Source: UNIVERSITY OF GEORGIA submitted to NRP
SUPPRESSION OF THE INSECT CELLULAR IMMUNE RESPONSE BY A POLYDNAVIRUS-CARRYING PARASITOID
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
0196684
Grant No.
2003-35302-13564
Cumulative Award Amt.
(N/A)
Proposal No.
2003-01358
Multistate No.
(N/A)
Project Start Date
Jul 15, 2003
Project End Date
Jul 14, 2008
Grant Year
2003
Program Code
[51.2]- (N/A)
Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
ENTOMOLOGY
Non Technical Summary
Many insect pests eliminate biological control agents like parasitoids via encapsulation and other immune responses. This project examines how certain virulence genes encoded by parasitoid-associated polydnaviruses suppress encapsulation and other host immune responses.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113110103010%
2113110109010%
2113110110110%
2113110111010%
2113110113010%
2114030103010%
2114030109010%
2114030110110%
2114030111010%
2114030113010%
Goals / Objectives
The most important insect immune response toward multicellular parasites is called encapsulation. The overall goal of this study is to understand how encapsulation by the lepidopteran Pseudoplusia includens is suppressed by Microplitis demolitor bracovirus (MdBV). Prior studies indicate that MdBV blocks encapsulation by inhibiting infected hemocytes from adhering to foreign surfaces. This effect involves surface proteins encoded for by the MdBV glc gene family. We hypothesize that Glc proteins disrupt hemocyte adhesion by interfering with the function of either integrins or the plasmatocyte spreading peptide receptor (PSPR) which are both involved in mediating hemocyte adhesion to foreign targets. Specific objectives of this proposal are to: 1) Characterize and produce recombinant Glc proteins, 2) Bioassay the binding and adhesion-blocking activity of Glc proteins, and 3) Examine how Glc proteins interact with host molecules that regulate encapsulation.
Project Methods
Objective 1 will be approached by determining whether MdBV Glc proteins are peripheral or membrane bound, identifying the glycan moieties present on these proteins, and producing recombinant Glc proteins. The goals of Objective 2 will be met by characterizing the distribution of Glc proteins on hemocytes and by conducting bioassays to determine which mutant recombinant proteins interfere with hemocyte adhesion. In Objective 3, we will determine whether Glc proteins bind directly to the plasmatocyte spreading peptide receptor or integrins expressed by hemocytes.

Progress 07/15/03 to 07/14/08

Outputs
OUTPUTS: Parasitoid wasps and flies are among the most important natural enemies of agricultural pests. This project focuses on the function of gene called glc1.8 expressed from Microplitis demolitor bracovirus (MdBV). Goals of this study were to characterize how MdBV and glc1.8 disable the insect cellular defense response by: a) characterizing recombinant Glc1.8 mutant proteins, b) bioassaying the immunosuppressive activity of these mutants, and c) examining how Glc1.8 interacts with pest insect molecules that regulate cellular immune defenses. Results of the study accomplished all major objectives of the project. Results of the study have also been disseminated through peer-reviewed scientific publications, national and international scientific meetings, and seminars presented in US and non-US universities. PARTICIPANTS: Participants: Dr. Markus H. Beck, Dr. Richard J. Suderman, Dr. Andrea Pruijssers, Ms. Shu Zhang, Ms. Jena Johnson, and Dr. Michael R. Strand TARGET AUDIENCES: Target audiences: Insect biology and insect pest management scientists, virologists, and immunologists with interests in insect-parasite or insect-pathogen interactions plus researchers studying the use of natural enemies for control of insect pest species. PROJECT MODIFICATIONS: No major changes in approach or goals were made during the course of the study.

Impacts
Major outcomes of the study included: a) development of multiple recombinant mutant proteins of Glc1.8, b) bioassays that characterized the effects of different mutations on protein function, and c) completion of functional studies indicating that Glc1.8 interferes with the ability of specific insect pattern recognition receptors to bind their cognate ligands. Mutational analysis indicated that the most important functional domain for Glc1.8 is its N-glycosylated extracellular domain. Characterization of the carbohydrate moieties in this domain further identified that the majority of glycans present are high mannose or paucimannose sugars.

Publications

  • Strand, M. R. 2008. Polydnaviruses: abrogation of the insect immune system. In: Encyclopedia of Virology, 2nd Ed. Volume 4, pp. 250-256. Elsevier, San Diego. Suderman, R. J., A. J. Pruijssers and M. R. Strand. 2008. Protein tyrosine phosphatase-H2 from a polydnavirus induces apoptosis of insect cells. J. Gen. Virol. 89, 5716-5721. Strand, M. R. 2008. The interactions between polydnavirus-carrying parasitoids and their lepidopteran hosts. In Molecular Biology of the Lepidoptera (M. Goldsmith and T. M Miller eds.) CRC Press, Boca Raton, FL. In press.


Progress 07/15/06 to 07/14/07

Outputs
Parasitoid wasps and flies are among the most important natural enemies of agricultural pests. This project focuses on the function of gene called glc1.8 expressed from Microplitis demolitor bracovirus (MdBV). Goals of this study are to characterize how MdBV and glc1.8 disable the insect cellular defense response. Progress during this year include production and characterization of multiple Glc1.8 mutants to assess domains required for functional activity. Analysis confirms that Glc1.8 is membrane bound due to a cytoplasmic anchor. Biological activity depends upon anchoring into the membrane, whereas the cytoplasmic tail is non-essential for function. Glycan analysis is still in progress but preliminary results indicate that all carbohydrate moities are N-linked. Expression of Glc1.8 interferes with multiple target cell receptors including Eater, dsC1 and alpha4/beta 1 integrin. Studies for the upcoming year will focus on completion of carbohydrate analyses and receptor-glc1.8 interactions.

Impacts
Deciphering host pathogen interactions is of utmost importance to the study of infectious diseases in mammals and insects. The proposed work will yield information of practical importance for the management of insect populations and on the function of immunosuppressive molecules that exhibit activity in multiple species. The interdisciplinary nature of the proposed project is also uniquely suited as a platform to train young scientists at the interface of parasitology, symbiosis, cell biology, and molecular processes.

Publications

  • Webb, B. A., M. R. Strand, S. E. Deborde, M. Beck, R. S. Hilgarth, K. Kadash, J. A. Kroemer, K. G. Lindstrom, W. Rattanadechakul, K. S. Shelby, L. Thoetkiattikul, M. W. Turnbull, W. E. Barney, and R. A. Witherell. 2006. Polydnavirus genomes reflect their dual roles as mutualists and pathogens. Virology 347, 160-174.
  • Pennacchio, F and M. R. Strand. 2006. Evolution of developmental strategies in parasitic Hymenoptera. Annu. Rev. Entomol. 51, 233-258.
  • Wertheim, B., A. R. Kraaijeveld, E. Schuster, E. Blanc, M. Hopkins, S. D. Pletcher, M. R. Strand, H. C. J. Godfray, and L. Partridge. 2005. Genome wide expression in response to parasitoid attack in Drosophila. Genome Biol. 6: R94 (doi:10.1186/gb-2005-2005-6-11-r94).


Progress 01/01/05 to 12/31/05

Outputs
The goal of this project is to characterize a viral protein called Glc1.8 which functions as an inhibitor of the insect encapsulation response. The overall goal of this project is to characterize this protein and its immunosuppressive functions. Progress during the current year include mutational studies that identify essential domains for the anti-capsule forming activity of the protein. These studies also identified that glc1.8 inhibits phagocytosis by insect immune cells. Stably transformed cell lines have now been developed that express wild-type and mutant forms of the protein. Assays also identify cell surface and signaling pathways that glc1.8 inhibit.

Impacts
This project focuses on basic science in the area of immunity and virology but our results provide important insights on how the insect immune system functions. Results also point to potential applications of this immunosuppressive molecule

Publications

  • Beck, M. and M. R. Strand. 2005. Glc1.8 from Microplitis demolitor bracovirus induces a loss of adhesion and phagocytosis by insect High Five and S2 cells. J. Virol. 79, 1861-1870.
  • Strand, M. R., M. H. Beck, M. D. Lavine, and K. D. Clark. 2005. Microplitis demolitor bracovirus inhibits phagocytosis by hemocytes from Pseudoplusia includens. Arch Insect Bich. Physiol. 61, 134-145.


Progress 01/01/04 to 12/31/04

Outputs
Parasitoid wasps and flies are among the most important natural enemies of insect agricultural pests. This project focuses on a gene called glc 1.8 expressed by a polydnavirus called MdBV. Progress during this year includes production of recombinant protein and transfected cell lines that stably express the Glc protein. Bioassays using these materials confirm that this molecule inhibits the ability of cells to bind foreign surfaces and to phagocytose pathogens. Additional mutagenesis studies identified functional domains of the protein required for adhesion blocking. Additional assays indicated that Glc proteins also block adhesion of immune cells in other insects and disrupt phagocytosis. These results identify a new class of virulence genes that suppress the insect immune system.

Impacts
These results identify a new class of molecules that suppress the insect immune system. Characterization of these factors could yield new materials for development of pest insect control products.

Publications

  • Webb B.A. and Strand M.R. 2005. The Biology and Genomics of Polydnaviruses. In Comprehensive Molecular Insect Science, Vol. 6 (Gilbert, L. I., Iatrou, and Gill, S. S., eds). Elsevier, San Diego, CA. pg. 323-360.
  • Beck, M. and M. R. Strand. 2005. Glc1.8 from Microplitis demolitor bracovirus induces a loss of adhesion and phagocytosis by Insect High Five and S2 cells. J. Virol. 79, 1861-1870.


Progress 01/01/03 to 12/31/03

Outputs
Parasitoid wasps and flies are among the most important natural enemies of insect agricultural pests. Parasitoids are usually killed in non-permissive hosts by encapsulation, whereas parasitoids survive in permissive hosts by either possessing surface features that passively protect the parasitoid from being encapsulated or by immunosuppressing the host's encapsulation response. Immunosuppression is mediated by symbiotic polydnaviruses (PDVs) such as Microplitis demolitor bracovirus (MdBV). Prior studies identified the Glc gene as a key immunosuppressive protein encoded by MdBV. Specific objectives of this project are to: 1) Produce recombinant Glc proteins, 2) Bioassay the binding and adhesion-blocking activity of Glc proteins, and 3) Examine how Glc proteins interact with host molecules that regulate encapsulation. Progress during this year includes production of recombinant protein and transfected cell lines that stably express the Glc protein. Bioassays using these materials confirm that this molecule completely inhibits the ability of cells to bind foreign surfaces and to phagocytose pathogens.

Impacts
These results identify a new class of molecules that suppress the insect immune system. Characterization of these factors could yield new materials for development of pest insect control products.

Publications

  • Kadash, K., J. A. Harvey and M. R. Strand. 2003. Cross-protection experiments with parasitoids in the genus Microplitis (Hymenoptera; Braconidae) suggest a high levelof specificity in their associated bracoviruses. J. Insect Physiol. 49: 473-482.
  • Beck, M. and M. R. Strand. 2003. RNA interference silences Microplitis demolitor bracovirus genes and implicates glc1.8 in blocking adhesion of infected host cells. Virology, 314, 521-535.