| 1. |
- Anders, Alfjorden, et al.
(författare)
-
Experimental challenge of Atlantic salmon (Salmo salar) with the diplomonad parasite Spironucleus salmonicida to characterize the infection cycle
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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. |
|
|
| 3. |
- Ástvaldsson, Ásgeir, 1981-
(författare)
-
Pathogenesis and Cell Biology of the Salmon Parasite Spironucleus salmonicida
- 2019
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
|
|
| 4. |
- Astvaldsson, Asgeir, 1981-, et al.
(författare)
-
Proximity Staining using Enzymatic Protein Tagging in Diplomonads
- 2019
-
Ingår i: mSphere. - 2379-5042. ; 4:2
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 5. |
- Santos, Rui, et al.
(författare)
-
Combined nanometric and phylogenetic analysis of unique endocytic compartments in Giardia lamblia sheds light on the evolution of endocytosis in Metamonada
- 2022
-
Ingår i: BMC Biology. - : BioMed Central (BMC). - 1741-7007. ; 20:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background Giardia lamblia, a parasitic protist of the Metamonada supergroup, has evolved one of the most diverged endocytic compartment systems investigated so far. Peripheral endocytic compartments, currently known as peripheral vesicles or vacuoles (PVs), perform bulk uptake of fluid phase material which is then digested and sorted either to the cell cytosol or back to the extracellular space. Results Here, we present a quantitative morphological characterization of these organelles using volumetric electron microscopy and super-resolution microscopy (SRM). We defined a morphological classification for the heterogenous population of PVs and performed a comparative analysis of PVs and endosome-like organelles in representatives of phylogenetically related taxa, Spironucleus spp. and Tritrichomonas foetus. To investigate the as-yet insufficiently understood connection between PVs and clathrin assemblies in G. lamblia, we further performed an in-depth search for two key elements of the endocytic machinery, clathrin heavy chain (CHC) and clathrin light chain (CLC), across different lineages in Metamonada. Our data point to the loss of a bona fide CLC in the last Fornicata common ancestor (LFCA) with the emergence of a protein analogous to CLC (GlACLC) in the Giardia genus. Finally, the location of clathrin in the various compartments was quantified. Conclusions Taken together, this provides the first comprehensive nanometric view of Giardia's endocytic system architecture and sheds light on the evolution of GlACLC analogues in the Fornicata supergroup and, specific to Giardia, as a possible adaptation to the formation and maintenance of stable clathrin assemblies at PVs.
|
|
| 6. |
- Stairs, Courtney W., et al.
(författare)
-
Oxygen induces the expression of invasion and stress response genes in the anaerobic salmon parasite Spironucleus salmonicida
- 2019
-
Ingår i: BMC Biology. - : BioMed Central (BMC). - 1741-7007. ; 17:1
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
