| 1. |
- Jenikova, Gabriela, et al.
(författare)
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alpha 1-giardin based live heterologous vaccine protects against Giardia lamblia infection in a murine model
- 2011
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Ingår i: Vaccine. - : Elsevier BV. - 0264-410X .- 1873-2518. ; 29:51, s. 9529-9537
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Tidskriftsartikel (refereegranskat)abstract
- Giardia lamblia is a leading protozoan cause of diarrheal disease worldwide, yet preventive medical strategies are not available. A crude veterinary vaccine has been licensed for cats and dogs, but no defined human vaccine is available. We tested the vaccine potential of three conserved antigens previously identified in human and murine giardiasis, alpha 1-giardin, alpha-enolase, and ornithine carbamoyl transferase, in a murine model of G. lamblia infection. Live recombinant attenuated Salmonella enterica Serovar Typhimurium vaccine strains were constructed that stably expressed each antigen, maintained colonization capacity, and sustained total attenuation in the host. Oral administration of the vaccine strains induced antigen-specific serum IgG, particularly IgG(2A), and mucosal IgA for alpha 1-giardin and alpha-enolase, but not for ornithine carbamoyl transferase. Immunization with the alpha 1-giardin vaccine induced significant protection against subsequent G. lamblia challenge, which was further enhanced by boosting with cholera toxin or sublingual alpha 1-giardin administration. The alpha-enolase vaccine afforded no protection. Analysis of at alpha 1-giardin from divergent assemblage A and B isolates of G. lamblia revealed >97% amino acid sequence conservation and immunological cross-reactivity, further supporting the potential utility of this antigen in vaccine development. Together. These results indicate that alpha 1-giardin is a suitable candidate antigen for a vaccine against giardiasis.
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| 2. |
- Morrison, Hilary G., et al.
(författare)
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Genomic minimalism in the early diverging intestinal parasite Giardia lamblia
- 2007
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 317:5846, s. 1921-1926
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Tidskriftsartikel (refereegranskat)abstract
- The genome of the eukaryotic protist Giardia lamblia, an important human intestinal parasite, is compact in structure and content, contains few introns or mitochondrial relics, and has simplified machinery for DNA replication, transcription, RNA processing, and most metabolic pathways. Protein kinases comprise the single largest protein class and reflect Giardia's requirement for a complex signal transduction network for coordinating differentiation. Lateral gene transfer from bacterial and archaeal donors has shaped Giardia's genome, and previously unknown gene families, for example, cysteine-rich structural proteins, have been discovered. Unexpectedly, the genome shows little evidence of heterozygosity, supporting recent speculations that this organism is sexual. This genome sequence will not only be valuable for investigating the evolution of eukaryotes, but will also be applied to the search for new therapeutics for this parasite.
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| 3. |
- Birkeland, Shanda R., et al.
(författare)
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Transcriptome analyses of the Giardia lamblia life cycle
- 2010
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Ingår i: Molecular and biochemical parasitology (Print). - : Elsevier BV. - 0166-6851 .- 1872-9428. ; 174:1, s. 62-65
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Tidskriftsartikel (refereegranskat)abstract
- 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 G. lamblia.
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| 4. |
- Davids, Barbara J., et al.
(författare)
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Polymeric immunoglobulin receptor in intestinal immune defense against the lumen-dwelling protozoan parasite Giardia
- 2006
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Ingår i: Journal of Immunology. - 0022-1767 .- 1550-6606. ; 177:9, s. 6281-6290
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Tidskriftsartikel (refereegranskat)abstract
- The polymeric Ig receptor (pIgR) is conserved in mammals and has an avian homologue, suggesting evolutionarily important functions in vertebrates. It transports multimeric IgA and IgM across polarized epithelia and is highly expressed in the intestine, yet little direct evidence exists for its importance in defense against common enteric pathogens. In this study, we demonstrate that pIgR can play a critical role in intestinal defense against the lumen-dwelling protozoan parasite Giardia, a leading cause of diarrheal disease. The receptor was essential for the eradication of Giardia when high luminal IgA levels were required. Clearance of Giardia muris, in which IgA plays a dominant role, was severely compromised in pIgR-deficient mice despite significant fecal IgA output at 10% of normal levels. In contrast, eradication of the human strain Giardia lamblia GS/M, for which adaptive immunity is less IgA dependent in mice, was unaffected by pIgR deficiency, indicating that pIgR had no physiologic role when lower luminal IgA levels were sufficient for parasite elimination. Immune IgA was greatly increased in the serum of pIgR-deficient mice, conferred passive protection against Giardia, and recognized several conserved giardial Ags, including ornithine carbamoyltransferase, arginine deiminase, alpha-enolase, and alpha- and beta-giardins, that are also detected in human giardiasis. Corroborative observations were made in mice lacking the J chain, which is required for pIgR-dependent transepithelial IgA transport. These results, together with prior data on pIgR-mediated immune neutralization of luminal cholera toxin, suggest that pIgR is essential in intestinal defense against pathogenic microbes with high-level and persistent luminal presence.
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| 5. |
- Ferella, Marcela, et al.
(författare)
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Gene expression changes during Giardia-host cell interactions in serum-free medium
- 2014
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Ingår i: Molecular and biochemical parasitology (Print). - : Elsevier BV. - 0166-6851 .- 1872-9428. ; 197:1-2, s. 21-23
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Tidskriftsartikel (refereegranskat)abstract
- Serial Analysis of Gene Expression (SAGE) was used to quantify transcriptional changes in Giardia intestinalis during its interaction with human intestinal epithelial cells (IECs, HT-29) in serum free M199 medium. Transcriptional changes were compared to those in trophozoites alone in M199 and in TYI-S-33 Giardia growth medium. In total, 90 genes were differentially expressed, mainly those involved in cellular redox homeostasis, metabolism and small molecule transport but also cysteine proteases and structural proteins of the giardin family. Only 29 genes changed their expression due to IEC interaction and the rest were due to M199 medium. Although our findings generated a small dataset, it was consistent with our earlier microarray studies performed under different interaction conditions. This study has confined the number of genes in Giardia to a small subset that specifically change their expression due to interaction with IECs.
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| 6. |
- Lauwaet, Tineke, et al.
(författare)
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Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation
- 2007
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Ingår i: Molecular and biochemical parasitology (Print). - : Elsevier BV. - 0166-6851 .- 1872-9428. ; 152:1, s. 80-89
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Manning, Gerard, et al.
(författare)
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The minimal kinome of Giardia lamblia illuminates early kinase evolution and unique parasite biology
- 2011
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Ingår i: Genome Biology. - : Springer Science and Business Media LLC. - 1465-6906 .- 1474-760X. ; 12:7, s. R66-
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Tidskriftsartikel (refereegranskat)abstract
- Background: The major human intestinal pathogen Giardia lamblia is a very early branching eukaryote with a minimal genome of broad evolutionary and biological interest. Results: To explore early kinase evolution and regulation of Giardia biology, we cataloged the kinomes of three sequenced strains. Comparison with published kinomes and those of the excavates Trichomonas vaginalis and Leishmania major shows that Giardia's 80 core kinases constitute the smallest known core kinome of any eukaryote that can be grown in pure culture, reflecting both its early origin and secondary gene loss. Kinase losses in DNA repair, mitochondrial function, transcription, splicing, and stress response reflect this reduced genome, while the presence of other kinases helps define the kinome of the last common eukaryotic ancestor. Immunofluorescence analysis shows abundant phospho-staining in trophozoites, with phosphotyrosine abundant in the nuclei and phosphothreonine and phosphoserine in distinct cytoskeletal organelles. The Nek kinase family has been massively expanded, accounting for 198 of the 278 protein kinases in Giardia. Most Neks are catalytically inactive, have very divergent sequences and undergo extensive duplication and loss between strains. Many Neks are highly induced during development. We localized four catalytically active Neks to distinct parts of the cytoskeleton and one inactive Nek to the cytoplasm. Conclusions: The reduced kinome of Giardia sheds new light on early kinase evolution, and its highly divergent sequences add to the definition of individual kinase families as well as offering specific drug targets. Giardia's massive Nek expansion may reflect its distinctive lifestyle, biphasic life cycle and complex cytoskeleton.
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| 8. |
- Reiner, David S., et al.
(författare)
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Synchronisation of Giardia lamblia : identification of cell cycle stage-specific genes and a differentiation restriction point
- 2008
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Ingår i: International Journal of Parasitology. - : Elsevier BV. - 0020-7519 .- 1879-0135. ; 38:8-9, s. 935-944
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Tidskriftsartikel (refereegranskat)abstract
- The intestinal parasite Giardia lamblia undergoes cell differentiations that entail entry into and departure from the replicative cell cycle. The pathophysiology of giardiasis depends directly upon the ability of the trophozoite form to replicate in the host upper small intestine. Thus, cell proliferation is tightly linked to disease. However, studies of cell cycle regulation in Giardia have been hampered by the inability to synchronise cultures. Here we report that Giardia isolates of the major human genotypes A and B can be synchronised using aphidicolin, a mycotoxin that reversibly inhibits replicative DNA polymerases in eukaryotic cells. Aphidicolin arrests Giardia trophozoites in the early DNA synthesis (S) phase of the cell cycle. We identified a set of cell cycle orthologues in the Giardia genome using bioinformatic analyses and showed that synchronised parasites express these genes in a cell cycle stage-specific manner. The synchronisation method also showed that during encystation, exit from the ordinary cell cycle occurs preferentially in GZ and defines a restriction point for differentiation. Synchronisation opens up possibilities for further molecular and cell biological studies of chromosome replication, mitosis and segregation of the complex cytoskeleton in Giardia.
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| 9. |
- Ringqvist, Emma, et al.
(författare)
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Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells
- 2008
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Ingår i: Molecular and biochemical parasitology (Print). - : Elsevier BV. - 0166-6851 .- 1872-9428. ; 159:2, s. 85-91
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Tidskriftsartikel (refereegranskat)abstract
- Giardia lamblia, an important cause of diarrheal disease, resides in the small intestinal lumen in close apposition to epithelial cells. Since the disease mechanisms underlying giardiasis are poorly understood, elucidating the specific interactions of the parasite with the host epithelium is likely to provide clues to understanding the pathogenesis. Here we tested the hypothesis that contact of Giardia lamblia with intestinal epithelial cells might lead to release of specific proteins. Using established co-culture models, intestinal ligated loops and a proteomics approach, we identified three G. lamblia proteins (arginine deiminase, ornithine carbamoyl transferase and enolase), previously recognized as immunodominant antigens during acute giardiasis. Release was stimulated by cell-cell interactions, since only small amounts of arginine deiminase and enolase were detected in the medium after culturing of G. lamblia alone. The secreted G. lamblia proteins were localized to the cytoplasm and the inside of the plasma membrane of trophozoites. Furthermore, in vitro studies with recombinant arginine deiminase showed that the secreted Giardia proteins can disable host innate immune factors such as nitric oxide production. These results indicate that contact of Giardia with epithelial cells triggers metabolic enzyme release, which might facilitate effective colonization of the human small intestine.
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