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Interaction between crayfish and some microorganisms; Effect of temperature
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology. (Söderhäll)
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Innate immunity, which constitutes the first line of defense in vertebrates, is the only immune system that invertebrates rely on to protect themselves from pathogens. The invertebrate immune system is composed of cellular and humoral components. Cellular immunity is phagocytosis, opsonization and encapsulation. The humoral part is mainly composed of the events taking place upon secretion of granules and the enzymes within that lead to the lysis of the pathogen by antimicrobial peptides (AMPs) and the melanization cascade. The Prophenoloxidase (proPO) activating system is an important pathway that is stored in the granules of semigranular and granular hemocytes (blood cells). These cells will degranulate and release the proPO system when activated upon pathogen recognition. This cascade results in the melanization reaction and to trap and eliminate pathogens. 

White spot syndrome virus (WSSV) is a deadly pathogen mainly targeting crustaceans and causing huge economic losses since its first emergence in 1992 in Taiwan. It is known that WSSV disables the immune system of the host by interfering with the proPO cascade. Temperature is a restricting factor for the WSSV infections however it is not known if its affects are on host immunity or on the virus itself.

With the aim of elucidating WSSV infection, we studied the virus entry mechanisms. By crosslinking WSSV with the hemocytes we showed that a new clip-domain serine protease (PlcSP) plays an important role during the WSSV infection in crayfish by means of interacting with WSSV envelope protein VP28. Moreover, we have shown that the viral entry is inhibited at cold temperatures due to temperature’s inhibitory effect on PlcSP expression. We also showed that by slowing down of the host’s metabolism hence proliferation in host tissue either by low temperature or cell cycle inhibitors, we could inhibit WSSV replication once it has entered the host cell. We tested if the temperature effects host or pathogen, or both, we investigated the mortalities, phagocytosis, bacterial clearance, total hemocyte counts, degranulation and melanization rate of crayfish under a cold and warm temperature by using two strains of gram-negative bacteria and LPS. It is apparent that the cellular immunity is more effective at low temperature while the humoral immunity can become overactivated and toxic for the host at higher temperature. Furthermore, we aimed to study the cleavage specificity for PlcSP since it is predicted to be secreted from hemocytes and takes part in the serine protease cascade during melanization reaction.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. , p. 50
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1688
National Category
Immunology
Identifiers
URN: urn:nbn:se:uu:diva-355147ISBN: 978-91-513-0374-1 (print)OAI: oai:DiVA.org:uu-355147DiVA, id: diva2:1225322
Public defence
2018-09-13, Lindahlsalen, Norbyvägen 14, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2018-08-21 Created: 2018-06-26 Last updated: 2018-08-27
List of papers
1. A Pacifastacus leniusculus serine protease interacts with WSSV
Open this publication in new window or tab >>A Pacifastacus leniusculus serine protease interacts with WSSV
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2017 (English)In: Fish and Shellfish Immunology, ISSN 1050-4648, E-ISSN 1095-9947, Vol. 68, p. 211-219Article in journal (Refereed) Published
Abstract [en]

Serine proteases are involved in many critical physiological processes including virus spread and replication. In the present study, we identified a new clip-domain serine protease (PIcSP) in the crayfish Pacifastacus leniusculus hemocytes, which can interact with the White Spot Syndrome Virus (WSSV) envelope protein VP28. It was characterized by a classic clip domain with six strictly conserved Cys residues, and contained the conserved His-Asp-Ser (H-D-S)motif in the catalytic domain. Furthermore, signal peptide prediction revealed that it has a 16-residue secretion signal peptide. Tissue distribution showed that it was mainly located in P. leniusculus hemocytes, and its expression was increased in hemocytes upon WSSV challenge. In vitro knock down of PIcSP decreased both the expression of VP28 and the WSSV copy number in hematopoietic stem (HPT) cells. Accordingly, these data suggest that the new serine protease may be of importance for WSSV infection into hematopoietic cells.

Keywords
Hematopoietic tissue, Invertebrate, Serine protease, Virus, WSSV
National Category
Immunology
Identifiers
urn:nbn:se:uu:diva-335858 (URN)10.1016/j.fsi.2017.07.026 (DOI)000411299500022 ()28705723 (PubMedID)
Funder
Swedish Research Council, 621-2012-2418
Available from: 2018-01-24 Created: 2018-01-24 Last updated: 2018-06-26Bibliographically approved
2. The effect of temperature on White Spot Disease progression in a crustacean, Pacifastacus leniusculus
Open this publication in new window or tab >>The effect of temperature on White Spot Disease progression in a crustacean, Pacifastacus leniusculus
2018 (English)In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 89, p. 7-13Article in journal (Refereed) Published
Abstract [en]

The effects of temperature on the progression of White Spot Disease (WSD) have been studied in the freshwater crayfish Pacifastacus leniusculus. In this study, we aimed to understand the reason for previously observed low mortalities with white spot syndrome virus (WSSV) infected crayfish at low temperatures. The susceptibility of freshwater crayfish to WSSV was studied at different temperatures. The mortality rate at 6°C was zero, meanwhile the animals kept at 22°C developed WSD symptoms and died in a few days after WSSV injections, however upon transfer of animals from 6°C to 22°C the mortality reached 100% indicating that the virus is not cleared. Moreover, the VP28 expression at 6°C was significantly lower compared to animals kept at 22°C. We injected animals with demecolcine, an inhibitor that arrests the cell cycle in metaphase, and observed a delayed mortality. Furthermore, the VP28 expression was found to be lower in these animals receiving both injections with WSSV and demecolcine since cell proliferation was inhibited by demecolcine. We quantified WSSV copy numbers and found that virus entry was blocked at 6°C, but not in demecolcine treatments. We supported this result by quantifying the expression of a clip domain serine protease (PlcSP) which plays an important role for WSSV binding, and we found that the PlcSP expression was inhibited at 6°C. Therefore, our hypothesis is that the WSSV needs proliferating cells to replicate, and an optimum temperature to enter the host hematopoietic stem cells successfully. 

Keywords
White spot syndrome virus, temperature, innate immunity, crustacean, serine protease
National Category
Immunology
Identifiers
urn:nbn:se:uu:diva-356485 (URN)10.1016/j.dci.2018.07.026 (DOI)000444932200002 ()30071208 (PubMedID)
Funder
Swedish Research Council, 621-2012-2418Swedish Research Council Formas, 2011-606
Available from: 2018-07-31 Created: 2018-07-31 Last updated: 2019-06-26Bibliographically approved
3. Expression of an active and inactive clip-domain Serine Protease Isolated from Signal Crayfish in an insect cell line
Open this publication in new window or tab >>Expression of an active and inactive clip-domain Serine Protease Isolated from Signal Crayfish in an insect cell line
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

In this study, we aimed to elucidate the cleavage specificity and endogenous substrate of a clip-domain serine protease from the crayfish Pacifastacus leniusculus (PlcSP). This serine protease can bind to white spot syndrome virus (WSSV) and is important for the entry of WSSV into the hematopoietic tissue (HPT). In order to understand its intrinsic role in the serine protease cascade, we wanted to use substrate phage display method to clarify cleavage specificity of PlcSP. Firstly, we aimed to make recombinant active and inactive forms of this PlcSP using human embryonic kidney cell line (HEK293) however PlcSP appeared to be toxic for mammalian cells and we were not able to express this crustacean protein. However, we were able to express these recombinantPlcSP both in their zymogenic and active forms in Sf9 insect cell line.

Keywords
Serine protease, Pacifastacus leniusculus, invertebrate immunity, Sf9, substrate phage display
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-356482 (URN)
Available from: 2018-07-31 Created: 2018-07-31 Last updated: 2018-08-02

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