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Two novel ficolin-like proteins act as pattern recognition receptors for invading pathogens in the freshwater crayfish Pacifastacus leniusculus
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
2011 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 11, no 11, 2249-2264 p.Article in journal (Refereed) Published
Abstract [en]

Abstract

To isolate pathogen-associated molecular patterns (PAMPs)-binding molecules, the bacterium, Staphylococcus aureus was used as an affinity matrix to find bacteria binding proteins in the plasma of the freshwater crayfish, Pacifastacus leniusculus. Two new bacteria binding ficolin-like proteins (FLPs) were identified by 2-DE and MS analysis. The FLPs have a fibrinogen-related domain (FReD) in their C-terminal and a repeat region in their N-terminal region with putative structural similarities to the collagen-like domain of vertebrate ficolins and mannose binding lectins (MBLs). Phylogenetic analysis shows that the newly isolated crayfish FLP1 and FLP2 cluster separately from other FReD containing proteins. A tissue distribution study showed that the mRNA expression of FLP occurred mainly in the hematopoietic tissue (Hpt) and in the hepatopancreas. Recombinant FLPs exhibited agglutination activity of Gram-negative bacteria Escherichia coli and Aeromonas hydrophila in the presence of Ca2+. FLPs were able to bind to A. hydrophila, E.coli and S.aureus as judged by bacteria adsorption. Moreover, the FLPs may help crayfish to clear Gram-negative bacteria, but not Gram-positive bacteria which had been injected into the hemolymph. When Gram-negative bacteria coated with FLPs were incubated with Hpt cells, a lower death rate of the cells was found compared to control treatment. Our results suggest that FLPs function as pattern recognition receptors in the immune response of crayfish.

Place, publisher, year, edition, pages
2011. Vol. 11, no 11, 2249-2264 p.
Keyword [en]
Aeromonas hydrophila, Animal proteomics, Ficolin-like protein, FReD domain, Pattern recognition receptor, Staphylococcus aureus
National Category
Immunology
Research subject
Biology with specialization in Comparative Physiology
Identifiers
URN: urn:nbn:se:uu:diva-146611DOI: 10.1002/pmic.201000728ISI: 000291087700009PubMedID: 21598394OAI: oai:DiVA.org:uu-146611DiVA: diva2:398553
Available from: 2011-02-18 Created: 2011-02-18 Last updated: 2017-12-11
In thesis
1. Innate Immune Proteins in a Crustacean Pacifastacus leniusculus
Open this publication in new window or tab >>Innate Immune Proteins in a Crustacean Pacifastacus leniusculus
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Hemocytes (blood cells) are important in the immune defense against pathogens in invertebrates. In crusteacean, the hemocytes and plasma components mount a strong innate immune response against different pathogens including bacteria and virus. This thesis is aimed to identify marker proteins associated with development of different hemocyte types, and to find a protein involved in the phenoloxidase-induced melanization and other innate immune reactions in freshwater crayfish Pacifastacus leniusculus.

In crustaceans, the hemocytes are produced and partly differentiated in the hematopoietic tissue (Hpt) before they are released into the hemolymph circulation. To investigate the connection between semigranular cells, granular cells and precursor cells in Hpt of P. leniusculus and possibly also in other crustaceans, two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) analysis was used to identify specific proteins expressed in different hemocytes. The specific expression was analyzed by RT-PCR and western blot. Moreover, RNA interference was used to study the hemocyte differentiation in vivo and in vitro.

Melanin formation is essential for host defence in arthopods, and it needs to be tightly regulated since unwanted production of quinone intermediates or melanization is also dangerous to the animal. By using western blot, 2-DE and MS, a melanization inhibiting protein (MIP) was found to have similar function as mealworm Tenebrio molitor MIP. Both of them interfere with the melanization reaction, but do not affect phenoloxidase activity.

In order to reveal the mechanism by which peptidoglycan (PGN) induces activation of the prophenoloxidase activating system in P. leniusculus, different forms of Lys-type PGN were used to pull down PGN recognition proteins (PGRPs) from plasma or hemocyte lysate supernatant of crayfish. The binding proteins were separated and then analyzed with MS. Results showed that two serine protease homologues are involved in this activation possibly by forming a complex with lipopolysaccharide and β-1,3-glucan binding protein (LGBP) and without a PGRP.

Besides, two ficolin-like proteins (FLPs) have been found from crayfish plasma by using different bacteria including Staphylocuccus aureus as an affinity matrix to pull down bacterial binding proteins, followed by the analysis with 2-DE and MS. Two FLPs can bind to bacteria, and may help crayfish to clear Gram-negative bacteria, but not Gram-positive bacteria injected into the crayfish hemolymph, which suggests that FLPs may function as pattern recognition receptors in the immune response of crayfish.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 72 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 807
Keyword
2-DE, hematopoiesis, marker protein, melaniztion inhibiting protein, proPO-system, pattern recognition protein, PGN, ficolin-like protein
National Category
Immunology
Research subject
Biology with specialization in Comparative Physiology
Identifiers
urn:nbn:se:uu:diva-147600 (URN)978-91-554-8015-8 (ISBN)
Public defence
2011-04-08, Lindahlsalen, Evolutionary Biology Centre, Norbyvägen 18A,, Uppsala, 10:00 (English)
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Supervisors
Note
Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 737Available from: 2011-03-16 Created: 2011-02-26 Last updated: 2011-05-04

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Söderhäll, KennethSöderhäll, Irene

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