Background
Major histocompatibility complex (MHC) class I and II molecules play a crucial role in the immune system by presenting antigens to T cells, thereby initiating immune responses. Until recently, it was believed that MHC genes and their associated immune components had been conserved since their evolutionary emergence in jawed fish. However, sequencing of the Atlantic cod (Gadus morhua) genome revealed a loss of MHC class II genes and an extreme expansion of MHC class I genes [1]. In mammals, MHC II deficiency leads to serious diseases and early mortality. However, how Atlantic cod, which naturally lacks MHC II, is able to fight infections is still unknown. It has been hypothesized that the loss of the MHC class II pathway coincided with a more versatile use of MHC class I, but this has not been experimentally demonstrated.
Sequencing of the Atlantic cod genome also revealed changes within the Toll-like receptor (TLR) repertoire with multiple losses and expansions. TLRs are receptors of the innate immune system that recognize pathogen-derived ligands. Their expansion is hypothesized to be correlated with the loss of the MHC II pathway and increased ligand repertoire. Nevertheless, limited knowledge exists on fish-specific TLRs and their ligand specificity.
Atlantic cod primary cells transfected with GFP- MHC I and mcherry-Rab7”.
Project
The aim of this project is to characterize the MHC I pathway in the Atlantic cod, as well as some of the fish-specific TLRs focusing on revealing their intracellular transport routes and response to infection. By characterizing both aspects of the Atlantic cod immune systems, we will improve our understanding of how Atlantic cod can live without MHC II.
Results from our group revealed that in Atlantic cod cells, MHC I resides in specialized intracellular acidic compartments named Atlantic cod MHC I compartments (cMICs) [2, 3]. This suggests that MHC I may bind exogenous peptides in these compartments for potential cross presentation. In this project, we want to further understand how MHC I is transported to cMIC in both Atlantic cod cell lines and primary leukocytes by using confocal live imaging. The intracellular localization of teleost specific TLRs will be determined by super-resolution confocal microscopy. To reveal the response of the selected TLRs to infection, gene silencing of TLRs will be performed in Atlantic cod cells using siRNAs or CRISPR followed by infection with Francisella noatunensis subsp. noatunensis (F.n.n.) and qRT-PCR.
Methods
- In this project, the master student will learn techniques in biochemistry, cellular and molecular biology, including:
- recombinant DNA technology
- RNA isolation and qRT-PCR
- Protein expression and purification
- Immunoprecipitation
- Western blotting
- Cell culture and isolation of primary cells
- Cell transfection
- RNA interference
- Infection assays
- Immunofluorescence and state-of-the-art microscopy techniques, using different microscopes available at the Oslo NorMIC Imaging Platform - Department of Biosciences.
- Quantitative image analysis using software such as ImageJ, Imaris, etc.
Workplace and environment
The project will be performed at the Department of Biosciences in the group of Cinzia Progida.
The group consists of researchers at different stages of their careers (Master, PhD students, post-docs, and an experienced technician).
If any questions, contact: c.a.m.progida@ibv.uio.no
Phone: 22854441
Room: 3626
References
[1] B. Star, A.J. Nederbragt, S. Jentoft, U. Grimholt, M. Malmstrom, T.F. Gregers, T.B. Rounge, J. Paulsen, M.H. Solbakken, A. Sharma, O.F. Wetten, A. Lanzen, R. Winer, J. Knight, J.H. Vogel, B. Aken, O. Andersen, K. Lagesen, A. Tooming-Klunderud, R.B. Edvardsen, K.G. Tina, M. Espelund, C. Nepal, C. Previti, B.O. Karlsen, T. Moum, M. Skage, P.R. Berg, T. Gjoen, H. Kuhl, J. Thorsen, K. Malde, R. Reinhardt, L. Du, S.D. Johansen, S. Searle, S. Lien, F. Nilsen, I. Jonassen, S.W. Omholt, N.C. Stenseth, K.S. Jakobsen, The genome sequence of Atlantic cod reveals a unique immune system, Nature, 477 (2011) 207-210.
[2] S.A. Bjornestad, M.H. Solbakken, K.S. Jakobsen, S. Jentoft, O. Bakke, C. Progida, Atlantic cod (Gadus morhua) MHC I localizes to endolysosomal compartments independently of cytosolic sorting signals, Front Cell Dev Biol, 11 (2023) 1050323.
[3] S.A. Bjornestad, M.H. Solbakken, P. Krokene, B. Thiede, K. Hylland, K.S. Jakobsen, S. Jentoft, O. Bakke, C. Progida, The Atlantic Cod MHC I compartment has the properties needed for cross-presentation in the absence of MHC II, Sci Rep, 14 (2024) 25404.