Combined nanometric and phylogenetic analysis of unique endocytic compartments in Giardia lamblia sheds light on the evolution of endocytosis in Metamonada

BACKGROUND: Giardia lamblia, a parasitic protist of the Metamonada supergroup, has evolved one of the most diverged endocytic compartment systems investigated so far. Peripheral endocytic compartments, currently known as peripheral vesicles or vacuoles (PVs), perform bulk uptake of fluid phase material which is then digested and sorted either to the cell cytosol or back to the extracellular space. RESULTS: Here, we present a quantitative morphological characterization of these organelles using volumetric electron microscopy and super-resolution microscopy (SRM). We defined a morphological classification for the heterogenous population of PVs and performed a comparative analysis of PVs and endosome-like organelles in representatives of phylogenetically related taxa, Spironucleus spp. and Tritrichomonas foetus. To investigate the as-yet insufficiently understood connection between PVs and clathrin assemblies in G. lamblia, we further performed an in-depth search for two key elements of the endocytic machinery, clathrin heavy chain (CHC) and clathrin light chain (CLC), across different lineages in Metamonada. Our data point to the loss of a bona fide CLC in the last Fornicata common ancestor (LFCA) with the emergence of a protein analogous to CLC (GlACLC) in the Giardia genus. Finally, the location of clathrin in the various compartments was quantified. CONCLUSIONS: Taken together, this provides the first comprehensive nanometric view of Giardia's endocytic system architecture and sheds light on the evolution of GlACLC analogues in the Fornicata supergroup and, specific to Giardia, as a possible adaptation to the formation and maintenance of stable clathrin assemblies at PVs

The influence of the design of removable dentures on patient's voice quality

Background: The protozoan parasite Giardia intestinalis and the pathogenic bacterium Helicobacter pylori are well known for their high prevalences in human hosts worldwide. The prevalence of both organisms is known to peak in densely populated, low resource settings and children are infected early in life. Different Giardia genotypes/assemblages have been associated with different symptoms and H. pylori with induction of cancer. Despite this, not much data are available from sub-Saharan Africa with regards to the prevalence of different G. intestinalis assemblages and their potential association with H. pylori infections. Methodology/Principal Findings: Fecal samples from 427 apparently healthy children, 0-12 years of age, living in urban Kampala, Uganda were analyzed for the presence of H. pylori and G. intestinalis. G. intestinalis was found in 86 (20.1%) out of the children and children age 1<5 years had the highest rates of colonization. H. pylori was found in 189 (44.3%) out of the 427 children and there was a 3-fold higher risk of concomitant G. intestinalis and H. pylori infections compared to non-concomitant G. intestinalis infection, OR = 2.9 (1.7-4.8). No significant association was found in the studied population with regard to the presence of Giardia and gender, type of toilet, source of drinking water or type of housing. A panel of 45 G. intestinalis positive samples was further analyzed using multi-locus genotyping (MLG) on three loci, combined with assemblage-specific analyses. Giardia MLG analysis yielded a total of five assemblage AII, 25 assemblage B, and four mixed assemblage infections. The assemblage B isolates were highly genetically variable but no significant association was found between Giardia assemblage type and H. pylori infection. Conclusions/Significance: This study shows that Giardia assemblage B dominates in children in Kampala, Uganda and that the presence of H. pylori is an associated risk factor for G. intestinalis infection

A genomic survey of the fish parasite Spironucleus salmonicida indicates genomic plasticity among diplomonads and significant lateral gene transfer in eukaryote genome evolution

BACKGROUND: Comparative genomic studies of the mitochondrion-lacking protist group Diplomonadida (diplomonads) has been lacking, although Giardia lamblia has been intensively studied. We have performed a sequence survey project resulting in 2341 expressed sequence tags (EST) corresponding to 853 unique clones, 5275 genome survey sequences (GSS), and eleven finished contigs from the diplomonad fish parasite Spironucleus salmonicida (previously described as S. barkhanus). RESULTS: The analyses revealed a compact genome with few, if any, introns and very short 3' untranslated regions. Strikingly different patterns of codon usage were observed in genes corresponding to frequently sampled ESTs versus genes poorly sampled, indicating that translational selection is influencing the codon usage of highly expressed genes. Rigorous phylogenomic analyses identified 84 genes – mostly encoding metabolic proteins – that have been acquired by diplomonads or their relatively close ancestors via lateral gene transfer (LGT). Although most acquisitions were from prokaryotes, more than a dozen represent likely transfers of genes between eukaryotic lineages. Many genes that provide novel insights into the genetic basis of the biology and pathogenicity of this parasitic protist were identified including 149 that putatively encode variant-surface cysteine-rich proteins which are candidate virulence factors. A number of genomic properties that distinguish S. salmonicida from its human parasitic relative G. lamblia were identified such as nineteen putative lineage-specific gene acquisitions, distinct mutational biases and codon usage and distinct polyadenylation signals. CONCLUSION: Our results highlight the power of comparative genomic studies to yield insights into the biology of parasitic protists and the evolution of their genomes, and suggest that genetic exchange between distantly-related protist lineages may be occurring at an appreciable rate in eukaryote genome evolution

Transcriptome analyses of the Giardia lamblia life cycle

Author Posting. © The Author(s), 2010. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Molecular and Biochemical Parasitology 174 (2010): 62-65, doi:10.1016/j.molbiopara.2010.05.010.We quantified mRNA abundance from 10 stages in the Giardia lamblia life cycle in vitro using Serial Analysis of Gene Expression (SAGE). 163 abundant transcripts were expressed constitutively. 71 transcripts were upregulated specifically during excystation and 42 during encystation. Nonetheless, the transcriptomes of cysts and trophozoites showed major differences. SAGE detected co-expressed clusters of 284 transcripts differentially expressed in cysts and excyzoites and 287 transcripts in vegetative trophozoites and encysting cells. All clusters included known genes and pathways as well as proteins unique to Giardia or diplomonads. SAGE analysis of the Giardia life cycle identified a number of kinases, phosphatases, and DNA replication proteins involved in excystation and encystation, which could be important for examining the roles of cell signaling in giardial differentiation. Overall, these data pave the way for directed gene discovery and a better understanding of the biology of Giardia lamblia.BJD, DSR, and FDG were supported by NIH grants AI42488, GM61896, DK35108, and AI051687. DP and SGS were supported by grants from the Swedish Natural Science Research Council, the Swedish Medical Research Council, and the Karolinska Institutet. AGM, SRB, SPP, and MJC were supported by NIH grant AI51089 and by the Marine Biological Laboratory’s Program in Global Infectious Diseases, funded by the Ellison Medical Foundation

Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation

Author Posting. © The Authors, 2006. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Molecular and Biochemical Parasitology 152 (2007): 80-89, doi:10.1016/j.molbiopara.2006.12.001.The ability of Giardia lamblia to undergo two distinct differentiations in response to physiologic stimuli is central to its pathogenesis. The giardial cytoskeleton changes drastically during encystation and excystation. However, the signal transduction pathways mediating these transformations are poorly understood. We tested the hypothesis that PP2A, a highly conserved serine/threonine protein phosphatase, might be important in giardial differentiation. We found that in vegetatively growing trophozoites, gPP2A-C protein localizes to basal bodies/centrosomes, and to cytoskeletal structures unique to Giardia: the ventral disk, and the dense rods of the anterior, posterior-lateral, and caudal flagella. During encystation, gPP2A-C protein disappears from only the anterior flagellar dense rods. During excystation, gPP2A-C localizes to the cyst wall in excysting cysts but is not found in the wall of cysts with emerging excyzoites. Transcriptome and immunoblot analyses indicated that gPP2A-C mRNA and protein are upregulated in mature cysts and during the early stage of excystation that models passage through the host stomach. Stable expression of gPP2A-C antisense RNA did not affect vegetative growth, but strongly inhibited the formation of encystation secretory vesicles (ESV) and water-resistant cysts. Moreover, the few cysts that formed were highly defective in excystation. Thus, gPP2A-C localizes to universal cytoskeletal structures and to structures unique to Giardia. It is also important for encystation and excystation, crucial giardial transformations that entail entry into and exit from dormancy.This work was funded by NIH grants GM61896, AI51687, AI42488, and DK35108. Dr. A.G. McArthur was supported by NIH grant AI51089 and the Marine Biological Laboratory’s Program in Global Infectious Diseases, funded by the Ellison Medical Foundation

Multilocus Genotyping of Human Giardia Isolates Suggests Limited Zoonotic Transmission and Association between Assemblage B and Flatulence in Children

Giardia intestinalis is a protozoan parasite found world-wide and it is a major cause of diarrhea in humans and other mammals. The genetic variability within G. intestinalis is high with eight distinct genotypes or assemblages (A-H). Here we performed sequence-based multilocus genotyping of around 200 human Giardia isolates. We found evidence of limited zoonotic transmission of certain A subtypes and an association between flatulence and assemblage B infection in children. This shows that it is important to investigate different assemblages and sub-assemblages of G. intestinalis in human infections in order to understand the clinical significance, zoonotic potential, sequence divergence, and transmission pathways of this parasite

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

<p>Abstract</p> <p>Background</p> <p><it>Giardia intestinalis </it>is a protozoan parasite that causes diarrhea in a wide range of mammalian species. To further understand the genetic diversity between the <it>Giardia intestinalis </it>species, we have performed genome sequencing and analysis of a wild-type <it>Giardia intestinalis </it>sample from the assemblage E group, isolated from a pig.</p> <p>Results</p> <p>We 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 <it>Giardia intestinalis </it>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 <it>Giardia </it>revealed differential rates of divergence among cellular processes.</p> <p>Conclusions</p> <p>Our results indicate that despite a well conserved core of genes there is significant genome variation between <it>Giardia </it>isolates, both in terms of gene content, gene polymorphisms, structural chromosomal variations and surface molecule repertoires. This study improves the annotation of the <it>Giardia </it>genomes and enables the identification of functionally important variation.</p

Draft Genome Sequencing of Giardia intestinalis Assemblage B Isolate GS: Is Human Giardiasis Caused by Two Different Species?

Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genotypes, assemblages A and B, infect humans. The genome of assemblage A parasite WB was recently sequenced, and the structurally compact 11.7 Mbp genome contains simplified basic cellular machineries and metabolism. We here performed 454 sequencing to 16× coverage of the assemblage B isolate GS, the only Giardia isolate successfully used to experimentally infect animals and humans. The two genomes show 77% nucleotide and 78% amino-acid identity in protein coding regions. Comparative analysis identified 28 unique GS and 3 unique WB protein coding genes, and the variable surface protein (VSP) repertoires of the two isolates are completely different. The promoters of several enzymes involved in the synthesis of the cyst-wall lack binding sites for encystation-specific transcription factors in GS. Several synteny-breaks were detected and verified. The tetraploid GS genome shows higher levels of overall allelic sequence polymorphism (0.5 versus <0.01% in WB). The genomic differences between WB and GS may explain some of the observed biological and clinical differences between the two isolates, and it suggests that assemblage A and B Giardia can be two different species

A Tomographic Measurement Technique for Irradiated Nuclear Fuel Assemblies

The fuel assemblies used at the Swedish nuclear power plants contain typically between 100 and 300 fuel rods. An experimental technique has been demanded for determining the relative activities of specific isotopes in individual fuel rods without dismantling the assemblies. The purpose is to validate production codes, which requires an experimental relative accuracy of &lt;2 % (1 σ). Therefore, a new, non-destructive tomographic measurement technique for irradiated nuclear fuel assemblies has been developed. The technique includes two main steps: (1) the gamma-ray flux distribution around the assembly is recorded, and (2) the interior gamma-ray source distribution in the assembly is reconstructed. The use of detailed gamma-ray transport calculations in the reconstruction procedure enables accurate determination of the relative rod-by-rod source distribution. To investigate the accuracy achievable, laboratory equipment has been constructed, including a fuel model with a well-known distribution of 137Cs. Furthermore, an instrument has been constructed and built for in-pool measurements on irradiated fuel assemblies at nuclear power plants. Using the laboratory equipment, a relative accuracy of 1.2 % was obtained (1 σ). The measurements on irradiated fuel resulted in a repeatability of 0.8 %, showing the accuracy that can be achieved using this instrument. The agreement between rod-by-rod data obtained in calculations using the POLCA–7 production code and measured data was 3.1 % (1 σ). Additionally, there is a safeguards interest in the tomographic technique for verifying that no fissile material has been diverted from fuel assemblies, i.e. that no fuel rods have been removed or replaced. The applicability has been demonstrated in a measurement on a spent fuel assembly. Furthermore, detection of both the removal of a rod as well as the replacement with a non-active rod has been investigated in detail and quantitatively established using the laboratory equipment