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Pathogenesis and Cell Biology of the Salmon Parasite Spironucleus salmonicida
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. (Staffan Svärd)
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 [en]
Diplomonads, Spironucleus, Giardia, genome, annexin, oxygen stress, APEX, pathology
National Category
Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-379671ISBN: 978-91-513-0604-9 (print)OAI: oai:DiVA.org:uu-379671DiVA, id: diva2:1297426
Public defence
2019-05-10, A1:111a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Funder
Swedish Research Council FormasAvailable from: 2019-04-17 Created: 2019-03-19 Last updated: 2019-06-18
List of papers
1. The genome of Spironucleus salmonicida highlights a fish pathogen adapted to fluctuating environments
Open this publication in new window or tab >>The genome of Spironucleus salmonicida highlights a fish pathogen adapted to fluctuating environments
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2014 (English)In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 10, no 2, p. e1004053-Article in journal (Refereed) Published
Abstract [en]

Spironucleus salmonicida causes systemic infections in salmonid fish. It belongs to the group diplomonads, binucleated heterotrophic flagellates adapted to micro-aerobic environments. Recently we identified energy-producing hydrogenosomes in S. salmonicida. Here we present a genome analysis of the fish parasite with a focus on the comparison to the more studied diplomonad Giardia intestinalis. We annotated 8067 protein coding genes in the ∼12.9 Mbp S. salmonicida genome. Unlike G. intestinalis, promoter-like motifs were found upstream of genes which are correlated with gene expression, suggesting a more elaborate transcriptional regulation. S. salmonicida can utilise more carbohydrates as energy sources, has an extended amino acid and sulfur metabolism, and more enzymes involved in scavenging of reactive oxygen species compared to G. intestinalis. Both genomes have large families of cysteine-rich membrane proteins. A cluster analysis indicated large divergence of these families in the two diplomonads. Nevertheless, one of S. salmonicida cysteine-rich proteins was localised to the plasma membrane similar to G. intestinalis variant-surface proteins. We identified S. salmonicida homologs to cyst wall proteins and showed that one of these is functional when expressed in Giardia. This suggests that the fish parasite is transmitted as a cyst between hosts. The extended metabolic repertoire and more extensive gene regulation compared to G. intestinalis suggest that the fish parasite is more adapted to cope with environmental fluctuations. Our genome analyses indicate that S. salmonicida is a well-adapted pathogen that can colonize different sites in the host.

National Category
Microbiology Genetics
Identifiers
urn:nbn:se:uu:diva-224545 (URN)10.1371/journal.pgen.1004053 (DOI)000332021500041 ()24516394 (PubMedID)
Available from: 2014-05-14 Created: 2014-05-14 Last updated: 2019-03-19Bibliographically approved
2. 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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2016 (English)In: MSphere, ISSN 2379-5042, Vol. 1, no 2, article id 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
Keywords
intestinal parasite, annexins, diplomonad, Spironucleus salmonicida, Giardia, proximity labeling, APEX
National Category
Cell Biology Microbiology Evolutionary Biology
Identifiers
urn:nbn:se:uu:diva-264537 (URN)10.1128/mSphere.00032-15 (DOI)000392584700008 ()
Funder
Swedish Research Council, 2012-3364Swedish Research Council Formas, 2013-910
Available from: 2015-10-14 Created: 2015-10-14 Last updated: 2019-03-19Bibliographically approved
3. Oxygen induces the expression of invasion and stress response genes in the anaerobic salmon parasite Spironucleus salmonicida
Open this publication in new window or tab >>Oxygen induces the expression of invasion and stress response genes in the anaerobic salmon parasite Spironucleus salmonicida
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2019 (English)In: BMC Biology, ISSN 1741-7007, E-ISSN 1741-7007, Vol. 17, no 1, article id 19Article in journal (Refereed) Published
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.

Keywords
Anaerobiosis, Diplomonads, Giardia, Lateral gene transfer, Oxygen stress, Parasitology, Protist, RNAseq, Spironucleosis, Spironucleus
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-378917 (URN)10.1186/s12915-019-0634-8 (DOI)000459961200001 ()
Funder
EU, European Research Council, 310039-PUZZLE_CELLSwedish Foundation for Strategic Research Swedish Research Council, 2015-04959Swedish Research Council Formas, 2016-00539
Available from: 2019-03-12 Created: 2019-03-12 Last updated: 2019-03-22Bibliographically approved
4. Proximity Staining using Enzymatic Protein Tagging in Diplomonads
Open this publication in new window or tab >>Proximity Staining using Enzymatic Protein Tagging in Diplomonads
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.

National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-379669 (URN)10.1128/mSphereDirect.00153-19 (DOI)
Funder
Swedish Research Council Formas, 2016-00539
Available from: 2019-03-19 Created: 2019-03-19 Last updated: 2020-02-20Bibliographically approved
5. Experimental challenge of Atlantic salmon (Salmo salar) with the diplomonad parasite Spironucleus salmonicida to characterize the infection cycle
Open this publication in new window or tab >>Experimental challenge of Atlantic salmon (Salmo salar) with the diplomonad parasite Spironucleus salmonicida to characterize the infection cycle
(English)Manuscript (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.

Keywords
Diplomonads, Spironucleus salmonis, Salmo salar, fish infection, pathology
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-378925 (URN)
Funder
Swedish Research Council Formas, 2016-00539
Available from: 2019-03-09 Created: 2019-03-09 Last updated: 2019-03-19
6. Dual transcriptomic analysis of Spironucleus salmonicida-infected salmon cells identifies putative virulence factors and host responses
Open this publication in new window or tab >>Dual transcriptomic analysis of Spironucleus salmonicida-infected salmon cells identifies putative virulence factors and host responses
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-379666 (URN)
Available from: 2019-03-19 Created: 2019-03-19 Last updated: 2019-03-19

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