by deleting the saturated positions. Its use is very simple. It goes through the whole process in several steps: data input - necessary choices - production of new alignments. CONCLUSION: SlowFaster is a user-friendly tool providing new alignments prepared with slow-fast analysis. These data can be used for further phylogenetic analyses with lower risk of long branch attraction artifacts.