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Innate Immune Proteins in a Crustacean Pacifastacus leniusculus
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
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 [en]
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: urn:nbn:se:uu:diva-147600ISBN: 978-91-554-8015-8OAI: oai:DiVA.org:uu-147600DiVA: diva2:400557
Public defence
2011-04-08, Lindahlsalen, Evolutionary Biology Centre, Norbyvägen 18A,, Uppsala, 10:00 (English)
Opponent
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
List of papers
1. Hemocyte lineage marker proteins in a crustacean, the freshwater crayfish, Pacifastacus leniusculus
Open this publication in new window or tab >>Hemocyte lineage marker proteins in a crustacean, the freshwater crayfish, Pacifastacus leniusculus
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2008 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 8, no 20, 4226-4235 p.Article in journal (Refereed) Published
Abstract [en]

To identify proteins associated with development of different hemocyte types in the freshwater crayfish Pacifastacus leniusculus, 2-DE followed by MS analysis was carried out with hematopoietic tissue (Hpt) cells, semigranular cells (SGC) and granular cells (GC). Within the hemocyte lineages one two-domain Kazal proteinase inhibitor (KPI) was found to be specific for SGC, while a superoxide dismutase (SOD) was specific for GC at protein as well as at mRNA level. The proliferation cell nuclear antigen (PCNA) was detected at the mRNA level in Hpt cells only. We also provide evidence that SGC and GC most likely differentiate to maturation as separate lineages. We found that after laminarin or lipopolysaccharide (LPS) injection into crayfish, the transcript levels of PCNA and SOD increased in the Hpt cells, whereas the KPI transcript never was present in Hpt regardless of any challenge. RNA interference of PCNA in the Hpt cells led to that most of the cells did not spread or attach to the tissue culture dish. These results suggest that PCNA, KPI and SOD can be used as markers for Hpt cells, SGC and GC, respectively, and in conjunction with these results, a model is proposed how the Hpt responds to a microbial challenge by proliferation and release of Hpt cells.

Keyword
Crustacean hematopoiesis, Hemocyte markers, Kazal, PCNA, SOD
National Category
Immunology
Research subject
Biology with specialization in Comparative Physiology; Immunology
Identifiers
urn:nbn:se:uu:diva-97412 (URN)10.1002/pmic.200800177 (DOI)000260717300010 ()18814328 (PubMedID)
Available from: 2008-08-18 Created: 2008-08-18 Last updated: 2016-05-13
2. A novel protein acts as a negative regulator of prophenoloxidase activation and melanization in the freshwater crayfish Pacifastacus leniusculus
Open this publication in new window or tab >>A novel protein acts as a negative regulator of prophenoloxidase activation and melanization in the freshwater crayfish Pacifastacus leniusculus
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2009 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, no 10, 6301-6310 p.Article in journal (Refereed) Published
Abstract [en]

Melanization is an important immune component of the innate immune system of invertebrates and is vital for defense as well as for wound healing. In most invertebrates melanin synthesis is achieved by the prophenoloxidase-activating system, a proteolytic cascade similar to vertebrate complement. Even though melanin formation is necessary for host defense in crustaceans and insects, the process needs to be tightly regulated because of the hazard to the animal of unwanted production of quinone intermediates and melanization in places where it is not suitable. In the present study we have identified a new melanization inhibition protein (MIP) from the hemolymph of the crayfish, Pacifastacus leniusculus. Crayfish MIP has a similar function as the insect MIP molecule we recently discovered in the beetle Tenebrio molitor but interestingly has a completely different sequence. Crayfish MIP as well as Tenebrio MIP do not affect phenoloxidase activity in itself but instead interfere with the melanization reaction from quinone compounds to melanin. Importantly, crayfish MIP in contrast to Tenebrio MIP contains a fibrinogen-like domain, most similar to the substrate recognition domain of vertebrate l-ficolins. Surprisingly, an Asp-rich region similar to that found in ficolins that is likely to be involved in Ca2+ binding is present in crayfish MIP. However, crayfish MIP did not show any hemagglutinating activity as is common for the vertebrate ficolins. A mutant form of MIP with a deletion lacking four Asp amino acids from the Asp-rich region lost most of its activity, implicating that this part of the protein is involved in regulating the prophenoloxidase activating cascade. Overall, a new negative regulator of melanization was identified in freshwater crayfish that shows interesting parallels with proteins (i.e. ficolins) involved in vertebrate immune response.

Keyword
Pacifastacus leniusculus, melanization inhibiting protein, crayfish, phenoloxidase
National Category
Immunology
Research subject
Immunology; Biology with specialization in Comparative Physiology
Identifiers
urn:nbn:se:uu:diva-111473 (URN)10.1074/jbc.M806764200 (DOI)000263742700033 ()19129195 (PubMedID)
Available from: 2009-12-15 Created: 2009-12-15 Last updated: 2016-05-13
3. Peptidoglycan activation of the proPO-system without a peptidoglycan receptor protein (PGRP)?
Open this publication in new window or tab >>Peptidoglycan activation of the proPO-system without a peptidoglycan receptor protein (PGRP)?
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2011 (English)In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 35, no 1, 51-61 p.Article in journal (Refereed) Published
Abstract [en]

Recognition of microbial polysaccharide by pattern recognition receptors triggers the prophenoloxidase (proPO) cascade, resulting in melanin synthesis and its deposition on the surface of invading pathogens. Several masquerade-like proteins and serine proteinase homologues have been shown to be involved in the proPO activation in insects. In this study, a novel serine proteinase homologue, Pl-SPH2, was found and isolated as a 30 kDa protein from hemocytes of the freshwater crayfish, Pacifastocus leniusculus, by its binding property to a partially lysozyme digested or TCA-treated insoluble Lysine (Lys)-type pepticloglycan (PGN) and soluble polymeric Lys-type PGN. Two other proteins, the Pl-SPH1 and lipopolysaccharide- and beta-1,3-glucan-bincling protein (LGBP) were also found in the several different PGN-binding assays. However no PGRP homologue was detected. Neither was any putative PGRP found after searching available crustacean sequence databases. If RNA interference of Pl-SPH2, Pl-SPH1 or LGBP in the crayfish hematopoietic tissue cell culture was performed, it resulted in lower PO activity following activation of the proPO-system by soluble Lys-type PGN. Taken together, we report for the first time that Lys-type PGN is a trigger of proPO-system activation in a crustacean and that two Pl-SPlis are involved in this activation possibly by forming a complex with LGBP and without a PGRP.

Keyword
Innate immunity, Lys-type PGN, PGRP, proPO, SPHs
National Category
Immunology
Research subject
Biology with specialization in Comparative Physiology
Identifiers
urn:nbn:se:uu:diva-140158 (URN)10.1016/j.dci.2010.08.005 (DOI)000285024400007 ()20713082 (PubMedID)
Available from: 2011-01-04 Created: 2011-01-04 Last updated: 2016-05-13
4. Two novel ficolin-like proteins act as pattern recognition receptors for invading pathogens in the freshwater crayfish Pacifastacus leniusculus
Open this publication in new window or tab >>Two novel ficolin-like proteins act as pattern recognition receptors for invading pathogens in the freshwater crayfish Pacifastacus leniusculus
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.

Keyword
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:nbn:se:uu:diva-146611 (URN)10.1002/pmic.201000728 (DOI)000291087700009 ()21598394 (PubMedID)
Available from: 2011-02-18 Created: 2011-02-18 Last updated: 2016-05-13

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