| 1. |
- Emery-Corbin, Samantha J., et al.
(författare)
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Proteomic diversity in a prevalent human-infective Giardia duodenalis sub-species
- 2018
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Ingår i: International Journal of Parasitology. - : ELSEVIER SCI LTD. - 0020-7519 .- 1879-0135. ; 48:11, s. 817-823
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Tidskriftsartikel (refereegranskat)abstract
- Giardia duodenalis a species complex of gastrointestinal protists, with assemblages A and B infective to humans. To date, post-genomic proteomics are largely derived from Assemblage A, biasing understanding of parasite biology. To address this gap, we quantitatively analysed the proteomes of trophozoites from the genome reference and two clinical Assemblage B isolates, revealing lower spectrum-to-peptide matches in non-reference isolates, resulting in significant losses in peptide and protein identifications, and indicating significant intra-assemblage variation. We also explored differential protein expression between in vitro cultured subpopulations putatively enriched for dividing and feeding cells, respectively. This data is an important proteomic baseline for Assemblage B, highlighting proteomic differences between physiological states, and unique differences relative to Assemblage A.
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| 2. |
- Emery, Samantha J., et al.
(författare)
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Differential protein expression and post-translational modifications in metronidazole-resistant Giardia duodenalis
- 2018
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Ingår i: GigaScience. - : OXFORD UNIV PRESS. - 2047-217X. ; 7:4
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Tidskriftsartikel (refereegranskat)abstract
- Background: Metronidazole (Mtz) is the frontline drug treatment for multiple anaerobic pathogens, including the gastrointestinal protist, Giardia duodenalis. However, treatment failure is common and linked to in vivo drug resistance. In Giardia, in vitro drug-resistant lines allow controlled experimental interrogation of resistance mechanisms in isogenic cultures. However, resistance-associated changes are inconsistent between lines, phenotypic data are incomplete, and resistance is rarely genetically fixed, highlighted by reversion to sensitivity after drug selection ceases or via passage through the life cycle. Comprehensive quantitative approaches are required to resolve isolate variability, fully define Mtz resistance phenotypes, and explore the role of post-translational modifications therein. Findings: We performed quantitative proteomics to describe differentially expressed proteins in 3 seminal Mtz-resistant lines compared to their isogenic, Mtz-susceptible, parental line. We also probed changes in post-translational modifications including protein acetylation, methylation, ubiquitination, and phosphorylation via immunoblotting. We quantified more than 1,000 proteins in each genotype, recording substantial genotypic variation in differentially expressed proteins between isotypes. Our data confirm substantial changes in the antioxidant network, glycolysis, and electron transport and indicate links between protein acetylation and Mtz resistance, including cross-resistance to deacetylase inhibitor trichostatin A in Mtz-resistant lines. Finally, we performed the first controlled, longitudinal study of Mtz resistance stability, monitoring lines after cessation of drug selection, revealing isolate-dependent phenotypic plasticity. Conclusions: Our data demonstrate understanding that Mtz resistance must be broadened to post-transcriptional and post-translational responses and that Mtz resistance is polygenic, driven by isolate-dependent variation, and is correlated with changes in protein acetylation networks.
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| 3. |
- Guadalupe Ortega-Pierres, M., et al.
(författare)
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Recent advances in the genomic and molecular biology of Giardia
- 2018
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Ingår i: Acta Tropica. - : ELSEVIER SCIENCE BV. - 0001-706X .- 1873-6254. ; 184, s. 67-72
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Tidskriftsartikel (refereegranskat)abstract
- Giardia duodenalis is the most common gastrointestinal protozoan parasite of humans and a significant contributor to the global burden of both diarrheal disease and post-infectious chronic disorders. Robust tools for analyzing gene function in this parasite have been developed and a range of genetic tools are now available. These together with public databases have provided insights on the function of different genes in Giardia. In this review we provide a current perspective on different molecular aspects of Giardia related to genomics, regulation of encystation, trophozoite transcriptional responses to physiological and xenobiotic (drug-induced) stress, and mechanisms of drug resistance. We also examine recent insights that have contributed to gain knowledge in the study of VSPs, antigenic variation, epigenetics, DNA repair and in the direct manipulation of gene function in Giardia, with a particular focus on the inducible Cre/loxP system.
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| 4. |
- Ma'ayeh, Showgy Y., et al.
(författare)
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Responses of the Differentiated Intestinal Epithelial Cell Line Caco-2 to Infection With the Giardia intestinalis GS Isolate
- 2018
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Ingår i: Frontiers in Cellular and Infection Microbiology. - : FRONTIERS MEDIA SA. - 2235-2988. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Giardia intestinalis is a parasitic protist that causes diarrhea in humans, affecting mainly children of the developing world, elderly and immunocompromised individuals. Humans are infected by two major Giardia assemblages (i.e. genetic subtypes), A and B, with the latter being the most common. So far, there is little information on molecular or cellular changes during infections with assemblage B. Here, we used RNA sequencing to study transcriptional changes in Caco-2 intestinal epithelial cells (IECs) co-incubated with assemblage B (GS isolate) trophozoites for 1.5, 3, and 4.5 h. We aimed to identify early molecular events associated with the establishment of infection and followed cellular protein changes up to 10h. IEC transcriptomes showed a dominance of immediate early response genes which was sustained across all time points. Transcription of inflammatory cytokines (e.g., cxcl1-3, ccl2, 1l1a, and il1b) peaked at 1.5 and 3 h of infection. Compared to co-incubation with assemblage A Giardia, we identified the induction of novel cytokines (cxcl8, cxcl10, csf1 , cx3cl1, il12a, il11) and showed that inflammatory signaling is mediated by Erk1/2 phosphorylation (mitogen activated protein kinase, MAPK), nuclear factor kappa B (NF kappa B) and adaptor protein-1 (AP-1). We also showed that GS trophozoites attenuate P38 (MAPK) phosphorylation in IECs. Low amounts of IL-8, CXCL1 and CCL20 proteins were measured in the interaction medium, which was attributed to cytokine degradation by trophozoite secreted proteases. Based on the transcriptome, the decay of cytokines mRNA mediated by zinc finger protein 36 might be another mechanism controlling cytokine levels at later time points. IEC transcriptomes suggested homeostatic responses to counter oxidative stress, glucose starvation, and disturbances in amino acid and lipid metabolism. A large group of differentially transcribed genes were associated with cell cycle arrest and induction of apoptosis, which was validated at protein level. IEC transcriptomes also suggested changes in tight junction's integrity, microvilli structure and the extracellular mucin layer. This is the first study to illuminate transcriptional and protein regulatory events underlying IECs responses and pathogenesis during Giardia assemblage B infection. It highlights differences compared to assemblage A infections which might account for the differences observed in human infections with the two assemblages.
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