| 1. |
- Emery-Corbin, Samantha J., et al.
(author)
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Eukaryote-Conserved Methylarginine Is Absent in Diplomonads and Functionally Compensated in Giardia
- 2020
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In: Molecular biology and evolution. - : OXFORD UNIV PRESS. - 0737-4038 .- 1537-1719. ; 37:12, s. 3525-3549
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Journal article (peer-reviewed)abstract
- Methylation is a common posttranslational modification of arginine and lysine in eukaryotic proteins. Methylproteomes are best characterized for higher eukaryotes, where they are functionally expanded and evolved complex regulation. However, this is not the case for protist species evolved from the earliest eukaryotic lineages. Here, we integrated bioinformatic, proteomic, and drug-screening data sets to comprehensively explore the methylproteome of Giardia duodenalis-a deeply branching parasitic protist. We demonstrate that Giardia and related diplomonads lack arginine-methyltransferases and have remodeled conserved RGG/RG motifs targeted by these enzymes. We also provide experimental evidence for methylarginine absence in proteomes of Giardia but readily detect methyllysine. We bioinformatically infer 11 lysine-methyltransferases in Giardia, including highly diverged Su(var)3-9, Enhancer-of-zeste and Trithorax proteins with reduced domain architectures, and novel annotations demonstrating conserved methyllysine regulation of eukaryotic elongation factor 1 alpha. Using mass spectrometry, we identifymore than 200methyllysine sites in Giardia, including in species-specific gene families involved in cytoskeletal regulation, enriched in coiled-coil features. Finally, we use known methylation inhibitors to show that methylation plays key roles in replication and cyst formation in this parasite. This study highlights reduced methylation enzymes, sites, and functions early in eukaryote evolution, including absent methylarginine networks in the Diplomonadida. These results challenge the view that arginine methylation is eukaryote conserved and demonstrate that functional compensation of methylarginine was possible preceding expansion and diversification of these key networks in higher eukaryotes.
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| 2. |
- Ansell, Brendan R. E., et al.
(author)
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Divergent Transcriptional Responses to Physiological and Xenobiotic Stress in Giardia duodenalis
- 2016
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In: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 60:10, s. 6034-6045
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Journal article (peer-reviewed)abstract
- Understanding how parasites respond to stress can help to identify essential biological processes. Giardia duodenalis is a parasitic protist that infects the human gastrointestinal tract and causes 200 to 300 million cases of diarrhea annually. Metronidazole, a major antigiardial drug, is thought to cause oxidative damage within the infective trophozoite form. However, treatment efficacy is suboptimal, due partly to metronidazole-resistant infections. To elucidate conserved and stress-specific responses, we calibrated sublethal metronidazole, hydrogen peroxide, and thermal stresses to exert approximately equal pressure on trophozoite growth and compared transcriptional responses after 24 h of exposure. We identified 252 genes that were differentially transcribed in response to all three stressors, including glycolytic and DNA repair enzymes, a mitogen-activated protein (MAP) kinase, high-cysteine membrane proteins, flavin adenine dinucleotide (FAD) synthetase, and histone modification enzymes. Transcriptional responses appeared to diverge according to physiological or xenobiotic stress. Downregulation of the antioxidant system and alpha-giardins was observed only under metronidazole-induced stress, whereas upregulation of GARP-like transcription factors and their subordinate genes was observed in response to hydrogen peroxide and thermal stressors. Limited evidence was found in support of stress-specific response elements upstream of differentially transcribed genes; however, antisense derepression and differential regulation of RNA interference machinery suggest multiple epigenetic mechanisms of transcriptional control.
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| 3. |
- Ansell, Brendan R. E., et al.
(author)
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Drug resistance in Giardia duodenalis
- 2015
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In: Biotechnology Advances. - : Elsevier BV. - 0734-9750 .- 1873-1899. ; 33:6, s. 888-901
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Research review (peer-reviewed)abstract
- Giardia duodenalis is a microaerophilic parasite of the human gastrointestinal tract and a major contributor to diarrheal and post-infectious chronic gastrointestinal disease world-wide. Treatment of G. duodenalis infection currently relies on a small number of drug classes. Nitroheterocyclics, in particular metronidazole, have represented the front line treatment for the last 40 years. Nitroheterocyclic-resistant G. duodenalis have been isolated from patients and created in vitro, prompting considerable research into the biomolecular mechanisms of resistance. These compounds are redox-active and are believed to damage proteins and DNA after being activated by oxidoreductase enzymes in metabolically active cells. In this review, we explore the molecular phenotypes of nitroheterocyclic-resistant G. duodenalis described to date in the context of the protisfs unusual glycolytic and antioxidant systems. We propose that resistance mechanisms are likely to extend well beyond currently described resistance-associated enzymes (i.e., pyruvate ferredoxin oxidoreductases and nitroreductases), to include NAD(P)H- and flavin-generating pathways, and possibly redox-sensitive epigenetic regulation. Mechanisms that allow G. duodenalis to tolerate oxidative stress may lead to resistance against both oxygen and nitroheterocyclics, with implications for clinical control. The present review highlights the potential for systems biology tools and advanced bioinformatics to further investigate the multifaceted mechanisms of nitroheterocyclic resistance in this important pathogen.
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| 4. |
- Ansell, Brendan R. E., et al.
(author)
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Time-Dependent Transcriptional Changes in Axenic Giardia duodenalis Trophozoites
- 2015
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In: PLoS Neglected Tropical Diseases. - : Public Library of Science (PLoS). - 1935-2727 .- 1935-2735. ; 9:12
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Journal article (peer-reviewed)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. Although G. duodenalis can be cultured axenically, significant gaps exist in our understanding of the molecular biology and metabolism of this pathogen. The present study employed RNA sequencing to characterize the mRNA transcriptome of G. duodenalis trophozoites in axenic culture, at log (48 h of growth), stationary (60 h), and declining (96 h) growth phases. Using similar to 400-times coverage of the transcriptome, we identified 754 differentially transcribed genes (DTGs), mainly representing two large DTG groups: 438 that were down-regulated in the declining phase relative to log and stationary phases, and 281 that were up-regulated. Differential transcription of prominent antioxidant and glycolytic enzymes implicated oxygen tension as a key factor influencing the transcriptional program of axenic trophozoites. Systematic bioinformatic characterization of numerous DTGs encoding hypothetical proteins of unknown function was achieved using structural homology searching. This powerful approach greatly informed the differential transcription analysis and revealed putative novel antioxidant-coding genes, and the presence of a nearcomplete two-component-like signaling system that may link cytosolic redox or metabolite sensing to the observed transcriptional changes. Motif searching applied to promoter regions of the two large DTG groups identified different putative transcription factor-binding motifs that may underpin global transcriptional regulation. This study provides new insights into the drivers and potential mediators of transcriptional variation in axenic G. duodenalis and provides context for static transcriptional studies.
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| 5. |
- Ansell, Brendan R. E., et al.
(author)
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Transcriptomics Indicates Active and Passive Metronidazole Resistance Mechanisms in Three Seminal Giardia Lines
- 2017
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In: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 8
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Journal article (peer-reviewed)abstract
- Giardia duodenalis is an intestinal parasite that causes 200-300 million episodes of diarrhoea annually. Metronidazole (Mtz) is a front-line anti-giardial, but treatment failure is common and clinical resistance has been demonstrated. Mtz is thought to be activated within the parasite by oxidoreductase enzymes, and to kill by causing oxidative damage. In G. duodenalis, Mtz resistance involves active and passive mechanisms. Relatively low activity of iron-sulfur binding proteins, namely pyruvate: ferredoxin oxidoreductase (PFOR), ferredoxins, and nitroreductase-1, enable resistant cells to passively avoid Mtz activation. Additionally, low expression of oxygen-detoxification enzymes can allow passive (non-enzymatic) Mtz detoxification via futile redox cycling. In contrast, active resistance mechanisms include complete enzymatic detoxification of the pro-drug by nitroreductase-2 and enhanced repair of oxidized biomolecules via thioredoxin-dependent antioxidant enzymes. Molecular resistance mechanisms may be largely founded on reversible transcriptional changes, as some resistant lines revert to drug sensitivity during drug-free culture in vitro, or passage through the life cycle. To comprehensively characterize these changes, we undertook strand-specific RNA sequencing of three laboratory-derived Mtz-resistant lines, 106-2ID(10), 713-M3, and WB-M3, and compared transcription relative to their susceptible parents. Common up-regulated genes encoded variant-specific surface proteins (VSPs), a high cysteine membrane protein, calcium and zinc channels, a Mad-2 cell cycle regulator and a putative fatty acid a alpha-oxidase. Down-regulated genes included nitroreductase-1, putative chromate and quinone reductases, and numerous genes that act proximal to PFOR. Transcriptional changes in 106-2ID(10) diverged from those in 713-r and WB-r (r <= 0.2), which were more similar to each other (r = 0.47). In 106-2ID(10), a nonsense mutation in nitroreductase-1 transcripts could enhance passive resistance whereas increased transcription of nitroreductase-2, and a MATE transmembrane pump system, suggest active drug detoxification and efflux, respectively. By contrast, transcriptional changes in 713-M3 and WB-M3 indicated a higher oxidative stress load, attributed to Mtz- and oxygen-derived radicals, respectively. Quantitative comparisons of orthologous gene transcription between Mtz-resistant G. duodenalis and Trichomonas vaginalis, a closely related parasite, revealed changes in transcripts encoding peroxidases, heat shock proteins, and FMN-binding oxidoreductases, as prominent correlates of resistance. This work provides deep insight into Mtz-resistant G. duodenalis, and illuminates resistance-associated features across parasitic species.
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| 6. |
- Emery-Corbin, Samantha J., et al.
(author)
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Proteomic diversity in a prevalent human-infective Giardia duodenalis sub-species
- 2018
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In: International Journal of Parasitology. - : ELSEVIER SCI LTD. - 0020-7519 .- 1879-0135. ; 48:11, s. 817-823
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Journal article (peer-reviewed)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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| 7. |
- Emery, Samantha J., et al.
(author)
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Differential protein expression and post-translational modifications in metronidazole-resistant Giardia duodenalis
- 2018
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In: GigaScience. - : OXFORD UNIV PRESS. - 2047-217X. ; 7:4
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Journal article (peer-reviewed)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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| 8. |
- Guadalupe Ortega-Pierres, M., et al.
(author)
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Recent advances in the genomic and molecular biology of Giardia
- 2018
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In: Acta Tropica. - : ELSEVIER SCIENCE BV. - 0001-706X .- 1873-6254. ; 184, s. 67-72
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Journal article (peer-reviewed)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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| 9. |
- Hansen, Eline P., et al.
(author)
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Exploration of extracellular vesicles from Ascaris suum provides evidence of parasite-host cross talk
- 2019
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In: Journal of Extracellular Vesicles. - : Wiley. - 2001-3078. ; 8:1
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Journal article (peer-reviewed)abstract
- The prevalent porcine helminth, Ascaris suum, compromises pig health and reduces farm productivity worldwide. The closely related human parasite, A. lumbricoides, infects more than 800 million people representing a disease burden of 1.31 million disability-adjusted life years. The infections are often chronic in nature, and the parasites have a profound ability to modulate their hosts' immune responses. This study provides the first in-depth characterisation of extracellular vesicles (EVs) from different developmental stages and body parts of A. suum and proposes the role of these vesicles in the host-parasite interplay. The release of EVs from the third- (L3) and fourth-stage (L4) larvae and adults was demonstrated by transmission electron microscopy (TEM), and sequencing of EV-derived RNA identified a number of microRNAs (miRNAs) and transcripts of potential host immune targets, such as IL-13, IL-25 and IL-33, were identified. Furthermore, proteomics of EVs identified several proteins with immunomodulatory properties and other proteins previously shown to be associated with parasite EVs. Taken together, these results suggest that A. suum EVs and their cargo may play a role in host-parasite interactions. This knowledge may pave the way to novel strategies for helminth infection control and knowledge of their immune modulatory potential.
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| 10. |
- Ma'ayeh, Showgy Y., et al.
(author)
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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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In: Frontiers in Cellular and Infection Microbiology. - : FRONTIERS MEDIA SA. - 2235-2988. ; 8
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Journal article (peer-reviewed)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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