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Comparative Cell Biology in Diplomonads
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The diplomonads are a diverse group of eukaryotic flagellates found in microaerophilic and anaerobic environments. The most studied diplomonad is the intestinal parasite Giardia intestinalis, which infects a variety of mammals and cause diarrheal disease. Less is known about Spironucleus salmonicida, a parasite of salmonid fish, known to cause systemic infections with high mortality.

We created a transfection system for S. salmonicida to study cellular functions and virulence in detail (Paper I). The system was applied to explore the mitochondrion-related organelle (MRO) in S. salmonicida. We showed that S. salmonicida possesses a hydrogenosome (Paper II) with a higher metabolic capacity than the corresponding MRO of Giardia, the mitosome. Evolutionary analysis of key hydrogenosomal proteins showed ancient origin, indicating their presence in the ancestral diplomonad and subsequent loss in Giardia. Annexins are of evolutionary interest since these proteins are found across all kingdoms. Annexin-like proteins are intriguingly expanded into multigene families in Giardia and Spironucleus. The annexins of S. salmonicida were characterized (Paper III) with distinct localizations to various cellular structures, including a putative adhesion structure anterior in the cell.

The disease-causing Giardia trophozoites differentiate into infectious cysts, a process essential for transmission and virulence of the parasite. Cysts are often spread via contaminated water and exposed to environmental stressors, such as UV irradiation. We studied the survival and transcriptional response to this stress factor (Paper IV) and results showed the importance of active DNA replication machinery for parasite survival after DNA damage. In addition, we studied transcriptional changes along the trajectory of encystation (Paper V), which revealed a coordinated cascade of gene regulation. This was observed for the entire transcriptome as well as putative regulators. Large transcriptional changes appeared late in the process with the majority of differentially regulated genes encoding hypothetical proteins. We studied the localizations of several of these to gain information of their possible function.

To conclude, the diplomonads are complex eukaryotic microbes with cellular processes adjusted to match their life styles. The work in this thesis has provided insight of their adaptations, differences and similarities, but also new interesting leads for future studies of diplomonad biology and virulence. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. , 84 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1303
Keyword [en]
Giardia intestinalis, Spironucleus salmonicida, intestinal parasite, hydrogenosome, encystation, gene regulation, transfection, diplomonad, antigenic variation, annexin
National Category
URN: urn:nbn:se:uu:diva-264541ISBN: 978-91-554-9374-5OAI: diva2:861070
Public defence
2015-12-04, A1:111a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Available from: 2015-11-12 Created: 2015-10-14 Last updated: 2015-11-13
List of papers
1. Stable transfection of the diplomonad parasite Spironucleus salmonicida
Open this publication in new window or tab >>Stable transfection of the diplomonad parasite Spironucleus salmonicida
2012 (English)In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 11, no 11, 1353-1361 p.Article in journal (Refereed) Published
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.

National Category
Microbiology Cell Biology
Research subject
Biology with specialization in Microbiology; Biology with specialization in Molecular Cell Biology
urn:nbn:se:uu:diva-182827 (URN)10.1128/EC.00179-12 (DOI)000310566400006 ()22983987 (PubMedID)
Available from: 2012-10-16 Created: 2012-10-16 Last updated: 2015-11-13Bibliographically approved
2. Hydrogenosomes in the diplomonad Spironucleus salmonicida
Open this publication in new window or tab >>Hydrogenosomes in the diplomonad Spironucleus salmonicida
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2013 (English)In: Nature Communications, ISSN 2041-1723, Vol. 4, 2493- p.Article in journal (Refereed) Published
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.

National Category
Natural Sciences
urn:nbn:se:uu:diva-210741 (URN)10.1038/ncomms3493 (DOI)000325534300002 ()
Available from: 2013-11-14 Created: 2013-11-14 Last updated: 2015-11-13Bibliographically approved
3. Comparative cell biology and evolution of Annexins in Diplomonads
Open this publication in new window or tab >>Comparative cell biology and evolution of Annexins in Diplomonads
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(English)Article in journal (Refereed) Submitted
Place, publisher, year, edition, pages
National Category
Cell Biology Microbiology Evolutionary Biology
urn:nbn:se:uu:diva-264537 (URN)
Available from: 2015-10-14 Created: 2015-10-14 Last updated: 2015-11-13
4. UV irradiation responses in Giardia intestinalis
Open this publication in new window or tab >>UV irradiation responses in Giardia intestinalis
2015 (English)In: Experimental parasitology, ISSN 0014-4894, E-ISSN 1090-2449, Vol. 154, 25-32 p.Article in journal (Refereed) Published
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.

UV stress, Giardia, Trophozoites, Cysts, Inactivation, DNA replication
National Category
Microbiology in the medical area
urn:nbn:se:uu:diva-256984 (URN)10.1016/j.exppara.2015.03.024 (DOI)000355640500005 ()25825252 (PubMedID)
Available from: 2015-06-30 Created: 2015-06-29 Last updated: 2015-11-13Bibliographically approved
5. Coordinated Changes in Gene Expression Throughout Encystation of Giardia intestinalis
Open this publication in new window or tab >>Coordinated Changes in Gene Expression Throughout Encystation of Giardia intestinalis
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2016 (English)In: PLoS Neglected Tropical Diseases, ISSN 1935-2727, E-ISSN 1935-2735, Vol. 11, no 3, e0004571Article in journal (Refereed) Published
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.

National Category
Cell Biology Microbiology
urn:nbn:se:uu:diva-264538 (URN)10.1371/journal.pntd.0004571 (DOI)000373272500076 ()
Swedish Research Council, 2012-3364
Available from: 2015-10-14 Created: 2015-10-14 Last updated: 2016-06-20Bibliographically approved

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