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The biology and characterization of NOD-like receptors in goldfish (Carassius auratus L.) macrophages exposed to bacterial pathogens Open Access


Other title
Gene expression
NOD-like receptors
Type of item
Degree grantor
University of Alberta
Author or creator
Xie, Jiasong
Supervisor and department
Belosevic, Miodrag (Biological Sciences)
Examining committee member and department
Stafford, James (Biological Sciences)
Chang, John (Biological Sciences)
Chadee, Kris (Microbiology, Immunology & Infectious Diseases at the University of Calgary)
Gallin, Warren (Biological Sciences)
Department of Biological Sciences
Physiology, Cell and Developmental Biology
Date accepted
Graduation date
2017-06:Spring 2017
Doctor of Philosophy
Degree level
The recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) is important for the initiation of the host defense against pathogens. NOD-like receptors (NLRs) are a family of PRRs that primarily detect the cytosolic PAMPs of the pathogens. The NLRs have been well studied in mammals, but are relatively less known in fish. The objective of my thesis was to identify and functionally characterize NLRs and their signaling molecules of goldfish macrophages in response to bacterial pathogens. The NOD1, NOD2 and NLRX1 of goldfish macrophages were functionally characterized. Treatment of macrophages with LPS, Poly I:C, muramyl dipeptide (MDP), peptidoglycan (PGN), heat-killed Aeromonas salmonicida or Mycobacterium marinum differentially altered the expression of the NOD-like receptors. These results indicated that goldfish NLRs were functionally highly conserved and that they played a pivotal role in recognition of fish pathogens such as A. salmonicida and M. marinum. The association between the NOD1/NOD2 receptors and the key adaptor protein (RIP2) of their signaling pathway was examined. Treatment of goldfish macrophages with LPS, PGN, MDP, Poly I:C, heat-killed and live M. marinum, and heat-killed A. salmonicida differentially changed the expression of RIP2 at both mRNA and protein levels. RIP2 was found to associate with NOD1 and NOD2 receptors in eukaryotic cells, and RIP2 over-expression resulted in the activation of the NF-κB. RIP2 was shown to play a central role in the production of pro-inflammatory cytokines TNFalpha-2 and IL-1beta1 by goldfish macrophages exposed to M. marinum. I identified and functionally characterized, for the first time, an NLRP inflammasome-like molecule (= NLRP3rel), and its adaptor protein (ASC) of bony fish macrophages. Treatment of goldfish macrophages with nigericin, an inducer of inflammasome pathway, up-regulated the expression of both NLRP3rel and ASC at mRNA and protein levels. Confocal microscopy and co-immunoprecipitation assays results indicated that goldfish NLRP3rel associated with ASC in eukaryotic cells. In addition, the results indicated that ASC associated with caspase-1 and RIP2, and ASC over-expression did not cause the activation of NF-κB, but down-regulated RIP2 ability to activate NF-κB. These results indicate that the goldfish NLRP3rel molecule has a similar inflammasome signaling pathway to that of mammals. An inflammasome signaling pathway downstream molecule, HMGB1, was also functionally characterized after exposure to bacteria. My results indicated that goldfish HMGB1, was a critical regulatory cytokine of inflammatory and antimicrobial responses of the goldfish. The results of my dissertation highlight the importance of NLRs in antimicrobial responses of bony fish.
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
Citation for previous publication
Xie, J., Hodgkinson, J.W., Katzenback, B.A., Kovacevic, N., and Belosevic, M., 2013. Characterization of three Nod-like receptors and their role in antimicrobial responses of goldfish (Carassius auratus L.) macrophages to Aeromonas salmonicida and Mycobacterium marinum. Developmental and Comparative Immunology, 39: 180-187Xie, J., Hodgkinson, J.W., Li, C., Kovacevic, N., and Belosevic, M., 2014. Identification and functional characterization of the goldfish (Carassius auratus L.) high mobility group box 1 (HMGB1) chromatin-binding protein. Developmental and Comparative immunology, 44: 245-253Xie, J., and Belosevic, M., 2015. Functional characterization of receptor-interacting serine/threonine kinase 2 (RIP2) of the goldfish (Carassius auratus L.). Developmental and Comparative Immunology, 48: 76-85Xie, J., and Belosevic, M., 2016. Functional characterization of apoptosis-associated spec-like protein (ASC) of the goldfish (Carassius auratus L.). Developmental and Comparative Immunology, 65: 201-210

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