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  • 1. Anders, Alfjorden
    et al.
    Astvaldsson, Asgeir
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eva, Jansson
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Experimental challenge of Atlantic salmon (Salmo salar) with the diplomonad parasite Spironucleus salmonicida to characterize the infection cycleManuscript (preprint) (Other academic)
    Abstract [en]

    Experimental infections were performed of Atlantic salmon (Salmo salar) from the Baltic Sea region with the Diplomonad fish parasite Spironucleus salmonicida in order to define the infection cycle, specifically the time-line and putative routes of transmission. An oral infection protocol using axenic parasites was developed, as were new diagnostic tools using PCR and specific antibodies. We also produced firefly luciferase expressing S. salmonicida parasites that could be identified in the infected fish using in vivo and ex vivo imaging. The new tools made it possible to follow the S. salmonicida infection cycle in detail. Three different stages of the infection were identified: one initial intestinal stage, followed by a blood stage and a final tissue stage. Parasites intubated into the intestine attached to the intestinal surface and were identified in the blood after 1-3 weeks. Skin lesions and infections of the muscles, internal organs and eyes were seen 4-10 weeks after initiation of infection. Several morphologically different forms of S. salmonicida cells were detected in ex vivo cell-cultures of biopsies from skin lesions. By this infection trial we have been able to show that S. salmonicida may use several alternative routes of transmission. One alternative is the fecal-oral route, similar to other Diplomonad parasites but the parasites can also be excreted directly into the surrounding water from the mucous layer of the skin or from an ulcerated skin lesion. This information can be used to prevent the transmission of the parasite in fish farms.

  • 2.
    Ankarklev, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Franzen, Oscar
    Karolinska Inst, Dept Cell & Mol Biol, SE-17177 Stockholm, Sweden. KISP, Sci Life Lab, S-17165 Solna, Sweden..
    Peirasmaki, Dimitra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Jerlstrom-Hultqvist, Jon
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lebbad, Marianne
    Publ Hlth Agcy Sweden, Dept Microbiol, SE-17182 Solna, Sweden..
    Andersson, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Andersson, Bjorn
    Karolinska Inst, Dept Cell & Mol Biol, SE-17177 Stockholm, Sweden.;KISP, Sci Life Lab, S-17165 Solna, Sweden..
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage A isolates2015In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 16, article id 697Article in journal (Refereed)
    Abstract [en]

    Background: The diarrhea-causing protozoan Giardia intestinalis makes up a species complex of eight different assemblages (A-H), where assemblage A and B infect humans. Comparative whole-genome analyses of three of these assemblages have shown that there is significant divergence at the inter-assemblage level, however little is currently known regarding variation at the intra-assemblage level. We have performed whole genome sequencing of two sub-assemblage AII isolates, recently axenized from symptomatic human patients, to study the biological and genetic diversity within assemblage A isolates. Results: Several biological differences between the new and earlier characterized assemblage A isolates were identified, including a difference in growth medium preference. The two AII isolates were of different sub-assemblage types (AII-1 [AS175] and AII-2 [AS98]) and showed size differences in the smallest chromosomes. The amount of genetic diversity was characterized in relation to the genome of the Giardia reference isolate WB, an assemblage AI isolate. Our analyses indicate that the divergence between AI and AII is approximately 1 %, represented by similar to 100,000 single nucleotide polymorphisms (SNP) distributed over the chromosomes with enrichment in variable genomic regions containing surface antigens. The level of allelic sequence heterozygosity (ASH) in the two AII isolates was found to be 0.25-0.35 %, which is 25-30 fold higher than in the WB isolate and 10 fold higher than the assemblage AII isolate DH (0.037 %). 35 protein-encoding genes, not found in the WB genome, were identified in the two AII genomes. The large gene families of variant-specific surface proteins (VSPs) and high cysteine membrane proteins (HCMPs) showed isolate-specific divergences of the gene repertoires. Certain genes, often in small gene families with 2 to 8 members, localize to the variable regions of the genomes and show high sequence diversity between the assemblage A isolates. One of the families, Bactericidal/ Permeability Increasing-like protein (BPIL), with eight members was characterized further and the proteins were shown to localize to the ER in trophozoites. Conclusions: Giardia genomes are modular with highly conserved core regions mixed up by variable regions containing high levels of ASH, SNPs and variable surface antigens. There are significant genomic variations in assemblage A isolates, in terms of chromosome size, gene content, surface protein repertoire and gene polymorphisms and these differences mainly localize to the variable regions of the genomes. The large genetic differences within one assemblage of G. intestinalis strengthen the argument that the assemblages represent different Giardia species.

  • 3.
    Ankarklev, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hestvik, Elin
    Lebbad, Marianne
    Lindh, Johan
    Kaddu-Mulindwa, Deogratias H.
    Andersson, Jan O.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Tylleskar, Thorkild
    Tumwine, James K.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Common Coinfections of Giardia intestinalis and Helicobacter pylori in Non-Symptomatic Ugandan Children2012In: PLOS Neglected Tropical Diseases, ISSN 1935-2735, Vol. 6, no 8, p. e1780-Article in journal (Refereed)
    Abstract [en]

    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.

  • 4.
    Ankarklev, Johan
    et al.
    Stockholm Univ, Dept Mol Biosci, SE-10691 Stockholm, Sweden.
    Lebbad, Marianne
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Einarsson, Elin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Franzen, Oscar
    Karolinska Inst, Integrated Cardio Metab Ctr, Novum, Box 285, SE-14157 Stockholm, Sweden.
    Ahola, Harri
    Natl Vet Inst, Dept Microbiol, SE-75189 Uppsala, Sweden.
    Troell, Karin
    Natl Vet Inst, Dept Microbiol, SE-75189 Uppsala, Sweden.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    A novel high-resolution multilocus sequence typing of Giardia intestinalis Assemblage A isolates reveals zoonotic transmission, clonal outbreaks and recombination2018In: Infection, Genetics and Evolution, ISSN 1567-1348, E-ISSN 1567-7257, Vol. 60, p. 7-16Article in journal (Refereed)
    Abstract [en]

    Molecular epidemiology and genotyping studies of the parasitic protozoan Giardia intestinalis have proven difficult due to multiple factors, such as low discriminatory power in the commonly used genotyping loci, which has hampered molecular analyses of outbreak sources, zoonotic transmission and virulence types. Here we have focused on assemblage A Giardia and developed a high-resolution assemblage-specific multilocus sequence typing (MLST) method. Analyses of sequenced G. intestinalis assemblage A genomes from different sub-assemblages identified a set of six genetic loci with high genetic variability. DNA samples from both humans (n = 44) and animals (n = 18) that harbored Giardia assemblage A infections, were PCR amplified (557-700 bp products) and sequenced at the six novel genetic loci. Bioinformatic analyses showed five to ten-fold higher levels of polymorphic sites than what was previously found among assemblage A samples using the classic genotyping loci. Phylogenetically, a division of two major clusters in assemblage A became apparent, separating samples of human and animal origin. A subset of human samples (n = 9) from a documented Giardia outbreak in a Swedish day-care center, showed full complementarity at nine genetic loci (the six new and the standard BG, TPI and GDH loci), strongly suggesting one source of infection. Furthermore, three samples of human origin displayed MLST profiles that were phylogenetically more closely related to MLST profiles from animal derived samples, suggesting zoonotic transmission. These new genotyping loci enabled us to detect events of recombination between different assemblage A isolates but also between assemblage A and E isolates. In summary, we present a novel and expanded MLST strategy with significantly improved sensitivity for molecular analyses of virulence types, zoonotic potential and source tracking for assemblage A Giardia.

  • 5.
    Ankarklev, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lebbad, Marianne
    Allelic sequence heterozygosity in single Giardia parasites2012In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, article id 65Article in journal (Refereed)
    Abstract [en]

    Background: Genetic heterogeneity has become a major inconvenience in the genotyping and molecular epidemiology of the intestinal protozoan parasite Giardia intestinalis, in particular for the major human infecting genotype, assemblage B. Sequence-based genotyping of assemblage B Giardia from patient fecal samples, where one or several of the commonly used genotyping loci (beta-giardin, triosephosphate isomerase and glutamate dehydrogenase) are implemented, is often hampered due to the presence of sequence heterogeneity in the sequencing chromatograms. This can be due to allelic sequence heterozygosity (ASH) and /or co-infections with parasites of different assemblage B sub-genotypes. Thus, two important questions have arisen; i) does ASH occur at the single cell level, and/or ii) do multiple sub-genotype infections commonly occur in patients infected with assemblage B, G. intestinalis isolates? Results: We used micromanipulation in order to isolate single Giardia intestinalis, assemblage B trophozoites (GS isolate) and cysts from human patients. Molecular analysis at the tpi loci of trophozoites from the GS lineage indicated that ASH is present at the single cell level. Analyses of assemblage B Giardia cysts from clinical samples at the bg and tpi loci also indicated ASH at the single cell level. Additionally, alignment of sequence data from several different cysts that originated from the same patient yielded different sequence patterns, thus suggesting the presence of multiple sub-assemblage infections in congruence with ASH within the same patient. Conclusions: Our results conclusively show that ASH does occur at the single cell level in assemblage B Giardia. Furthermore, sequence heterogeneity generated during sequence-based genotyping of assemblage B isolates may possess the complexity of single cell ASH in concurrence with co-infections of different assemblage B sub-genotypes. These findings explain the high abundance of sequence heterogeneity commonly found when performing sequence based genotyping of assemblage B Giardia, and illuminates the necessity of developing new G. intestinalis genotyping tools.

  • 6.
    Ansell, Brendan R. E.
    et al.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    Baker, Louise
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia..
    Emery, Samantha J.
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia..
    McConville, Malcolm J.
    Univ Melbourne, Mol Sci & Biotechnol Inst Bio21, Melbourne, Vic, Australia..
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Gasser, Robin B.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    Jex, Aaron R.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia.;Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia..
    Transcriptomics Indicates Active and Passive Metronidazole Resistance Mechanisms in Three Seminal Giardia Lines2017In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, article id 398Article in journal (Refereed)
    Abstract [en]

    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.

  • 7.
    Ansell, Brendan R. E.
    et al.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    McConville, Malcolm J.
    Univ Melbourne, Bio21 Mol Sci & Biotechnol Inst, Melbourne, Vic, Australia..
    Baker, Louise
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia.;Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun, Melbourne, Vic, Australia..
    Korhonen, Pasi K.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    Emery, Samantha J.
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun, Melbourne, Vic, Australia..
    Svärd, Staffan G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Gasser, Robin B.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    Jex, Aaron R.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia.;Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun, Melbourne, Vic, Australia..
    Divergent Transcriptional Responses to Physiological and Xenobiotic Stress in Giardia duodenalis2016In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 60, no 10, p. 6034-6045Article in journal (Refereed)
    Abstract [en]

    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.

  • 8.
    Ansell, Brendan R. E.
    et al.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    McConville, Malcolm J.
    Univ Melbourne, Mol Sci & Biotechnol Inst Bio21, Parkville, Vic 3052, Australia..
    Baker, Louise
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Korhonen, Pasi K.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Young, Neil D.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Hall, Ross S.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Rojas, Cristian A. A.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Gasser, Robin B.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Jex, Aaron R.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Time-Dependent Transcriptional Changes in Axenic Giardia duodenalis Trophozoites2015In: PLoS Neglected Tropical Diseases, ISSN 1935-2727, E-ISSN 1935-2735, Vol. 9, no 12, article id e0004261Article in journal (Refereed)
    Abstract [en]

    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.

  • 9.
    Ansell, Brendan R. E.
    et al.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3010, Australia..
    McConville, Malcolm J.
    Univ Melbourne, Inst Bio21, Parkville, Vic 3010, Australia..
    Ma'ayeh, Showgy Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Dagley, Michael J.
    Univ Melbourne, Inst Bio21, Parkville, Vic 3010, Australia..
    Gasser, Robin B.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3010, Australia..
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Jex, Aaron R.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3010, Australia..
    Drug resistance in Giardia duodenalis2015In: Biotechnology Advances, ISSN 0734-9750, E-ISSN 1873-1899, Vol. 33, no 6, p. 888-901Article, review/survey (Refereed)
    Abstract [en]

    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.

  • 10.
    Astvaldsson, Asgeir
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hultenby, Kjell
    Karolinska Inst, Dept Lab Med, Stockholm, Sweden.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala Univ, BMC, Dept Cell & Mol Biol, Uppsala, Sweden.
    Jerlström-Hultqvist, Jon
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Proximity Staining Using Enzymatic Protein Tagging in Diplomonads2019In: MSPHERE, ISSN 2379-5042, Vol. 4, no 2, article id e00153-19Article in journal (Refereed)
    Abstract [en]

    The diplomonads are a group of understudied eukaryotic flagellates whose most prominent member is the human pathogen Giardia intestinalis. Methods commonly used in other eukaryotic model systems often require special optimization in diplomonads due to the highly derived character of their cell biology. We have optimized a proximity labeling protocol using pea ascorbate peroxidase (APEX) as a reporter for transmission electron microscopy (TEM) to enable the study of ultrastructural cellular details in diplomonads. Currently available TEM-compatible tags require light-induced activation (1, 2) or are inactive in many cellular compartments (3), while ascorbate peroxidase has not been shown to have those limitations. Here, we have optimized the in vivo activities of two versions of pea ascorbate peroxidase (APX(W41F) and APEX) using the diplomonad fish parasite Spironucleus salmonicida, a relative of G. intestinalis. We exploited the well-known peroxidase substrates, Amplex UltraRed and 3,3'-diaminobenzidine (DAB), to validate the activity of the two tags and argue that APEX is the most stable version to use in Spironucleus salmonicida. Next, we fused APEX to proteins with established localization to evaluate the activity of APEX in different cellular compartments of the diplomonad cell and used Amplex UltraRed as well as antibodies along with superresolution microscopy to confirm the protein-APEX localization. The ultrastructural details of protein-APEX fusions were determined by TEM, and we observed marker activity in all cellular compartments tested when using the DAB substrate. Finally, we show that the optimized conditions established for S. salmonicida can be used in the related diplomonad G. intestinalis. IMPORTANCE The function of many proteins is intrinsically related to their cellular location. Novel methods for ascertainment of the ultrastructural location of proteins have been introduced in recent years, but their implementation in protists has so far not been readily realized. Here, we present an optimized proximity labeling protocol using the APEX system in the salmon pathogen Spironucleus salmonicida. This protocol was also applicable to the human pathogen Giardia intestinalis. Both organisms required extraneous addition of hemin to the growth medium to enable detectable peroxidase activity. Further, we saw no inherent limitation in labeling efficiency coupled to the cellular compartment, as evident with some other proximity labeling systems. We anticipate that the APEX proximity labeling system might offer a great resource to establish the ultrastructural localization of proteins across genetically tractable protists but might require organism-specific labeling conditions.

  • 11.
    Astvaldsson, Asgeir
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hultenby, Kjell
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Jerlström-Hultqvist, Jon
    Proximity Staining using Enzymatic Protein Tagging in Diplomonads2019In: mSphere, E-ISSN 2379-5042, Vol. 4, no 2, article id e00153-19Article in journal (Refereed)
    Abstract [en]

    The diplomonads are a group of understudied eukaryotic flagellates whose most prominent member is the human pathogen Giardia intestinalis. Methods commonly used in other eukaryotic model systems often require special optimization in diplomonads due to the highly derived character of their cell biology. We have optimized a proximity labeling protocol using pea ascorbate peroxidase (APEX) as a reporter for transmission electron microscopy (TEM), to enable study of ultrastructural cellular details in diplomonads. Currently available TEM-compatible tags requires light-induced activation (1, 2) or are inactive in many cellular compartments (3) while ascorbate peroxidase has not been shown to have those limitations. Here we have optimized the in vivo activity of two versions of pea ascorbate peroxidase (APXW41F and APEX) using the diplomonad fish parasite Spironucleus salmonicida, a relative of G. intestinalis. We exploited the well-known peroxidase substrates, Amplex UltraRed and 3,3’-diaminobenzidine (DAB), to validate the activity of the two tags and argue that APEX is the most stable version to use in Spironucleus salmonicida. Next, we fused APEX to proteins with established localization to evaluate the activity of APEX in different cellular compartments of the diplomonad cell and used Amplex UltraRed as well as antibodies along with super-resolution microscopy to confirm the protein-APEX localization. The ultrastructural details of protein-APEX fusions were determined by TEM and we observed marker activity in all cellular compartments tested when using the DAB substrate. Finally, we show that the optimized conditions established for S. salmonicida can be used in the related diplomonad G. intestinalis.

  • 12.
    Astvaldsson, Asgeir
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Stairs, Courtney W.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Xu, Feifei
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Haag, Lars
    Alfjorden, Anders
    Jansson, Eva
    Ettema, Thijs
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Dual transcriptomic analysis of Spironucleus salmonicida-infected salmon cells identifies putative virulence factors and host responsesManuscript (preprint) (Other academic)
  • 13.
    Caccio, Simone M.
    et al.
    Ist Super Sanita, Dept Infect Dis, Viale Regina Elena 299, I-00161 Rome, Italy.
    Lalle, Marco
    Ist Super Sanita, Dept Infect Dis, Viale Regina Elena 299, I-00161 Rome, Italy.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Host specificity in the Giardia duodenalis species complex2018In: Infection, Genetics and Evolution, ISSN 1567-1348, E-ISSN 1567-7257, Vol. 66, p. 335-345Article, review/survey (Refereed)
    Abstract [en]

    Giardia duodenalis is a unicellular flagellated parasite that infects the gastrointestinal tract of a wide range of mammalian species, including humans. Investigations of protein and DNA polymorphisms revealed that G. duodenalis should be considered as a species complex, whose members, despite being morphologically indistinguishable, can be classified into eight groups, or Assemblages, separated by large genetic distances. Assemblages display various degree of host specificity, with Assemblages A and B occurring in humans and many other hosts, Assemblage C and D in canids, Assemblage E in hoofed animals, Assemblage F in cats, Assemblage G in rodents, and Assemblage H in pinnipeds. The factors determining host specificity are only partially understood, and clearly involve both the host and the parasite. Here, we review the results of in vitro and in vivo experiments, and clinical observations to highlight relevant biological and genetic differences between Assemblages, with a focus on human infection.

  • 14.
    Carranza, Pedro G.
    et al.
    Univ Catolica Cordoba, Fac Med, Lab Bioquim & Biol Mol, Cordoba, Argentina.;CITSE, Santiago Del Estero, Argentina..
    Gargantini, Pablo R.
    Univ Catolica Cordoba, Fac Med, Lab Bioquim & Biol Mol, Cordoba, Argentina.;Consejo Nacl Invest Cient & Tecn, Ctr Invest & Desarrollo Inmunol & Enfermedades In, Cordoba, Argentina..
    Prucca, Cesar G.
    Univ Catolica Cordoba, Fac Med, Lab Bioquim & Biol Mol, Cordoba, Argentina.;Ctr Invest Quim Biol Cordoba CIQUIBIC, Cordoba, Argentina..
    Torri, Alessandro
    Univ Catolica Cordoba, Fac Med, Lab Bioquim & Biol Mol, Cordoba, Argentina..
    Saura, Alicia
    Univ Catolica Cordoba, Fac Med, Lab Bioquim & Biol Mol, Cordoba, Argentina.;Consejo Nacl Invest Cient & Tecn, Ctr Invest & Desarrollo Inmunol & Enfermedades In, Cordoba, Argentina..
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lujan, Hugo D.
    Univ Catolica Cordoba, Fac Med, Lab Bioquim & Biol Mol, Cordoba, Argentina.;Consejo Nacl Invest Cient & Tecn, Ctr Invest & Desarrollo Inmunol & Enfermedades In, Cordoba, Argentina..
    Specific histone modifications play critical roles in the control of encystation and antigenic variation in the early-branching eukaryote Giardia lamblia2016In: International Journal of Biochemistry and Cell Biology, ISSN 1357-2725, E-ISSN 1878-5875, Vol. 81, p. 32-43Article in journal (Refereed)
    Abstract [en]

    During evolution, parasitic microorganisms have faced the challenges of adapting to different environments to colonize a variety of hosts. Giardia lamblia, a common cause of intestinal disease, has developed fascinating strategies to adapt both outside and inside its host's intestine, such as trophozoite differentiation into cyst and the switching of its major surface antigens. How gene expression is regulated during these adaptive processes remains undefined. Giardia lacks some typical eukaryotic features, like canonical transcription factors, linker histone H1, and complex promoter regions; suggesting that post transcriptional and translational control of gene expression is essential for parasite survival. However, epigenetic factors may also play critical roles at the transcriptional level. Here, we describe the most common post -translational histone modifications; characterize enzymes involved in these reactions, and analyze their association with the Giardia's differentiation processes. We present evidence that NAD(+)-dependent and NAD(+)-independent histone deacetylases regulate encystation; however, a unique NAD(+)-independent histone deacetylase modulate antigenic switching. The rates of acetylation of H4K8 and H4K16 are critical for encystation, whereas a decrease in acetylation of H4K8 and methylation of H3K9 occur preferentially during antigenic variation. These results show the complexity of the mechanisms regulating gene expression in this minimalistic protozoan parasite.

  • 15.
    Davids, Barbara J.
    et al.
    Univ Calif San Diego, Dept Med, La Jolla, CA 92093 USA.
    Liu, Ching M.
    Univ Calif San Diego, Dept Med, La Jolla, CA 92093 USA.
    Hanson, Elaine M.
    Univ Calif San Diego, Dept Med, La Jolla, CA 92093 USA.
    Le, Christine H. Y.
    Univ Calif San Diego, Dept Med, La Jolla, CA 92093 USA.
    Ang, Jonathan
    Univ Calif San Diego, Dept Med, La Jolla, CA 92093 USA.
    Hanevik, Kurt
    Univ Bergen, Dept Clin Sci, Bergen, Norway.
    Fischer, Marvin
    Univ Bergen, Dept Clin Sci, Bergen, Norway.
    Radunovic, Matej
    Univ Calif San Diego, Dept Med, La Jolla, CA 92093 USA.
    Langeland, Nina
    Univ Bergen, Dept Clin Sci, Bergen, Norway.
    Ferella, Marcela
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Ghassemian, Majid
    Univ Calif San Diego, Dept Chem & Biochem, La Jolla, CA 92093 USA.
    Miyamoto, Yukiko
    Univ Calif San Diego, Dept Med, La Jolla, CA 92093 USA.
    Eckmann, Lars
    Univ Calif San Diego, Dept Med, La Jolla, CA 92093 USA.
    Identification of Conserved Candidate Vaccine Antigens in the Surface Proteome of Giardia lamblia2019In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 87, no 6, article id e00219-19Article in journal (Refereed)
    Abstract [en]

    Giardia lamblia, one of the most common protozoal infections of the human intestine, is an important worldwide cause of diarrheal disease, malabsorption, malnutrition, delayed cognitive development in children, and protracted postinfectious syndromes. Despite its medical importance, no human vaccine is available against giardiasis. A crude veterinary vaccine has been developed, and experimental vaccines based on expression of multiple variant-specific surface proteins have been reported, but poorly defined vaccine components and excessive antigen variability are problematic for pharmaceutical vaccine production. To expand the repertoire of antigen candidates for vaccines, we reasoned that surface proteins may provide an enriched source of such antigens since key host effectors, such as secretory IgA, can directly bind to such antigens in the intestinal lumen and interfere with epithelial attachment. Here, we have applied a proteomics approach to identify 23 novel surface antigens of G. lamblia that show >90% amino acid sequence identity between the two human-pathogenic genetic assemblages (A and B) of the parasite. Surface localization of a representative subset of these proteins was confirmed by immunostaining. Four selected proteins, uridine phosphorylase-like protein-1, protein 21.1 (GL50803_ 27925), alpha 1-giardin, and alpha 11-giardin, were subsequently produced in recombinant form and shown to be immunogenic in mice and G. lamblia-infected humans and confer protection against G. lamblia infection upon intranasal immunization in rodent models of giardiasis. These results demonstrate that identification of conserved surface antigens provides a powerful approach for overcoming a key rate-limiting step in the design and construction of an effective vaccine against giardiasis.

  • 16.
    Einarsson, Elin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Ma'ayeh, Showgy Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    An up-date on Giardia and giardiasis2016In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 34, p. 47-52Article, review/survey (Refereed)
    Abstract [en]

    Giardia intestinalis is a non-invasive protozoan parasite infecting the upper small intestine causing acute, watery diarrhea or giardiasis in 280 million people annually. Asymptomatic infections are equally common and recent data have suggested that infections even can be protective against other diarrhea! diseases. Most symptomatic infections resolve spontaneously but infections can lead to chronic disease and treatment failures are becoming more common world-wide. Giardia infections can also result in irritable bowel syndrome (IBS) and food allergies after resolution. Until recently not much was known about the mechanism of giardiasis or the cause of post-giardiasis syndromes and treatment failures, but here we will describe the recent progress in these areas.

  • 17.
    Einarsson, Elin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Troell, Karin
    UV irradiation responses in Giardia intestinalis2015In: Experimental parasitology, ISSN 0014-4894, E-ISSN 1090-2449, Vol. 154, p. 25-32Article in journal (Refereed)
    Abstract [en]

    The response to ultraviolet light (UV) radiation, a natural stressor to the intestinal protozoan parasite Giardia intestinalis, was studied to deepen the understanding of how the surrounding environment affects the parasite during transmission. UV radiation at 10 mJ/cm(2) kills Giardia cysts effectively whereas trophozoites and encysting parasites can recover from UV treatment at 100 mJ/cm(2) and 50 mJ/cm(2) respectively. Staining for phosphorylated histone H2A showed that UV treatment induces double-stranded DNA breaks and flow cytometry analyses revealed that UV treatment of trophozoites induces DNA replication arrest. Active DNA replication coupled to DNA repair could be an explanation to why UV light does not kill trophozoites and encysting cells as efficiently as the non-replicating cysts. We also examined UV-induced gene expression responses in both trophozoites and cysts using RNA sequencing (RNA seq). UV radiation induces small overall changes in gene expression in Giardia but cysts show a stronger response than trophozoites. Heat shock proteins, kinesins and Nek kinases are up-regulated, whereas alpha-giardins and histones are down-regulated in UV treated trophozoites. Expression of variable surface proteins (VSPs) is changed in both trophozoites and cysts. Our data show that Giardia cysts have limited ability to repair UV-induced damage and this may have implications for drinking- and waste-water treatment when setting criteria for the use of UV disinfection to ensure safe water.

  • 18.
    Einarsson, Elin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Troell, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Natl Vet Inst, Dept Microbiol, S-75007 Uppsala, Sweden.
    Höppner, Marc
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Univ Kiel, Inst Clin Mol Biol, Kiel, Germany.
    Grabherr, Mannfred
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ribacke, Ulf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Coordinated Changes in Gene Expression Throughout Encystation of Giardia intestinalis2016In: PLoS Neglected Tropical Diseases, ISSN 1935-2727, E-ISSN 1935-2735, Vol. 11, no 3, article id e0004571Article in journal (Refereed)
    Abstract [en]

    Differentiation into infectious cysts through the process of encystation is crucial for transmission and survival of the intestinal protozoan parasite Giardia intestinalis. Hitherto the majority of studies have focused on the early events, leaving late encystation poorly defined. In order to further study encystation, focusing on the later events, we developed a new encystation protocol that generates a higher yield of mature cysts compared to standard methods. Transcriptome changes during the entire differentiation from trophozoites to cysts were thereafter studied using RNA sequencing (RNA-seq). A high level of periodicity was observed for up-and down-regulated genes, both at the level of the entire transcriptome and putative regulators. This suggests the trajectory of differentiation to be coordinated through developmentally linked gene regulatory activities. Our study identifies a core of 13 genes that are consistently up-regulated during initial encystation. Of these, two constitute previously uncharacterized proteins that we were able to localize to a new type of encystation-specific vesicles. Interestingly, the largest transcriptional changes were seen in the late phase of encystation with the majority of the highly up-regulated genes encoding hypothetical proteins. Several of these were epitope-tagged and localized to further characterize these previously unknown genetic components of encystation and possibly excystation. Finally, we also detected a switch of variant specific surface proteins (VSPs) in the late phase of encystation. This occurred at the same time as nuclear division and DNA replication, suggesting a potential link between the processes.

  • 19.
    Einarsson, Elin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Ástvaldsson, Ásgeir
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hultenby, Kjell
    Karolinska Inst, Dept Lab Med, Stockholm, Sweden.
    Andersson, Jan O.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Jerlstrom-Hultqvist, Jon
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Comparative cell biology and evolution of Annexins in Diplomonads2016In: MSphere, ISSN 2379-5042, Vol. 1, no 2, article id e00032-15Article in journal (Refereed)
    Abstract [en]

    Annexins are multifunctional, calcium-binding proteins found in organisms across all kingdoms. Most studies of annexins from single-celled eukaryotes have focused on the alpha-giardins, proteins assigned to the group E annexins, expressed by the diplomonad Giardia intestinalis. We have characterized the annexin gene family in another diplomonad parasite, Spironucleus salmonicida, by phylogenetic and experimental approaches. We constructed a comprehensive phylogeny of the diplomonad group E annexins and found that they are abundant across the group with frequent gene duplications and losses. The annexins of S. salmonicida were found to be related to alpha-giardins but with better-preserved type II Ca2+ coordination sites. Two annexins were confirmed to bind phospholipids in a Ca2+-dependent fashion but with different specificities. Superresolution and confocal microscopy of epitope-tagged S. salmonicida annexins revealed localization to distinct parts of the cytoskeleton and membrane. The ultrastructural details of the localization of several annexins were determined by proximity labeling and transmission electron microscopy. Two annexins localize to a novel cytoskeletal structure in the anterior of the cell. Our results show that the annexin gene family is expanded in diplomonads and that these group E annexins are associated mostly with cytoskeletal and membrane structures. IMPORTANCE Annexins are proteins that associate with phospholipids in a Ca2+-dependent fashion. These proteins have been intensely studied in animals and plants because of their importance in diverse cellular processes, yet very little is known about annexins in single-celled eukaryotes, which represent the largest diversity of organisms. The human intestinal parasite Giardia intestinalis is known to have more annexins than humans, and they contribute to its pathogenic potential. In this study, we investigated the annexin complement in the salmon pathogen Spironucleus salmonicida, a relative of G. intestinalis. We found that S. salmonicida has a large repertoire of annexins and that the gene family has expanded separately across diplomonads, with members showing sequence diversity similar to that seen across kingdom-level groups such as plants and animals. S. salmonicida annexins are prominent components of the cytoskeleton and membrane. Two annexins are associated with a previously unrecognized structure in the anterior of the cell.

  • 20.
    Emery, Samantha J.
    et al.
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia.
    Baker, Louise
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia;Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia.
    Ansell, Brendan R. E.
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia;Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia.
    Mirzaei, Mehdi
    Macquarie Univ, Fac Sci, Chem & Biomol Sci, N Ryde, NSW, Australia;Macquarie Univ, Australian Proteome Anal Facil, N Ryde, NSW, Australia.
    Haynes, Paul A.
    Macquarie Univ, Fac Sci, Chem & Biomol Sci, N Ryde, NSW, Australia.
    McConville, Malcom J.
    Univ Melbourne, Mol Sci & Biotechnol Inst Bio21, Melbourne, Vic, Australia.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Jex, Aaron R.
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia;Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia.
    Differential protein expression and post-translational modifications in metronidazole-resistant Giardia duodenalis2018In: GigaScience, ISSN 2047-217X, E-ISSN 2047-217X, Vol. 7, no 4Article in journal (Refereed)
    Abstract [en]

    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.

  • 21.
    Emery-Corbin, Samantha J.
    et al.
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia.
    Vuong, Daniel
    Microbial Screening Technol, Smithfield, NSW, Australia.
    Lacey, Ernest
    Microbial Screening Technol, Smithfield, NSW, Australia;Macquarie Univ, Fac Sci, Chem & Biomol Sci, N Ryde, NSW, Australia.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Ansell, Brendan R. E.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Jex, Aaron R.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia.
    Proteomic diversity in a prevalent human-infective Giardia duodenalis sub-species2018In: International Journal of Parasitology, ISSN 0020-7519, E-ISSN 1879-0135, Vol. 48, no 11, p. 817-823Article in journal (Refereed)
    Abstract [en]

    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. 

  • 22.
    Ettema, Thijs J. G.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Lindas, Ann-Christin
    Hjort, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Poplawski, Andrzej B.
    Kaessmann, Henrik
    Grogan, Dennis W.
    Kelman, Zvi
    Andersson, Anders F.
    Pelve, Erik A.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Rolf Bernander (1956-2014): pioneer of the archaeal cell cycle Obituary2014In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 92, no 5, p. 903-909Article in journal (Refereed)
    Abstract [en]

    On 19 January 2014 Rolf (Roffe') Bernander passed away unexpectedly. Rolf was a dedicated scientist; his research aimed at unravelling the cell biology of the archaeal domain of life, especially cell cycle-related questions, but he also made important contributions in other areas of microbiology. Rolf had a professor position in the Molecular Evolution programme at Uppsala University, Sweden for about 8 years, and in January 2013 he became chair professor at the Department of Molecular Biosciences, The Wenner-Gren Institute at Stockholm University in Sweden. Rolf was an exceptional colleague and will be deeply missed by his family and friends, and the colleagues and co-workers that he leaves behind in the scientific community. He will be remembered for his endless enthusiasm for science, his analytical mind, and his quirky sense of humour.

  • 23.
    Ferella, Marcela
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Davids, Barbara J
    Cipriano, Michael J
    Birkeland, Shanda R
    Palm, Daniel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Gillin, Frances D
    McArthur, Andrew G
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Gene expression changes during Giardia-host cell interactions in serum-free medium2014In: Molecular and biochemical parasitology (Print), ISSN 0166-6851, E-ISSN 1872-9428, Vol. 197, no 1-2, p. 21-23Article in journal (Refereed)
    Abstract [en]

    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.

  • 24.
    Franzén, Oscar
    et al.
    Karolinska Institutet.
    Jerlström-Hultqvist, Jon
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Einarsson, Elin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Ankarklev, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Ferella, Marcela
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Andersson, Björn
    Karolinska Institutet.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Transcriptome Profiling of Giardia intestinalis Using Strand-specific RNAseq2013In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 9, no 3, article id e1003000Article in journal (Refereed)
    Abstract [en]

    Giardia intestinalis is a common cause of diarrheal disease and it consists of eight genetically distinct genotypes or assemblages (A-H). Only assemblages A and B infect humans and are suggested to represent two different Giardia species. Correlations exist between assemblage type and host-specificity and to some extent symptoms. Phenotypical differences have been documented between assemblages and genome sequences are available for A, B and E. We have characterized and compared the polyadenylated transcriptomes of assemblages A, B and E. Four genetically different isolates were studied (WB (AI), AS175 (AII), P15 (E) and GS (B)) using paired-end, strand-specific RNA-seq. Most ofthe genome was transcribed in trophozoites grown in vitro, but at vastly different levels.RNA-seq confirmed many of the present annotations and refined the current genome annotation. Gene expression divergence was found to recapitulate the known phylogeny, and uncovered lineage-specific differences in expression. Polyadenylation sites were mapped for over 70% of the genes and revealed many examples of conserved and unexpectedly long 3' UTRs. 28 open reading frames were found in a non-transcribed gene cluster on chromosome 5 of the WB isolate. Analysis of allele-specific expression revealed a correlation between allele-dosage and allele expression in the GS isolate. Previously reported cis-splicing events were confirmed and global mapping of cis-splicing identified only one novel intron. These observations can possibly explain differences in host-preference and symptoms, and it will be the basis for further studies of Giardia pathogenesis and biology.

  • 25.
    Guadalupe Ortega-Pierres, M.
    et al.
    IPN, Ctr Invest & Estudios Avanzados, Dept Genet & Mol Biol, Mexico City, DF, Mexico..
    Jex, Aaron R.
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Parkville, Vic, Australia.;Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic, Australia..
    Ansell, Brendan R. E.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic, Australia..
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Recent advances in the genomic and molecular biology of Giardia2018In: Acta Tropica, ISSN 0001-706X, E-ISSN 1873-6254, Vol. 184, p. 67-72Article in journal (Refereed)
    Abstract [en]

    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.

  • 26.
    Hanevik, Kurt
    et al.
    Univ Bergen, Dept Clin Sci, Lab Bldg 8 Floor, N-5021 Bergen, Norway.;Haukeland Hosp, Ctr Trop Infect Dis, Bergen, Norway..
    Kristoffersen, Einar
    Univ Bergen, Dept Clin Sci, Lab Bldg 8 Floor, N-5021 Bergen, Norway.;Haukeland Hosp, Dept Immunol Transfus Med, Bergen, Norway..
    Morch, Kristine
    Univ Bergen, Dept Clin Sci, Lab Bldg 8 Floor, N-5021 Bergen, Norway.;Haukeland Hosp, Ctr Trop Infect Dis, Bergen, Norway..
    Rye, Kristin Paulsen
    Univ Bergen, Dept Clin Sci, Lab Bldg 8 Floor, N-5021 Bergen, Norway..
    Sornes, Steinar
    Univ Bergen, Dept Clin Sci, Lab Bldg 8 Floor, N-5021 Bergen, Norway..
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Bruserud, Oystein
    Univ Bergen, Dept Clin Sci, Lab Bldg 8 Floor, N-5021 Bergen, Norway..
    Langeland, Nina
    Univ Bergen, Dept Clin Sci, Lab Bldg 8 Floor, N-5021 Bergen, Norway.;Haukeland Hosp, Ctr Trop Infect Dis, Bergen, Norway..
    Giardia-specific cellular immune responses in post-giardiasis chronic fatigue syndrome2017In: BMC Immunology, ISSN 1471-2172, E-ISSN 1471-2172, Vol. 18, article id 5Article in journal (Refereed)
    Abstract [en]

    Background: The role of pathogen specific cellular immune responses against the eliciting pathogen in development of post-infectious chronic fatigue syndrome (PI-CFS) is not known and such studies are difficult to perform. The aim of this study was to evaluate specific anti-Giardia cellular immunity in cases that developed CFS after Giardia infection compared to cases that recovered well. Patients reporting chronic fatigue in a questionnaire study three years after a Giardia outbreak were clinically evaluated five years after the outbreak and grouped according to Fukuda criteria for CFS and idiopathic chronic fatigue. Giardia specific immune responses were evaluated in 39 of these patients by proliferation assay, T cell activation and cytokine release analysis. 20 Giardia exposed non-fatigued individuals and 10 healthy unexposed individuals were recruited as controls. Results: Patients were clinically classified into CFS (n = 15), idiopathic chronic fatigue (n = 5), fatigue from other causes (n = 9) and recovered from fatigue (n = 10). There were statistically significant antigen specific differences between these Giardia exposed groups and unexposed controls. However, we did not find differences between the Giardia exposed fatigue classification groups with regard to CD4 T cell activation, proliferation or cytokine levels in 6 days cultured PBMCs. Interestingly, sCD40L was increased in patients with PI-CFS and other persons with fatigue after Giardia infection compared to the non-fatigued group, and correlated well with fatigue levels at the time of sampling. Conclusion: Our data show antigen specific cellular immune responses in the groups previously exposed to Giardia and increased sCD40L in fatigued patients.

  • 27. Hanevik, Kurt
    et al.
    Kristoffersen, Einar
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Bruserud, Oystein
    Ringqvist, Emma
    Sornes, Steinar
    Langeland, Nina
    Human Cellular Immune Response Against Giardia lamblia 5 Years After Acute Giardiasis2011In: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 204, no 11, p. 1779-1786Article in journal (Refereed)
    Abstract [en]

    Background. Clinical and epidemiological studies have suggested the development of acquired immunity in individuals previously infected with Giardia lamblia. However, there are no data on the long-term cellular immunity and genotype cross-reactivity. An outbreak of assemblage B giardiasis in a nonendemic area made it possible to evaluate the long-term cellular mediated immunity and its specificity toward the 2 Giardia assemblages known to infect humans.

    Methods. Peripheral blood mononuclear cells from 19 individuals infected with Giardia assemblage B 5 years previously and from 10 uninfected controls were cultured with antigens from assemblage A and B Giardia trophozoites for 6 days. Cell-mediated immunity was measured by a (3)H-thymidine proliferation assay and flow cytometric analysis of activation markers HLA-DR, CD45RO, CD25, and CD26 in T-cell subsets.

    Results. Proliferation responses were significantly elevated in the group previously exposed to Giardia for nearly all Giardia antigens tested. Individual responses toward Giardia trophozoite whole cell, cytosolic, and excretory-secretory antigens from both assemblages correlated well. Activation marker responses were mainly seen in CD4 T cells.

    Conclusions. G. lamblia infection induces long-term, albeit variable, cellular immune responses that are not assemblage specific and that are largely driven by CD4 T-cell activation.

  • 28. Hertz, H. M.
    et al.
    von Hofsten, O.
    Bertilson, M.
    Vogt, U.
    Holmberg, A.
    Reinspach, J.
    Martz, D.
    Selin, M.
    Christakou, A. E.
    Jerlstrom-Hultqvist, Joel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Laboratory cryo soft X-ray microscopy2012In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 177, no 2, p. 267-272Article in journal (Refereed)
    Abstract [en]

    Lens-based water-window X-ray microscopy allows two- and three-dimensional (2D and 3D) imaging of intact unstained cells in their near-native state with unprecedented contrast and resolution. Cryofixation is essential to avoid radiation damage to the sample. Present cryo X-ray microscopes rely on synchrotron radiation sources, thereby limiting the accessibility for a wider community of biologists. In the present paper we demonstrate water-window cryo X-ray microscopy with a laboratory-source-based arrangement. The microscope relies on a lambda = 2.48-nm liquid-jet high-brightness laser-plasma source, normal-incidence multilayer condenser optics, 30-nm zone-plate optics, and a cryo sample chamber. We demonstrate 2D imaging of test patterns, and intact unstained yeast, protozoan parasites and mammalian cells. Overview 3D information is obtained by stereo imaging while complete 3D microscopy is provided by full tomographic reconstruction. The laboratory microscope image quality approaches that of the synchrotron microscopes, but with longer exposure times. The experimental image quality is analyzed from a numerical wave-propagation model of the imaging system and a path to reach synchrotron-like exposure times in laboratory microscopy is outlined.

  • 29. Jenikova, Gabriela
    et al.
    Hruz, Petr
    Andersson, Mattias K.
    Tejman-Yarden, Noa
    Ferreira, Patricia C. D.
    Andersen, Yolanda S.
    Davids, Barbara J.
    Gillin, Frances D.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Curtiss, Roy, III
    Eckmann, Lars
    alpha 1-giardin based live heterologous vaccine protects against Giardia lamblia infection in a murine model2011In: Vaccine, ISSN 0264-410X, E-ISSN 1873-2518, Vol. 29, no 51, p. 9529-9537Article in journal (Refereed)
    Abstract [en]

    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.

  • 30.
    Jerlström-Hultqvist, Jon
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Einarsson, Elin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Stable transfection of the diplomonad parasite Spironucleus salmonicida2012In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 11, no 11, p. 1353-1361Article in journal (Refereed)
    Abstract [en]

    Eukaryotic microbes are highly diverse and many lineages remain poorly studied. One such lineage, the diplomonads, a group of binucleate heterotrophic flagellates, has mainly been studied due to the impact of Giardia intestinalis, an intestinal, diarrhea-causing parasite in humans and animals. Here we describe the development of a stable transfection system for use in Spironucleus salmonicida, a diplomonad casuing systemic spironucleosis in salmonid fish. We designed vectors in cassette format carrying epitope tags for localization (3xHA, 2xOLLAS, 3xMYC) and purification of proteins (2xStrepII-FLAG or SBP-GST) under the control of native or constitutive promoters. Three selectable markers, puromycin acetyltransferase (pac), blasticidin S-deaminase (bsr) or neomycin phosphotransferase (nptII) were successfully applied for generation of stable transfectants. Site-specific integration on the S. salmonicida chromosome was shown to be possible using the bsr resistance gene. We epitope-tagged six proteins and confirmed their expression by Western Blot. Next, we demonstrated the utility of these vectors by recording the sub-cellular localizations of the six proteins by laser scanning confocal microscopy. Finally, we describe the creation of a S. salmonicida double transfectant suitable for co-localization studies. The transfection system described herein and the imminent completion of the S. salmonicida genome will make it possible to use comparative genomics as an investigative tool to explore specific as well as general diplomonad traits, benefiting research on both Giardia and Spironucleus.

  • 31.
    Jerlström-Hultqvist, Jon
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Einarsson, Elin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Xu, Feifei
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Hjort, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ek, Bo
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Steinhauf, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hultenby, Kjell
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Andersson, Jan O.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hydrogenosomes in the diplomonad Spironucleus salmonicida2013In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 4, p. 2493-Article in journal (Refereed)
    Abstract [en]

    Acquisition of the mitochondrion is a key event in the evolution of the eukaryotic cell, but diversification of the organelle has occurred during eukaryotic evolution. One example of such mitochondria-related organelles (MROs) are hydrogenosomes, which produce ATP by substrate- level phosphorylation with hydrogen as a byproduct. The diplomonad parasite Giardia intestinalis harbours mitosomes, another type of MRO. Here we identify MROs in the salmon parasite Spironucleus salmonicida with similar protein import and Fe-S cluster assembly machineries as in Giardia mitosomes. We find that hydrogen production is prevalent in the diplomonad genus Spironucleus, and that S. salmonicida MROs contain enzymes characteristic of hydrogenosomes. Evolutionary analyses of known hydrogenosomal components indicate their presence in the diplomonad ancestor, and subsequent loss in Giardia. Our results suggest that hydrogenosomes are metabolic adaptations predating the split between parabasalids and diplomonads, which is deeper than the split between animals and fungi in the eukaryotic tree.

  • 32.
    Jerlström-Hultqvist, Jon
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Stadelmann, Britta
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Birkestedt, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome2012In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 11, no 7, p. 864-873Article in journal (Refereed)
    Abstract [en]

    In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide-glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG-tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes.

  • 33. Lauwaet, Tineke
    et al.
    Davids, Barbara J.
    Torres-Escobar, Ascención
    Birkeland, Shanda R.
    Cipriano, Michael J.
    Preheim, Sarah P.
    Palm, Daniel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    McArthur, Andrew G.
    Gillin, Frances D.
    Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation2007In: Molecular and biochemical parasitology (Print), ISSN 0166-6851, E-ISSN 1872-9428, Vol. 152, no 1, p. 80-89Article in journal (Refereed)
    Abstract [en]

    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.

  • 34.
    Li, Zhiqiang
    et al.
    Swedish Univ Agr Sci, Dept Biomed Sci & Vet Publ Hlth, SE-75007 Uppsala, Sweden.
    Peirasmaki, Dimitra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Åbrink, Magnus
    Swedish Univ Agr Sci, Dept Biomed Sci & Vet Publ Hlth, SE-75007 Uppsala, Sweden.
    Giardia excretory-secretory proteins modulate the enzymatic activities of mast cell chymase and tryptase2019In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 114, p. 535-544Article in journal (Refereed)
    Abstract [en]

    Background

    Mast cells are involved in the host immune response controlling infection with the non-invasive intestinal protozoan parasite Giardia intestinalis. Experimental infections in rodents with G. intestinalis showed increased intestinal expression of mucosal and connective mast cell specific proteases suggesting that both mucosal and connective tissue mast cells are recruited and activated during infection. During infection Giardia excretory-secretory proteins (ESPs) with immunomodulatory capacity are released. However, studies investigating potential interactions between Giardia ESPs and the connective tissue mast cell specific serine proteases, i.e. human chymase and mouse mast cell protease (mMCP)-4 and, human and mouse tryptase (mMCP-6) remain scarce.

    Results

    We first investigated if soluble Giardia proteins (sGPs), which over-lap extensively in protein content with ESP fractions, from the isolates GS, WB and H3, could induce mast cell activation. sGPs induced a minor activation of bone marrow derived mucosal-like mast cells, as indicated by increased IL-6 secretion and no degranulation. Furthermore, sGPs were highly resistant to degradation by human tryptase while human chymase degraded a 65 kDa sGP and, wild-type mouse ear tissue extracts degraded several protein bands in the 10 to 75 kDa range. In striking contrast, sGPs and ESPs were found to increase the enzymatic activity of human and mouse tryptase and to reduce the activity of human and mouse chymase.

    Conclusion

    Our finding suggests that Giardia ssp. via enhancement or reduction of mast cell protease activity may modulate mast cell-driven intestinal immune responses. ESP-mediated modulation of the mast cell specific proteases may also increase degradation of tight junctions, which may be beneficial for Giardia ssp. during infection.

  • 35.
    Liu, Jingyi
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Fu, Zhirong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Cleavage specificity of recombinant Giardia intestinalis cysteine proteases: Degradation of immunoglobulins and defensins2019In: Molecular and biochemical parasitology (Print), ISSN 0166-6851, E-ISSN 1872-9428, Vol. 227, p. 29-38Article in journal (Refereed)
    Abstract [en]

    Giardia intestinalis is a protozoan parasite and the causative agent of giardiasis, a common diarrheal disease. Cysteine protease (CP) activities have been suggested to be involved in Giardia's pathogenesis and we have recently identified and characterized three secreted Giardia CPs; CP14019, CP16160 and CP16779. Here we have studied the cleavage specificity of these CPs using substrate phage display and recombinant protein substrates. The phage display analyses showed that CP16160 has both chymase and tryptase activity and a broad substrate specificity. This was verified using recombinant protein substrates containing different variants of the cleavage sites. Phage display analyses of CP14019 and CP16779 failed but the substrate specificity of CP14019 and CP16779 was tested using the recombinant substrates generated for CP16160. CP16160 and CP14019 showed similar substrate specificity, while CP16779 has a slightly different substrate specificity. The consensus sequence for cleavage by CP16160, obtained from phage display analyses, was used in an in silico screen of the human intestinal proteome for detection of potential targets. Immunoglobulins, including IgA and IgG and defensins (α-HD6 and β-HD1) were predicted to be targets and they were shown to be cleaved by the recombinant CPs in vitro. Our results suggest that the secreted Giardia CPs are key players in the interaction with host cells during Giardia infections since they can cleave several components of the human mucosal defense machinery.

  • 36.
    Liu, Jingyi
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Ma'ayeh, Showgy Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Peirasmaki, Dimitra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lundstrom-Stadelmann, Britta
    Univ Bern, Vetsuisse Fac, Inst Parasitol, Bern, Switzerland.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Secreted Giardia intestinalis cysteine proteases disrupt intestinal epithelial cell junctional complexes and degrade chemokines2018In: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 9, no 1, p. 879-894Article in journal (Refereed)
    Abstract [en]

    Giardiasis is a common diarrheal disease caused by the protozoan parasite Giardia intestinalis. Cysteine proteases (CPs) are acknowledged as virulence factors in Giardia but their specific role in the molecular pathogenesis of disease is not known. Herein, we aimed to characterize the three main secreted CPs (CP14019, CP16160 and CP16779), which were identified by mass spectrometry in the medium during interaction with intestinal epithelial cells (IECs) in vitro. First, the CPs were epitope-tagged and localized to the endoplasmic reticulum and cytoplasmic vesicle-like structures. Second, we showed that recombinant CPs, expressed in Pichia pastoris, are more active in acidic environment (pH 5.5-6) and we determined the kinetic parameters using fluorogenic substrates. Third, excretory-secretory proteins (ESPs) from Giardia trophozoites affect the localization of apical junctional complex (AJC) proteins and recombinant CPs cleave or re-localize the AJC proteins (claudin-1 and -4, occludin, JAM-1, beta-catenin and E-cadherin) of IECs. Finally, we showed that the ESPs and recombinant CPs can degrade several chemokines, including CXCL1, CXCL2, CXCL3, IL-8, CCL2, and CCL20, which are up-regulated in IECs during Giardia-host cell interactions. This is the first study that characterizes the role of specific CPs secreted from Giardia and our results collectively indicate their roles in the disruption of the intestinal epithelial barrier and modulating immune responses during Giardia infections.

  • 37.
    Liu, Jingyi
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Klotz, Christian
    Robert Koch Inst, Dept Mycot & Parasit Agents & Mycobacteria FG16, Seestr 10, D-13353 Berlin, Germany.
    Giardia intestinalis cystatin is a potent inhibitor of papain, parasite cysteine proteases and, to a lesser extent, human cathepsin B2019In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 593, no 12, p. 1313-1325Article in journal (Refereed)
    Abstract [en]

    Cystatins are important regulators of papain-like cysteine proteases. In the protozoan parasite Giardia intestinalis, papain-like cysteine proteases play an essential role in the parasite's biology and pathogenicity. Here, we characterized a cysteine protease inhibitor of G. intestinalis that belongs to type-I-cystatins. The parasite cystatin is shown to be a strong inhibitor of papain (K-i approximate to 0.3 nm) and three parasite cysteine proteases (CP14019, CP16160 and CP16779, K-i approximate to 0.9-5.8 nm), but a weaker inhibitor of human cathepsin B (K-i approximate to 79.9 nm). The protein localizes mainly in the cytoplasm. Together, these data suggest that cystatin of G. intestinalis plays a role in the regulation of cysteine protease activities in the parasite and, possibly, in the interaction with the host.

  • 38.
    Ma'ayeh, Showgy Y.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Knörr, Livia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Sköld, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Garnham, Alexandra
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Parkville, Vic, Australia.
    Ansell, Brendan R. E.
    Univ Melbourne, Fac Vet Sci, Parkville, Vic, Australia;Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Parkville, Vic, Australia.
    Jex, Aaron R.
    Univ Melbourne, Fac Vet Sci, Parkville, Vic, Australia;Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Parkville, Vic, Australia.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Responses of the Differentiated Intestinal Epithelial Cell Line Caco-2 to Infection With the Giardia intestinalis GS Isolate2018In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 8, article id 244Article in journal (Refereed)
    Abstract [en]

    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.

  • 39.
    Ma'ayeh, Showgy Y.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Knörr, Livia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Transcriptional profiling of Giardia intestinalis in response to oxidative stress2015In: International Journal of Parasitology, ISSN 0020-7519, E-ISSN 1879-0135, Vol. 45, no 14, p. 925-938Article in journal (Refereed)
    Abstract [en]

    Giardia intestinalis is a microaerophilic parasite that infects the human upper small intestine, an environment that is fairly aerobic with reactive oxygen species being produced to fight off the parasite. It is quite perplexing how Giardia, lacking conventional eukaryotic antioxidant machinery (e.g. catalase, superoxide dismutase and glutathione peroxidase), can cope with the oxidative stress in this environment. We used transcriptomics (RNA sequencing and quantitative PCR) to study giardial gene expression changes in response to oxygen (O-2; 1 h) and hydrogen peroxide (H2O2; 150 mu M, 500 mu M and 1 mM for 1 h). The results showed phenotypic and transcriptional differences between Giardia isolates of different genotypes (WB, assemblage A and GS, assemblage B), with GS being more tolerant to H2O2 and exhibiting higher basic transcript levels of antioxidant genes (e.g. NADH oxidase lateral transfer candidate, peroxiredoxin 1 (Prxl) and thioredoxin (Trx)-like proteins). Cysteine is a major antioxidant in Giardia and its role in oxidative defense could be highlighted here by the up-regulation of gene transcripts encoding the cysteine-rich variable surface proteins (VSPs) and high cysteine membrane proteins (HCMPs). Genes in the thioredoxin system (Prxl, Trx and Trx reductase) occupied a central role in the gene expression response to oxidative stress, together with genes encoding metabolic (NADPH-producing enzymes, glutathione and glycerol biosynthetic enzymes) and O-2-consuming nitric oxide detoxification enzymes (e.g. nitroreductase, flavohemoprotein and a flavodiiron protein). This study reveals the intricate network of genes associated with the oxidative stress response in Giardia, and provides a stepping-stone towards future studies at the protein level.

  • 40.
    Ma'ayeh, Showgy Y.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Liu, Jingyi
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Peirasmaki, Dimitra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hörnaeus, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Bergström Lind, Sara K.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Grabherr, Manfred
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Characterization of the Giardia intestinalis secretome during interaction with human intestinal epithelial cells: The impact on host cells2017In: PLoS Neglected Tropical Diseases, ISSN 1935-2727, E-ISSN 1935-2735, Vol. 11, no 12, article id e0006120Article in journal (Refereed)
    Abstract [en]

    BACKGROUND:

    Giardia intestinalis is a non-invasive protozoan parasite that causes giardiasis in humans, the most common form of parasite-induced diarrhea. Disease mechanisms are not completely defined and very few virulence factors are known.

    METHODOLOGY:

    To identify putative virulence factors and elucidate mechanistic pathways leading to disease, we have used proteomics to identify the major excretory-secretory products (ESPs) when Giardia trophozoites of WB and GS isolates (assemblages A and B, respectively) interact with intestinal epithelial cells (IECs) in vitro.

    FINDINGS:

    The main parts of the IEC and parasite secretomes are constitutively released proteins, the majority of which are associated with metabolism but several proteins are released in response to their interaction (87 and 41 WB and GS proteins, respectively, 76 and 45 human proteins in response to the respective isolates). In parasitized IECs, the secretome profile indicated effects on the cell actin cytoskeleton and the induction of immune responses whereas that of Giardia showed anti-oxidation, proteolysis (protease-associated) and induction of encystation responses. The Giardia secretome also contained immunodominant and glycosylated proteins as well as new candidate virulence factors and assemblage-specific differences were identified. A minor part of Giardia ESPs had signal peptides (29% for both isolates) and extracellular vesicles were detected in the ESPs fractions, suggesting alternative secretory pathways. Microscopic analyses showed ESPs binding to IECs and partial internalization. Parasite ESPs reduced ERK1/2 and P38 phosphorylation and NF-κB nuclear translocation. Giardia ESPs altered gene expression in IECs, with a transcriptional profile indicating recruitment of immune cells via chemokines, disturbances in glucose homeostasis, cholesterol and lipid metabolism, cell cycle and induction of apoptosis.

    CONCLUSIONS:

    This is the first study identifying Giardia ESPs and evaluating their effects on IECs. It highlights the importance of host and parasite ESPs during interactions and reveals the intricate cellular responses that can explain disease mechanisms and attenuated inflammatory responses during giardiasis.

  • 41. Manning, Gerard
    et al.
    Reiner, David S.
    Lauwaet, Tineke
    Dacre, Michael
    Smith, Alias
    Zhai, Yufeng
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Gillin, Frances D.
    The minimal kinome of Giardia lamblia illuminates early kinase evolution and unique parasite biology2011In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 12, no 7, p. R66-Article in journal (Refereed)
    Abstract [en]

    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.

  • 42. Palm, Daniel
    et al.
    Weiland, Malin
    McArthur, Andrew G
    Winiecka-Krusnell, Jadwiga
    Cipriano, Michael J
    Birkeland, Shanda R
    Pacocha, Sarah E
    Davids, Barbara
    Gillin, Frances
    Linder, Ewert
    Svärd, Staffan
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Mikrobiologi.
    Developmental changes in the adhesive disk during Giardia differentiation.2005In: Mol Biochem Parasitol, ISSN 0166-6851, Vol. 141, no 2, p. 199-207Article in journal (Refereed)
  • 43.
    Peirasmaki, Dimitra
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Ma'ayeh, Showgy Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Xu, Feifei
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Ferella, Marcela
    Eukaryotic Single Cell Genomics Platform, Karolinska Institute, SciLifeLab, Sweden.
    Campos, Sara
    Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Liu, Jingyi
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    High Cysteine Proteins are up-regulated during Giardia-host cell interaction.Manuscript (preprint) (Other academic)
  • 44.
    Peirasmaki, Dimitra
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Xia, Dong
    The Royal Veterinary College, London, United Kingdom.
    Attree, Elizabeth
    The Royal Veterinary College, London, United Kingdom.
    Ferella, Marcela
    Eukaryotic Single Cell Genomics Platform, Karolinska Institute, SciLifeLab, Sweden.
    Ma'ayeh, Showgy Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wastling, Jonathan
    Faculty of Natural Sciences, University of Keele, Newcastle-under-Lyme, United Kingdom.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Proteome analyses of Giardia–host cell interactions in vitro.Manuscript (preprint) (Other academic)
  • 45. Roxström-Lindquist, Katarina
    et al.
    Ringqvist, Emma
    Palm, Daniel
    Svärd, Staffan
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Mikrobiologi.
    Giardia lamblia-induced changes in gene expression in differentiated Caco-22005In: Infect Immun, ISSN 0019-9567, Vol. 73, no 12, p. 8204-8Article in journal (Other scientific)
  • 46.
    Saghaug, Christina Skar
    et al.
    Haukeland Hosp, Dept Med, Natl Ctr Trop Infect Dis, N-5021 Bergen, Norway.;Univ Bergen, Dept Clin Sci, Bergen, Norway..
    Sornes, Steinar
    Univ Bergen, Dept Clin Sci, Bergen, Norway..
    Peirasmaki, Dimitra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Langeland, Nina
    Haukeland Hosp, Dept Med, Natl Ctr Trop Infect Dis, N-5021 Bergen, Norway.;Univ Bergen, Dept Clin Sci, Bergen, Norway..
    Hanevik, Kurt
    Haukeland Hosp, Dept Med, Natl Ctr Trop Infect Dis, N-5021 Bergen, Norway.;Univ Bergen, Dept Clin Sci, Bergen, Norway..
    Human Memory CD4+ T Cell Immune Responses against Giardia lamblia2016In: Clinical and Vaccine Immunology, ISSN 1556-6811, E-ISSN 1556-679X, Vol. 23, no 1, p. 11-18Article in journal (Refereed)
    Abstract [en]

    The intestinal protozoan parasite Giardia lamblia may cause severe prolonged diarrheal disease or pass unnoticed as an asymptomatic infection. T cells seem to play an important role in the immune response to Giardia infection, and memory responses may last years. Recently, T(H)17 responses have been found in three animal studies of Giardia infection. The aim of this study was to characterize the human CD4+ T cell responses to Giardia. Peripheral blood mononuclear cells (PBMCs) were obtained from 21 returning travelers with recent or ongoing giardiasis and 12 low-risk healthy controls and stimulated in vitro with Giardia lamblia proteins. Production of tumor necrosis factor alpha (TNF-alpha), gamma interferon, interleukin-17A (IL-17A), IL-10, and IL-4 was measured in CD4+ effector memory (EM) T cells after 24 h by flow cytometry. After 6 days of culture, activation and proliferation were measured by flow cytometry, while an array of inflammatory cytokine levels in supernatants were measured with multiplex assays. We found the number of IL-17A-producing CD4+ EM T cells, as well as that of cells simultaneously producing both IL-17A and TNF-alpha, to be significantly elevated in the Giardia-exposed individuals after 24 h of antigen stimulation. In supernatants of PBMCs stimulated with Giardia antigens for 6 days, we found inflammation-associated cytokines, including 1L-17A, as well as CD4+ T cell activation and proliferation, to be significantly elevated in the Giardia-exposed individuals. We conclude that symptomatic Giardia infection in humans induces a CD4+ EM T cell response of which IL-17A production seems to be an important component.

  • 47.
    Stadelmann, Britta
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hanevik, Kurt
    Andersson, Mattias K.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Bruserud, Oystein
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    The role of arginine and arginine-metabolizing enzymes during Giardia - host cell interactions in vitro2013In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 13, p. 256-Article in journal (Refereed)
    Abstract [en]

    Background: Arginine is a conditionally essential amino acid important in growing individuals and under non-homeostatic conditions/disease. Many pathogens interfere with arginine-utilization in host cells, especially nitric oxide (NO) production, by changing the expression of host enzymes involved in arginine metabolism. Here we used human intestinal epithelial cells (IEC) and three different isolates of the protozoan parasite Giardia intestinalis to investigate the role of arginine and arginine-metabolizing enzymes during intestinal protozoan infections. Results: RNA expression analyses of major arginine-metabolizing enzymes revealed the arginine-utilizing pathways in human IECs (differentiated Caco-2 cells) grown in vitro. Most genes were constant or down-regulated (e.g. arginase 1 and 2) upon interaction with Giardia, whereas inducible NO synthase (iNOS) and ornithine decarboxylase (ODC) were up-regulated within 6 h of infection. Giardia was shown to suppress cytokine-induced iNOS expression, thus the parasite has both iNOS inducing and suppressive activities. Giardial arginine consumption suppresses NO production and the NO-degrading parasite protein flavohemoglobin is up-regulated in response to host NO. In addition, the secreted, arginine-consuming giardial enzyme arginine deiminase (GiADI) actively reduces T-cell proliferation in vitro. Interestingly, the effects on NO production and T cell proliferation could be reversed by addition of external arginine or citrulline. Conclusions: Giardia affects the host's arginine metabolism on many different levels. Many of the effects can be reversed by addition of arginine or citrulline, which could be a beneficial supplement in oral rehydration therapy.

  • 48.
    Stadelmann, Britta
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Merino, Maria C.
    Persson, Lo
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Arginine Consumption by the Intestinal Parasite Giardia intestinalis Reduces Proliferation of Intestinal Epithelial Cells2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 9, p. e45325-Article in journal (Refereed)
    Abstract [en]

    In the field of infectious diseases the multifaceted amino acid arginine has reached special attention as substrate for the hosts production of the antimicrobial agent nitric oxide (NO). A variety of infectious organisms interfere with this part of the host immune response by reducing the availability of arginine. This prompted us to further investigate additional roles of arginine during pathogen infections. As a model we used the intestinal parasite Giardia intestinalis that actively consumes arginine as main energy source and secretes an arginine-consuming enzyme, arginine deiminase (ADI). Reduced intestinal epithelial cell (IEC) proliferation is a common theme during bacterial and viral intestinal infections, but it has never been connected to arginine-consumption. Our specific question was thereby, whether the arginine-consumption by Giardia leads to reduced IEC proliferation, in addition to NO reduction. In vitro cultivation of human IEC lines in arginine-free or arginine/citrulline-complemented medium, as well as in interaction with different G. intestinalis isolates, were used to study effects on host cell replication by MTT assay. IEC proliferation was further analyzed by DNA content analysis, polyamine measurements and expressional analysis of cell cycle regulatory genes. IEC proliferation was reduced upon arginine-withdrawal and also in an arginine-dependent manner upon interaction with G. intestinalis or addition of Giardia ADI. We show that arginine-withdrawal by intestinal pathogens leads to a halt in the cell cycle in IECs through reduced polyamine levels and upregulated cell cycle inhibitory genes. This is of importance with regards to intestinal tissue homeostasis that is affected through reduced cell proliferation. Thus, the slower epithelial cell turnover helps the pathogen to maintain a more stable niche for colonization. This study also shows why supplementation therapy of diarrhea patients with arginine/citrulline is helpful and that citrulline especially should gain further attention in future treatment strategies.

  • 49.
    Stairs, Courtney W.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Kokla, Anna
    Astvaldsson, Asgeir
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Jerlström-Hultqvist, Jon
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Ettema, Thijs J. G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Oxygen induces the expression of invasion and stress response genes in the anaerobic salmon parasite Spironucleus salmonicida2019In: BMC Biology, ISSN 1741-7007, E-ISSN 1741-7007, Vol. 17, no 1, article id 19Article in journal (Refereed)
    Abstract [en]

    Background: Spironucleus salmonicida is an anaerobic parasite that can cause systemic infections in Atlantic salmon. Unlike other diplomonad parasites, such as the human pathogen Giardia intestinalis, Spironucleus species can infiltrate the blood stream of their hosts eventually colonizing organs, skin and gills. How this presumed anaerobe can persist and invade oxygenated tissues, despite having a strictly anaerobic metabolism, remains elusive.

    Results: To investigate how S. salmonicida response to oxygen stress, we performed RNAseq transcriptomic analyses of cells grown in the presence of oxygen or antioxidant-free medium. We found that over 20% of the transcriptome is differentially regulated in oxygen (1705 genes) and antioxidant-depleted (2280 genes) conditions. These differentially regulated transcripts encode proteins related to anaerobic metabolism, cysteine and Fe-S cluster biosynthesis, as well as a large number of proteins of unknown function. S. salmonicida does not encode genes involved in the classical elements of oxygen metabolism (e.g., catalases, superoxide dismutase, glutathione biosynthesis, oxidative phosphorylation). Instead, we found that genes encoding bacterial-like oxidoreductases were upregulated in response to oxygen stress. Phylogenetic analysis revealed some of these oxygen-responsive genes (e.g., nadh oxidase, rubrerythrin, superoxide reductase) are rare in eukaryotes and likely derived from lateral gene transfer (LGT) events into diplomonads from prokaryotes. Unexpectedly, we observed that many host evasion- and invasion-related genes were also upregulated under oxidative stress suggesting that oxygen might be an important signal for pathogenesis.

    Conclusion: While oxygen is toxic for related organisms, such as G. intestinalis, we find that oxygen is likely a gene induction signal for host invasion- and evasion-related pathways in S. salmonicida. These data provide the first molecular evidence for how S. salmonicida could tolerate oxic host environments and demonstrate how LGT can have a profound impact on the biology of anaerobic parasites.

  • 50. Tellez, A
    et al.
    Palm, D
    Weiland, M
    Alemán, J
    Winiecka-Krusnell, J
    Linder, E
    Svärd, S
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Mikrobiologi.
    Secretory antibodies against Giardia intestinalis in lactating Nicaraguan women.2005In: Parasite Immunol, ISSN 0141-9838, Vol. 27, no 5, p. 163-9Article in journal (Other scientific)
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