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Comparative cell biology and evolution of Annexins in Diplomonads
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. (Staffan Svärd)
Karolinska Inst, Dept Lab Med, Stockholm, Sweden.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.ORCID iD: 0000-0003-1154-8218
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2016 (English)In: MSphere, ISSN 2379-5042, Vol. 1, no 2, e00032-15Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Uppsala, 2016. Vol. 1, no 2, e00032-15
Keyword [en]
intestinal parasite, annexins, diplomonad, Spironucleus salmonicida, Giardia, proximity labeling, APEX
National Category
Cell Biology Microbiology Evolutionary Biology
Identifiers
URN: urn:nbn:se:uu:diva-264537DOI: 10.1128/mSphere.00032-15ISI: 000392584700008OAI: oai:DiVA.org:uu-264537DiVA: diva2:860873
Funder
Swedish Research Council, 2012-3364Swedish Research Council Formas, 2013-910
Available from: 2015-10-14 Created: 2015-10-14 Last updated: 2017-03-06Bibliographically approved
In thesis
1. Comparative Cell Biology in Diplomonads
Open this publication in new window or tab >>Comparative Cell Biology in Diplomonads
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1303
Keyword
Giardia intestinalis, Spironucleus salmonicida, intestinal parasite, hydrogenosome, encystation, gene regulation, transfection, diplomonad, antigenic variation, annexin
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-264541 (URN)978-91-554-9374-5 (ISBN)
Public defence
2015-12-04, A1:111a, BMC, Husargatan 3, Uppsala, 09:15 (English)
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Supervisors
Available from: 2015-11-12 Created: 2015-10-14 Last updated: 2015-11-13

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Einarsson, ElinÁstvaldsson, ÁsgeirAndersson, Jan O.Svärd, Staffan G.Jerlstrom-Hultqvist, Jon

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