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Caspase-1-like regulation of the proPO-system and role of ppA and caspase-1-like cleaved peptides from proPO in innate immunity
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.
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2014 (English)In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 10, no 4, e1004059- p.Article in journal (Refereed) Published
Abstract [en]

Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO) by a proteolytic cascade finalized by the proPO-activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1 proteolysis.

Place, publisher, year, edition, pages
2014. Vol. 10, no 4, e1004059- p.
National Category
URN: urn:nbn:se:uu:diva-229571DOI: 10.1371/journal.ppat.1004059ISI: 000342033600027PubMedID: 24722332OAI: oai:DiVA.org:uu-229571DiVA: diva2:736959
Swedish Research Council
Available from: 2014-08-11 Created: 2014-08-11 Last updated: 2016-04-29Bibliographically approved
In thesis
1. Melanization and Hemocyte Homeostasis  in the Freshwater Crayfish, Pacifastacus leniusculus
Open this publication in new window or tab >>Melanization and Hemocyte Homeostasis  in the Freshwater Crayfish, Pacifastacus leniusculus
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Blood cells or hemocytes play important roles in immunity. They are a major source of many immune-related molecules such as antibodies in adaptive immunity of vertebrates and prophenoloxidase (proPO) in invertebrates. In the crayfish Pacifastacus leniusculus, the proPO-system has been reported to be an important component of immune responses against microorganisms. In this study, several mutant strains of Aeromonas hydrophila were used to reveal that LPS (lipopolysaccharide) is an important factor for the pathogenicity of A. hydrophila, strongly inducing the proPO system and melanization. This proPO activating system is a multistep process, which has to be tightly controlled to avoid the harmful side effects of toxic intermediates. Many regulating factors have been reported to fine-tune the proPO-system. In this study, the cleavage of caspase-1-like activity was shown to be a novel negative regulator of PO activity in crayfish. Moreover, the fragments obtained by cleavage of proPO by the proPO-activating enzyme and caspase-1-like protein increased bacterial clearance. Thus, the peptides generated also have important biological functions.

In addition to being a source of immune proteins, hemocytes also participate in phagocytosis, encapsulation, and nodulation. An infection normally causes a reduction of hemocyte numbers. Consequently, hemocyte homeostasis is important for maintaining appropriate hemocyte numbers in the circulation of the animal. This study shows that the reactive oxygen species level in the anterior proliferation center of crayfish hematopoietic tissue (HPT), together with cell proliferation, was increased during infection. Pl-β-thymosins were proposed to be involved in hemocyte homeostasis by increasing stem cell migration and thus increasing the circulating hemocyte number. Crayfish hemocyte numbers, as well astakine (Ast1 and Ast2) expression in hemocytes and HPT, were previously shown to be under circadian regulation. Here, we show that Ast1, Ast2, and proPO exhibit rhythmic expression in the crayfish brain similarly to their orthologs, prokineticin 1, prokineticin 2 and tyrosinase, respectively, in the zebrafish brain. Tyrosinase expression was detected in zebrafish brain cells while PO-positive cells were identified as hemocytes that had infiltrated into the crayfish brain. Therefore, this information suggests a close relationship between crayfish hemocytes and the crayfish brain as well as vertebrate neurons.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 48 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1086
melanization, prophenoloxidase, caspase, hematopoiesis, thymosin, astakine, circadian rhythm, reactive oxygen species, tyrosinase, prokineticin
National Category
urn:nbn:se:uu:diva-209209 (URN)978-91-554-8774-4 (ISBN)
Public defence
2013-11-28, Lindahlssalen, EBC, Norbyvägen 18A, Uppsala, 10:00 (English)
Available from: 2013-11-06 Created: 2013-10-15 Last updated: 2016-05-09

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