Eva Nohýnková

Genome analysis and comparative genomics of a Giardia intestinalis assemblage E isolate.

BackgroundGiardia intestinalis is a protozoan parasite that causes diarrhea in a wide range of mammalian species. To further understand the genetic diversity between the Giardia intestinalis species, we have performed genome sequencing and analysis of a wild-type Giardia intestinalis sample from the assemblage E group, isolated from a pig.ResultsWe identified 5012 protein coding genes, the majority of which are conserved compared to the previously sequenced genomes of the WB and GS strains in terms of microsynteny and sequence identity. Despite this, there is an unexpectedly large number of chromosomal rearrangements and several smaller structural changes that are present in all chromosomes. Novel members of the VSP, NEK Kinase and HCMP gene families were identified, which may reveal possible mechanisms for host specificity and new avenues for antigenic variation. We used comparative genomics of the three diverse Giardia intestinalis isolates P15, GS and WB to define a core proteome for this species complex and to identify lineage-specific genes. Extensive analyses of polymorphisms in the core proteome of Giardia revealed differential rates of divergence among cellular processes.ConclusionsOur results indicate that despite a well conserved core of genes there is significant genome variation between Giardia isolates, both in terms of gene content, gene polymorphisms, structural chromosomal variations and surface molecule repertoires. This study improves the annotation of the Giardia genomes and enables the identification of functionally important variation.

Cell Division of Giardia intestinalis: Flagellar Developmental Cycle Involves Transformation and Exchange of Flagella between Mastigonts of a Diplomonad Cell

ABSTRACT 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.

Localization of gamma-tubulin in interphase and mitotic cells of a unicellular eukaryote, Giardia intestinalis.

Giardia intestinalis, a bi-nucleated amitochondrial flagellate, possesses a complex cytoskeleton based on several microtubular systems (flagella, adhesive disk, median body, funis, mitotic spindles). MTOCs of the individual systems have not been fully defined. By using monoclonal antibodies against a conserved synthetic peptide from the C-terminus of human gamma-tubulin we investigated occurrence and distribution of gamma-tubulin in interphase and mitotic Giardia cells. On the immunoblots of Giardia cytoskeletal extracts the antibodies bound to a single polypeptide of approximately 50 kDa. Immunostaining of the interphase cell demonstrated gamma-tubulin as four bright spots at the basis of four out of eight flagella. Gamma-tubulin label was associated with perikinetosomal areas of the ventral and posterolateral pairs of flagella which are formed de novo during cell division. Basal body regions of the anterolateral and caudal pairs of flagella which persist during the division and are integrated into the flagellar systems of the daughter cells did not show gamma-tubulin staining. At early mitosis, gamma-tubulin spots disappeared reappearing again at late mitosis in accord with reorientation of parent flagella and reorganization of flagellar apparatus during cell division. The antibody-detectable gamma-tubulin epitope was absent at the poles of both mitotic spindles. Albendazole-treated Giardia, in which spindle assembly was completely inhibited, showed the same gamma-tubulin staining pattern thus confirming that the fluorescent label is exclusively located in the basal body regions. Our results point to a role of gamma-tubulin in nucleation of microtubules of newly formed flagella and indicate unusual mitotic spindle assembly. Moreover, the demonstration of gamma-tubulin in Giardia shows ubiquity of this protein through the evolutionary history of eukaryotes.

Cytogenetic evidence for diversity of two nuclei within a single diplomonad cell of Giardia

Giardia intestinalis is an ancient protist that causes the most commonly reported human diarrheal disease of parasitic origin worldwide. An intriguing feature of the Giardia cell is the presence of two morphologically similar nuclei, generally considered equivalent, in spite of the fact that their karyotypes are unknown. We found that within a single cell, the two nuclei differ both in the number and the size of chromosomes and that representatives of two major genetic groups of G. intestinalis possess different karyotypes. Odd chromosome numbers indicate aneuploidy of Giardia nuclei, and their stable occurrence is suggestive of a long-term asexuality. A semi-open type of Giardia mitosis excludes a chromosome interfusion between the nuclei. Differences in karyotype and DNA content, and cell cycle-dependent asynchrony are indicative of diversity of the two Giardia nuclei.

Amebic Encephalitis Caused by Balamuthia mandrillaris in a Czech Child : Description of the First Case from Europe

We describe a fatal case of amebic encephalitis caused by Balamuthia mandrillaris in a 3-year-old Czech boy who had never traveled abroad. This is the first such infection reported in Europe. The diagnosis was established by brain biopsy, in which abundant trophozoites and a few round amebic cysts were identified. The presence of multiple nucleoli in some trophozoites suggested the organism to be Balamuthia mandrillaris and this was confirmed by indirect immunofluorescence. The amebae invaded brain tissue, including neurons, and blood vessel walls, causing thrombovasculitis. The tissue reaction was a subacute necrotizing and granulomatous encephalitis (GAE) with an infiltrate of CD4- and CD8-positive T-lymphocytes, B-lymphocytes, plasma cells and macrophages. The child, in whom no underlying immunodeficiency was demonstrated, died after 45 days. The mode of infection was not established. Postmortem examination of the brain revealed massive areas of necrosis and microscopic findings like those in the surgical specimen. In vitro isolation of B. mandrillaris was unsuccessful.

Mouse genetic model for clinical and immunological heterogeneity of leishmaniasis

Abstract. Systematic assessment of the role of host genes in clinico-pathological and immunological manisfestations of Leishmania major-induced disease in mice was performed using 20 recombinant congenic (RC) strains. As the RC strains are homozygous and each carries a different, random set of 12.5% genes from the resistant strain, STS/A, and 87.5% genes from the susceptible strain, BALB/cHeA, they allowed us to study the pathological and immunological characteristics of infected hosts in 20 fixed different random combinations of BALB/c and STS genes. The 20 RC strains differ widely in expression of different symptoms of disease and in immunological characteristics. Disease or healing in different strains occurred in association with different components of immune response – with the exception of a frequently occurring correlation between the disease and IgE levels. Moreover, some parameters of the immune response were highly correlated in some strains but not at all in others. This shows that several patterns of the immune response may be associated with the same clinical outcome, depending on the host genotype. Our data also suggest that despite the complexity of regulation, when a sufficient number of controlling loci is known, the prediction of a phenotype is possible. Combining functional and clinical information with multilocus genotyping may improve our ability to predict the progression of the disease and to optimize the treatment.

Preparation of highly infective Leishmania promastigotes by cultivation on SNB-9 biphasic medium

Protozoan hemoflagellates Leishmania are causative agents of leishmaniases and an important biological model for study of host-pathogen interaction. A wide range of methods of Leishmania cultivation on both biphasic and liquid media is available. Biphasic media are considered to be superior for initial isolation of the parasites and obtaining high promastigote infectivity; however, liquid media are more suitable for large-scale experiments. The aim of the present study was the adaptation and optimization of the cultivation of Leishmania promastigotes on a biphasic SNB-9 (saline-neopeptone-blood 9) medium that was originally developed for Trypanosoma cultivation and combines the advantages of biphasic and liquid media. SNB-9 medium is characterized with a large volume of the liquid phase, which facilitates the manipulation with the culture and provides parasite yields comparable to parasite yields on such liquid medium as Schneiders Insect Medium. We demonstrate that SNB-9 very considerably surpasses Schneiders Insect Medium in in vitro infectivity of the parasites. Additionally, we show that the ratio of apoptotic parasites, which are important for the infectivity of the inoculum, in Leishmania culture in SNB-9 is higher than in Leishmania culture in Schneiders Insect Medium. Thus, we demonstrate that the cultivation of Leishmania on SNB-9 reliably yields highly infective promastigotes suitable for experimental infection.

Ultrastructure of Cochlosoma anatis Kotlán, 1923 and taxonomic position of the family Cochlosomatidae (Parabasala: Trichomonadida)

Summary The fine structure of Cochlosoma anatis is described from scanning and transmission electron microscopy. These flagellates are asymmetrical and uninucleate, with a conspicuous adhesive disc formed from the modified pelta. Six flagella arise from the anterior end. Four of them emerge anterolaterally from the lateral groove. The fifth flagellum is recurrent and adheres to the cell body by a well developed undulating membrane, formed by a cytoplasmic fold with the marginal lamella. The kinetosome of the recurrent flagellum is situated at an angle to the kinetosomes of the anterior flagella. This kinetosomal complex is associated with fibrillar appendages characteristic of a trichomonad mastigont. The kinetosome of the sixth flagellum is located outside the complex of the other kinetosomes, on the dorsal side of the protozoan body. The undulating membrane is supported by the prominent costa with Type B periodicity and passes throughout the lateral groove extending to the end of the parasite’s body. There is a parabasal apparatus composed of a Golgi complex and a single parabasal fiber. The axostyle, formed from a single layer of microtubules, passes through the body, protruding caudally in a short projection. Anteriorly it overlaps the inside of the pelta. An extranuclear mitotic spindle is formed during the nuclear division. The cytoplasm contains double membrane bound organelles similar to hydrogenosomes. These observations show high degree of homology of Cochlosoma with Trichomonadida Kirby, 1947 warranting inclusion of the family Cochlosomatidae Tyzzer, 1930 into this order.

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.

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.