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Proximity Staining using Enzymatic Protein Tagging in Diplomonads
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-7392-1746
2019 (English)In: mSphere, E-ISSN 2379-5042, Vol. 4, no 2, article id e00153-19Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2019. Vol. 4, no 2, article id e00153-19
National Category
Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-379669DOI: 10.1128/mSphereDirect.00153-19OAI: oai:DiVA.org:uu-379669DiVA, id: diva2:1297268
Funder
Swedish Research Council Formas, 2016-00539Available from: 2019-03-19 Created: 2019-03-19 Last updated: 2020-02-20Bibliographically approved
In thesis
1. Pathogenesis and Cell Biology of the Salmon Parasite Spironucleus salmonicida
Open this publication in new window or tab >>Pathogenesis and Cell Biology of the Salmon Parasite Spironucleus salmonicida
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Spironucleus species are classified as diplomonad organisms, diverse eukaryotic flagellates found in oxygen-deprived environments. Members of Spironucleus are parasitic and can infect a variety of hosts, such as mice and birds, while the majority are found to infect fish. Massive outbreaks of severe systemic infection caused by a Spironucleus member, Spironucleus salmonicida (salmonicida = salmon killer), have been reported in farmed salmonids resulting in large economic impacts for aquaculture.

In this thesis, the S. salmonicida genome was sequenced and compared to the genome of its diplomonad relative, the mammalian pathogen G. intestinalis (Paper I). Our analyses revealed large genomic differences between the two parasites that collectively suggests that S. salmonicida is more capable of adapting to different environments. As S. salmonicida can infiltrate different host tissues, we provide molecular evidence for how the parasite can tolerate oxygenated environments and suggest oxygen as a potential regulator of virulence factors (Paper III). To further investigate the molecular responses of the parasite and in addition, its host, during infection we set up an interaction system of S. salmonicida and ASK (Atlantic salmon kidney) cells (Paper VI).

To study the cell biology in S. salmonicida we optimized an enzymatic proximity labeling method using ascorbate peroxidase (APEX) as a reporter for transmission electron microscopy (TEM) (Paper IV). As the system is robust and versatile, we showed the localization and performed ultrastructural characterization of numerous proteins in S. salmonicida and G. intestinalis. We furthermore utilized the APEX system to study the annexin protein family in S. salmonicida (Paper II). Super resolution microscopy and TEM were applied to show that the annexins are mostly associated with cytoskeletal and membranous structures. In addition, we performed phylogenetic analyses concluding that the annexin gene family is expanded in diplomonads.

We performed experimental infection in Atlantic salmon and derived a potential model for the route of infection (Paper V). The results suggested multiple routes of transmission between hosts for the parasite.

To conclude, the comprehensive work in this thesis has provided valuable insights into the pathogenesis and cell biology of the highly adaptable diplomonad parasite S. salmonicida.      

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 70
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1785
Keywords
Diplomonads, Spironucleus, Giardia, genome, annexin, oxygen stress, APEX, pathology
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-379671 (URN)978-91-513-0604-9 (ISBN)
Public defence
2019-05-10, A1:111a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Funder
Swedish Research Council Formas
Available from: 2019-04-17 Created: 2019-03-19 Last updated: 2019-06-18

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Astvaldsson, AsgeirSvärd, Staffan

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