Source: KANSAS STATE UNIV submitted to
HEMOLYMPH PROTEINS IN INSECT IMMUNE RESPONSES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0181996
Grant No.
(N/A)
Project No.
KS516
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 1999
Project End Date
Sep 30, 2005
Grant Year
(N/A)
Project Director
Kanost, M. R.
Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506
Performing Department
BIOCHEMISTRY
Non Technical Summary
Although some microorganisms are well Adapted as insect pathogens, insects can defend themselves against most infections by microorganisms such as bacteria, fungi, and viruses. In order to make better use of insect pathogens as agents for microbial control of insect pests it would be helpful to learn how the immune responses of insects work. This research is aimed at understanding proteins that exist in insect blood, which aid an insect in recognizing infections and in killing bacteria and fungi that have entered the insect body.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21131101000100%
Goals / Objectives
Infection in insects provokes defensive responses by hemocytes, synthesis of antimicrobial proteins by fat body, and activation of pro-phenol oxidase. However, it is not understood how recognition of microorganisms triggers pro-phenol oxidase activation or produces a signal that is transmitted to fat body cells and hemocytes to stimulate their responses. The hypothesis of this proposal is that binding of polysaccharides from microbial cell walls by lectins in hemolymph is a mechanism for recognition of infection that results in activation of serine proteases in insect hemolymph; proteolytic processing of specific plasma proteins then produces signaling molecules, which stimulate biochemical and cellular protective responses. Parasites and pathogens adapted for using specific insects as hosts might have evolved mechanisms to avoid activating such protease response systems. Activation of proteases also leads to activation of hemolymph phenoloxidase and the subsequent production of melanin that encapsulates parasites and coats wound sites. We propose to identify and characterize lectins and proteases in hemolymph of Manduca sexta that participate in responses to wounding and infection and to gain an understanding of how the proteases are activated and regulated. Our goal of characterizing lectins and proteases in insect hemolymph is addressed in the following objectives. 1. Identify and characterize lectins present in M. sexta plasma. 2. Identify and characterize a M. sexta serine protease that activates the prophenol oxidase activating protease zymogen. 3. Use antibodies and cDNA clones to M. sexta lectins, prophenol oxidase and plasma proteinases to begin to characterize homologous proteins from the Indian meal moth, Plodia interpunctella.
Project Methods
1. Identify and characterize lectins present in M. sexta plasma. We have used affinity chromatography to isolate M. sexta lectins that bind to a variety of carbohydrates, including monosaccharides, disaccharides, and polysaccharides. These purified proteins will be used as antigens for producing antisera, and their amino-terminal sequences will be determined. cDNA clones for the lectins will be isolated from fat body expression libraries by screening with the antibodies or by polymerase chain reaction techniques. Recombinant lectins will be produced either in E. coli or in a baculovirus expression system. The recombinant proteins will be used in experiments to define the binding specificity of the lectins and to evaluate the roles of the lectins in activation of plasma serine protease zymogens. 2. Identify and characterize a serine protease that activates the prophenol oxidase activating protease zymogen. We have isolated a cDNA clone for a protease that activates prophenol oxidase (prophenol oxidase activating protease; (PAP). This protease is itself made as a zymogen that is activated by a specific proteolytic cleavage. We will use recombinant proPAP (produced in a baculovirus expression system) as a substrate to monitor purification of its activating protease from M. sexta plasma. Amino acid sequence information and antibodies produced to this protein will be used to screen cDNA libraries. Recombinant proPAP acitivating enzyme will be produced using a baculovirus expression system. The set of reagents produced (clones, antibodies, recombinant proteins) will be used in experiments to work out the pathway that leads to activation of pro-phenol oxidase, and to understand the regulation of this pathway by plasma serpins. 3. Use antibodies and cDNA clones to M. sexta lectins, prophenol oxidase and plasma proteinases to begin to characterize homologous proteins from the Indian meal moth, Plodia interpunctella. Western and Northern blot experiments will determine which molecular probes will be useful for detecting P. interpunctella plasma proteins. These will be used for screening P. interpunctella cDNA libraries. These experiments will be a start toward comparing the structure and function of plasma proteins involved in innate immune responses of two lepidopteran species, including a one that is a pest of stored grain.

Progress 10/01/99 to 09/30/05

Outputs
Our goal was to understand how recognition of microorganisms triggers pro-phenol oxidase activation, an innate immune response of insects. Our hypothesis was that binding of polysaccharides from microbial cell walls by proteins in hemolymph is a mechanism for recognition of infection that results in activation of serine proteases, which lead to activation of hemolymph phenoloxidase and the subsequent production of melanin that encapsulates parasites and coats wound sites. Our objectives were to identify and characterize carbohydrate-binding proteins and proteases in hemolymph of the tobacco hornworm, Manduca sexta, that participate in responses to wounding and infection and to gain an understanding of how the proteases are activated and regulated. We identified five types of proteins in hemolymph of Manduca, which bind to microbial polysaccharides. These include (A) four C-type lectins, named immulectins, which bind to the surface of bacteria and stimulate activation of the prophenoloxidase pathway, (B) hemolin, a protein composed of four immunoglobulin domains, which binds to bacterial lipopolysaccharide and lipoteichoic acid, (C) two proteins which bind to beta-1,3-glucans and lipoteichoic acid, and (D) a peptidoglycan- binding protein related to bacteriophage lysozyme, a protein composed of leucine-rich repeats, which binds to lipopolysaccharaide. We have isolated cDNA clones for all of these proteins and expressed them as recombinant proteins in E. coli or baculovirus systems to assess their physiological functions in insect immunity. A protein homologous to the beta-1,3-glucan binding protein was purified and its cDNA cloned from the Indianmeal moth, Plodia interpunctella. This protein binds to beta-1,3-glucan in the cell wall of fungi. The Manduca and Plodia glucan binding proteins are composed of two domains. A carboxyl-terminal domain is similar in sequence to bacterial glucanases. The amino-terminal domain, which is similar only to other arthropod glucan-binding proteins, has been produced in a recombinant form. The isolated amino-terminal domain binds tightly to glucans. Circular dichroism spectroscopy indicates that it is composed primarily of alpha helices. Study of this domain by nuclear magnetic resonance spectroscopy has been initiated. Interaction of the immulectins or glucan-binding proteins with microbial cell surfaces triggers activation of the serine protease cascade that activates phenoloxidase. We identified more than twenty serine proteases that are present in Manduca hemolymph as well as four serine protease homologs, which lack proteolytic activity but are required as cofactors for prophenoxidase activation. cDNAs for these proteins have been cloned, and are being used to produce recombinant forms of the proteins, for investigating the protease cascade pathways which are activated upon recognition of microbial infection. Prophenoloxidase activation appears to result from formation of a protein complex, which assembles on a microbial surface. We have begun to use proteomics techniques to characterize the set of proteins taking part in this process.

Impacts
Biochemical characterization of the molecules involved in insect immune responses aids in understanding how such defenses are regulated and may in the future suggest new methods to interfere with or stimulate these responses. The immune system represents a potential new target for strategies to control populations of insect pests and for disrupting transfer of diseases from insects to humans, animals, and plants.

Publications

  • Barillas-Mury, C., Paskewitz, S., and Kanost, M.R. 2005. Immune responses of vectors. In The Biology of Disease Vectors (Marquardt, W.C., Black, W.C., Freier, J., Hagedorn, H., Hemingway, J., Higgs, S., James, A.A., Kondratieff, B., Moore, C.G. eds.) Elsevier/Academic Press. pp. 363-376.
  • Kanost, M.R. and Clarke, T. 2005. Proteases. In Comprehensive Molecular Insect Science (L.I. Gilbert, K. Iatrou, S. Gill eds) Elsevier.Vol. 4: pp. 247-266.
  • Kanost, M.R. 2005. Serpins in a Lepidopteran Insect, Manduca sexta. In The Serpinopathies: Molecular and Cellular Aspects of Serpins and their Disorders, (G,A. Silverman, ed.) World Scientific Publishing Co., In press.
  • Levin, D., Breuer, L.N., Zhuang, S., Anderson, S.A., Nardi, J.B. and Kanost, M.R. 2005. A hemocyte-specific integrin required for hemocytic encapsulation in the tobacco hornworm, Manduca sexta. Insect Biochem. Mol. Biol. 35: 369-380.
  • Tong, Y. and Kanost, M.R. 2005. Manduca sexta serpin-4 and serpin-5 inhibit the prophenoloxidase activation pathway. cDNA cloning, protein expression, and characterization. J. Biol. Chem. 280: 14923-14931
  • Tong, Y., Jiang, H. and Kanost, M.R. 2005. Identification of plasma proteases inhibited by Manduca sexta serpin-4 and serpin-5 and their association with components of the prophenol oxidase activation pathway. J. Biol. Chem. 280: 14932-14942.
  • Jiang, H., Wang, Y., Gu, Y., Guo, X., Zou, Z., Scholz, F., Trenczek, T.E. and Kanost, M.R. 2005. Molecular identification of a bevy of serine proteinases in Manduca sexta hemolymph. Insect Biochem. Molec. Biol. 35: 931-943.


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

Outputs
Objectives are to identify and characterize microbial carbohydrate-binding proteins and proteases in hemolymph of the tobacco hornworm, Manduca sexta, and the Indianmeal moth, Plodia interpunctella, that participate in responses to wounding and infection and to understand how these are regulated. Proteins from hemolymph of both species that bind to lipopolysaccharide from gram negative bacteria and to beta-1,3-glucans from fungal cell walls have been purified, and their cDNAs have been cloned. Interaction of these proteins with microbial cell surfaces triggers activation of the serine protease cascade that activates phenoloxidase. The amino-terminal domain of the glucan binding proteins, which is unique to these pattern recognition molecules from invertebrates, strongly binds to glucans such as laminarin and curdlan, and this binding stimulates activation of the proteases in the phenoloxidase pathway. We have produced this amino-terminal domain in quantity sufficient for structural analysis by NMR and have begun studies to determine the structure of this new type of domain. We have expressed Manduca hemolymph proteinases (HP) 1 and 2 and LPS-binding immulectin-2 as recombinant proteins in stably transformed Drosophila S2 cell lines and have developed procedures for purifying the recombinant proteins. The purified recombinant proteins will be used to investigate the mechanisms of proteinase activation in experiments to reconstitute a system in which interaction of microbial elicitors such as LPS with plasma proteins (immulectin-2 or glucan recognition protein) stimulate activation of a proteinase that triggers a protease cascade pathway. In other studies, we have established that RNA interference can be used to decrease expression of a gene for tryptophan oxygenase in embryos of Plodia (resulting in loss of eye pigment), indicating that this technique may also be useful for investigating function of immune genes in this species. We have also determined that infection in Manduca eggs can stimulate expression of genes for antimicrobial peptides and proteins, primarily in the yolk cells rather than in the embryo itself.

Impacts
Understanding interactions between insects and microorganisms is relevant to potential development of new methods for managing populations of insect pests and for disrupting transfer of diseases from insects to humans, animals, and plants. Detailed bichemical characterization of the molecules involved will aid in understanding how such defenses are regulated and may in the future suggest new methods to interfere with or stimulate these responses.

Publications

  • Gorman, M.J., Kankanala, P. And Kanost, M.R. 2004. Bacterial challenge stimulates innate immune responses in extra-embryonic tissues of tobacco hornworm eggs. Insect Mol. Biol. 13: 19-24.
  • Dittmer, N., Suderman, R., Jiang, H., Zhu, Y. Gorman, M., Kramer, K., and Kanost, M.R. 2004. Cloning and developmental expression of three cDNAs encoding putative laccases from the tobacco hornworm, Manduca sexta, and the malaria mosquito, Anopheles gambiae. Insect Biochem. Molec. Biol. 34: 29-41.
  • Jiang, H., Ma, C., Lu, Z., and Kanost, M.R. 2004. beta-1,3-Glucan recognition protein-2 (beta GRP-2) from Manduca sexta: an acute-phase protein that binds beta-1,3-glucan and lipoteichoic acid to aggregate fungi and bacteria. Insect Biochem. Molec. Biol. 34: 89-100.
  • Fabrick, J.A., Baker, J.E. and Kanost, M.R. 2004. Innate immunity in a pyralid moth: functional evaluation of domains from a beta-1,3-glucan recognition protein. J. Biol. Chem. 279: 26605-26611.
  • Yu, X.-Q. And Kanost, M.R. 2004. Immulectin-2, a pattern recognition receptor that stimulates hemocyte encapsulation and melanization in the tobacco hornworm, Manduca sexta. Dev. Comp. Immunol. 9: 891-900.
  • Fabrick, J.A., Kanost, M.R. and Baker, J.E. 2004. RNAi-induced silencing of embryonic tryptophan oxygenase in the pyralid moth, Plodia interpunctella. J. Insect Sci. 4:15, Available online: insectscience.org/4.15.
  • Kanost, M.R, Jiang, H., and Yu, X.-Q. 2004. Innate immune responses of a Lepidopteran insect, Manduca sexta. Immunological Reviews. 198: 97-105.


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

Outputs
Objectives are to identify and characterize microbial carbohydrate-binding proteins and proteases in hemolymph of the tobacco hornworm, Manduca sexta, and the Indianmeal moth, Plodia interpunctella, that participate in responses to wounding and infection and to understand how these are regulated. We have characterized proteins from hemolymph of both species that bind to beta-1,3-glucans from fungal cell walls and trigger activation of a serine protease cascade that results in activation of phenoloxidase. Phenoloxidase participates in synthesis of melanin in a protective response to infection. Experiments have demonstrated that the glucan-binding proteins are composed of two domains. The amino-terminal domain is unique to invertebrate glucan binding proteins and strongly binds to glucans such as laminarin and curdlan. Surface plasmon resonance experiments have demonstrated that the carboxyl-terminal glucanase-like domain also binds to laminarin, although more weakly than the amino terminal domain. The amino-terminal domain alone can bind to curdlan and stimulate activation of the proteases in the phenoloxidase pathway. We have used subtractive hybridization techniques and classical biochemical purification methods to identify and characterize proteases and protease inhibitors in hemolymph that participate in immune defense responses. We have characterized two additional proteases that can activate prophenoloxidase, serine protease homologs that act as cofactors in this reaction, and a new protease inhibitor from the serpin family that inhibits these proteases to regulate their activity. Interactions between the serine protease homologs and a C-type lectin that binds to bacterial lipopolysaccharide have been demonstrated, suggesting a model in which a protein complex assembles on the surface of pathogens to localize the melanization response to infection.

Impacts
Understanding interactions between insects and microorganisms is relevant to potential development of new methods for managing populations of insect pests and for disrupting transfer of diseases from insects to humans, animals, and plants. Detailed bichemical characterization of the molecules involved will aid in understanding how such defenses are regulated and may in the future suggest new methods to interfere with or stimulate these responses.

Publications

  • Yu, X.-Q., Jiang, H., Wang, Y., and Kanost, M.R. 2003. Nonproteolytic serine proteinase homologs involved in phenoloxidase activation in the tobacco hornworm, Manduca sexta. Insect Biochem. Molec. Biol. 33: 197-208.
  • Jiang, H., Wang, Y., Yu, X.-Q., and Kanost, M.R. 2003. Prophenoloxidase-activating proteinase-2 (PAP-2) from hemolymph of Manduca sexta: a bacteria-inducible serine proteinase containing two clip domains. J. Biol. Chem. 278: 3552-3561.
  • Ross, J., Jiang, H., Kanost, M.R., and Wang, Y. 2003. Serine proteinases and their homologs from the Drosophila melanogaster genome: an initial analysis of sequence conservation and phylogenetic relationship. Gene. 304:117-131.
  • Yu, X.-Q. and Kanost, M.R. 2003. Manduca sexta lipopolysaccharide-specific immulectin-2 protects larvae from bacterial infection. Dev. Comp. Immunol. 27: 189 - 196.
  • Zhu, Y., Johnson, T., Myers, A. and Kanost, M.R. 2003. Identification by subtractive suppression hybridization of bacteria-induced genes expressed in Manduca sexta fat body. Insect Biochem. Molec. Biol. 33, 541-559.
  • Suderman, R.J., Andersen S.O., Hopkins, T.L., Kanost, M.R. and Kramer, K.J. 2003. Characterization and cDNA cloning of three major proteins from pharate pupal cuticle of Manduca sexta. Insect Biochem. Mol. Biol. 33: 331-343.
  • Fabrick, J.A., Baker, J.E. and Kanost, M.R. 2003. Properties of a beta-1,3-glucan recognition protein from an insect, Plodia interpunctella. Insect Biochem. Molec. Biol. 33: 579 - 594.
  • Jiang, H., Wang, Y., Yu, X.-Q., Zhu, Y., and Kanost, M.R. 2003. Prophenoloxidase-activating proteinase-3 (PAP-3) from Manduca sexta hemolymph: a clip-domain serine proteinase regulated by serpin-1J and serine proteinase homologs. Insect Biochem. Molec. Biol. 33: 1049-1060.
  • Zhu, Y., Wang, Y., Gorman, M.J.. Jiang, H., and Kanost, M. R. 2003. Manduca sexta serpin-3 regulates prophenoloxidase activation in response to infection by inhibiting prophenoloxidase-activating proteinases. J. Biol. Chem. 278: 46,556-46,564.


Progress 01/01/02 to 12/31/02

Outputs
We aim to understand how infection of insects triggers immune responses. Our hypothesis is that proteins in hemolymph bind to microbial cell wall polysaccharides, and this interaction causes activation of serine proteases in hemolymph. Subsequent proteolytic processing of specific plasma proteins then produces signaling molecules, which stimulate biochemical and cellular protective responses. Objectives are to identify and characterize carbohydrate-binding proteins and proteases in hemolymph of the tobacco hornworm, Manduca sexta, and the Indianmeal moth, Plodia interpunctella, that participate in responses to wounding and infection and to understand how these are regulated. We have identified proteins in hemolymph of M. sexta, which bind to microbial polysaccharides. These include: four C-type lectins, named immulectins, which bind to the surface of bacteria and stimulate activation of the prophenoloxidase pathway; hemolin, a protein composed of four immunoglobulin domains, which binds to bacterial lipopolysaccharide and lipoteichoic acid; two proteins related to bacterial glucanases, which bind to beta-1,3-glucans and lipoteichoic acid; and a peptidoglycan-binding protein. New studies have focused on carbohydrate recognition domain-2 (CRD2) of M. sexta immulectin-2 and the biological functions of IML-2 in immune responses. Binding of recombinant CRD2 to LPS stimulated activation of phenoloxidase in plasma. Injection of antiserum against immulectin-2 into M. sexta larvae inhibited clearance of a Gram-negative bacterial pathogen and decreased survival of infection. This suggests that immulectin-2 plays a major role in the immune system of M. sexta, and helps to protect them from Gram-negative bacterial infections. We cloned cDNAs for two serine proteinase homologs (SPHs) from M. sexta hemolymph that interact with phenoloxidase activating proteases (PAP). The SPHs bind to IML-2. PAP alone cannot efficiently activate prophenoloxidase, but a mixture of SPHs and PAP is highly active for prophenoloxidase activation. IML-2, prophenoloxidase, and PAP in hemolymph bound to the recombinant proteinase-like domain of SPH-1, indicating that a protein complex containing these proteins may exist in hemolymph. Since IML-2 binds to surface carbohydrates on pathogens, such a protein complex may localize activation of phenoloxidase on the surface of pathogens. A beta-1,3-glucan-binding protein has been purified and its cDNA cloned from P. interpunctella. This protein binds to the surface of fungi and bacteria. We expressed the full-length protein and its two domains separately as recombinant proteins and studied their binding to isolated beta-1,3-glucan, a component of fungal cell walls. Both domains bind to soluble glucan, but only the amino-terminal domain binds to insoluble glucan. The amino-terminal domain alone stimulates activation of prophenoloxidase in response to beta-1,3 glucan. New research will determine how interaction of the carbohydrate-binding domain stimulates activation of serine proteases that function in immune responses.

Impacts
Insects respond to infections with protective responses, but the molecular mechanisms by which they detect microbial invasion have been unclear. We are beginning to characterize insect proteins which bind specifically to polysaccharides in bacterial and fungal cell walls and that are involved triggering antimicrobial responses. Understanding of the biochemistry of these proteins may lead to new strategies for microbial control of insect pests.

Publications

  • Yu, X.-Q. and Kanost, M.R. 2002. Manduca sexta lipopolysaccharide-specific immulectin-2 protects larvae from bacterial infection. Dev. Comp. Immunol. In press.
  • Yu,X.-Q., Zhu, Y.-F., Ma, C, Fabrick, J.A. and Kanost, M.R. 2002. Pattern recognition proteins in Manduca sexta plasma. Insect Biochem. Molec. Biol. 32: 1287-1293.
  • Yu, X.-Q. and Kanost, M.R. 2002. Binding of hemolin to microbial lipopolysaccharide and lipoteichoic acid: an immunoglobulin superfamily member from insects as a pattern recognition receptor. Eur. J. Biochem. 269: 1827-1834.
  • Ross, J., Jiang, H., Kanost, M.R., and Wang, Y. 2002. Serine proteinases and their homologs from the Drosophila melanogaster genome: an initial analysis of sequence conservation and phylogenetic relationship. Gene. In Press.
  • Yu, X.-Q., Jiang, H., Wang, Y., and Kanost, M.R. 2002. Nonproteolytic serine proteinase homologs involved in phenoloxidase activation in the tobacco hornworm, Manduca sexta. Insect Biochem. Molec. Biol. In Press.
  • Jiang, H., Wang, Y., Yu, X.-Q., and Kanost, M.R. 2002. Prophenoloxidase-activating proteinase-2 (PAP-2) from hemolymph of Manduca sexta: a bacteria-inducible serine proteinase containing two clip domains. J. Biol. Chem. In Press.


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

Outputs
Our goal is to understand how infection of insects by microorganisms triggers pro-phenol oxidase activation or produces a signal that is transmitted to fat body cells and hemocytes to stimulate their responses. Our hypothesis is that binding of polysaccharides from microbial cell walls by proteins in hemolymph is a mechanism for recognition of infection that results in activation of serine proteases in insect hemolymph; proteolytic processing of specific plasma proteins then produces signaling molecules, which stimulate biochemical and cellular protective responses. One such response is the activation of proteases which lead to activation of hemolymph phenoloxidase and the subsequent production of melanin that encapsulates parasites and coats wound sites. Our objectives are to identify and characterize carbohydrate-binding proteins and proteases in hemolymph of the tobacco hornworm, Manduca sexta and the Indianmeal moth, Plodia interpunctella, that participate in responses to wounding and infection and to gain an understanding of how the proteases are activated and regulated. We have identified four types of proteins in hemolymph of M. sexta, which bind to microbial polysaccharides. These include (A) four C-type lectins, named immulectins, which bind to the surface of bacteria and stimulate activation of the prophenoloxidase pathway, (B) hemolin, a protein composed of four immunoglobulin domains, which binds to bacterial lipopolysaccharide and lipoteichoic acid, (C) two proteins related to bacterial glucanases, which bind to beta-1,3-glucans and lipoteichoic acid, and (D) a peptidoglycan- binding protein related to bacteriophage lysozyme. We have isolated cDNA clones for all of these proteins and expressed them as recombinant proteins in E. coli or baculovirus systems. These proteins will be used to further assess their physiological functions in insect immunity. A protein homologous to the beta-1,3-glucan protein has been purified and its cDNA cloned from the Indianmeal moth, Plodia interpunctella. This protein bind to the surface of fungi and bacteria. It binds very tightly to isolated beta-1,3-glucan, a component of fungal cell walls. The protein is composed of two domains. A carboxyl-terminal domain is similar in sequence to bacterial glucanases. The amino-terminal domain, which is similar only to other arthropod glucan-binding proteins, has been produced in a recombinant form. The isolated amino-terminal domain binds tightly to glucans. Circular dichroism spectroscopy indicates that it is composed primarily of alpha helices. Future research will map the carbohydrate-binding site of this protein and test its function in immunity to fungal infection.

Impacts
Insects respond to infections with protective responses, but the molecular mechanisms by which they detect microbial invasion have been unclear. We are beginning to characterize insect proteins which bind specifically to polysaccharides in bacterial and fungal cell walls and that are involved triggering antimicrobial responses. Understanding of the biochemistry of these proteins may lead to new strategies for microbial control of insect pests.

Publications

  • Kanost, M. R., Jiang, H., Wang, Y., Yu, X., Ma, C. C., and Zhu, Y. 2001. Hemolymph proteinases in immune responses of Manduca sexta. Adv. Exp. Med. Biol. 484:319-328.
  • Yu, X., and Kanost, M. R. 2001. A family of C-type lectins in Manduca sexta. Adv. Exp. Med. Biol. 484:191-194.
  • Ma, C. and Kanost, M. R. 2001. A beta-1,3-glucan-binding protein from Manduca sexta. Adv. Exp. Med. Biol. 484:309-312.
  • Yu, X., Zhu, Y., Ma, C., Fabrick, J. A. and Kanost, M. R. 2001. Pattern recognition proteins in Manduca sexta plasma. Insect Biochem. Molec. Biol. In press.


Progress 01/01/00 to 12/31/00

Outputs
Innate immune responses of insects have many apparent similarities to those of mammals. Our goal is to understand how recognition of microorganisms triggers pro-phenol oxidase activation or produces a signal that is transmitted to fat body cells and hemocytes to stimulate their responses. Our hypothesis is that binding of polysaccharides from microbial cell walls by proteins in hemolymph is a mechanism for recognition of infection that results in activation of serine proteases in insect hemolymph; proteolytic processing of specific plasma proteins then produces signaling molecules, which stimulate biochemical and cellular protective responses. One such response is the activation of proteases which lead to activation of hemolymph phenoloxidase and the subsequent production of melanin that encapsulates parasites and coats wound sites. Our objectives are to identify and characterize carbohydrate-binding proteins and proteases in hemolymph of the tobacco hornworm, Manduca sexta, that participate in responses to wounding and infection and to gain an understanding of how the proteases are activated and regulated. A family of four C-type lectins in Manduca hemolymph has been characterized. These proteins, named immulectins, bind to the surface of bacteria and stimulate activation of the prophenoloxidase pathway. We have purified four immulectins, cloned cDNAs which encode them, and expressed immulectins 1 and 2 as recombinant proteins. Recent studies have demonstrated that immulectins bind to lipopolysaccharides from gram-negative bacteria. Each immulectin is composed of two carbohydrate-recognition domains. Current studies involve expression and characterization of individual domains from the immulectins and experiments to clarify their roles in immune responses. Two related beta-1,3-glucan binding proteins have been purified from Manduca hemolymph, and their cDNAs have been cloned. A homologous protein has been purified and its cDNA cloned from the Indianmeal moth, Plodia interpunctella. These proteins bind to the surface of fungi and trigger activation of the prophenoloxidase pathway. We have identified two types of proteins in insect hemolymph, which function in recognition of microbial infection. The C-type lectins recognize Gram-negative bacterial infections, and the beta-1,3-glucan-binding proteins recognize fungal infection. Both types of proteins activate the prophenoloxidase pathway, and they may also trigger other types of protective responses. U.S. provisional patent application: Lipopolysaccharide-specific C-type lectin and methods for its use, filed 7/12/00. Docket No. P30562.

Impacts
Insects respond to infections with protective responses, but the molecular mechanisms by which they detect microbial invasion have been unclear. We are beginning to characterize insect proteins which bind specifically to polysaccharides in bacterial and fungal cell walls and that are involved triggering antimicrobial responses. Understanding of the biochemistry of these proteins may lead to new strategies for microbial control of insect pests.

Publications

  • Jiang, H. and Kanost, M. R.. 2000. The clip-domain family of serine proteinases in arthropods. Insect Biochem. Molec. Biol. 30: 95-105.
  • Ma, C. and Kanost, M. R.. 2000. A beta,1-3-glucan-recognition protein from an insect, Manduca sexta, agglutinates microorganisms and activates the phenoloxidase cascade. J. Biol. Chem. 275: 7505-7514.
  • Yu, X. Q. and Kanost, M. R. 2000. Immulectin-2, a lipopolysaccharide-specific lectin from an insect, Manduca sexta, is induced in response to Gram-negative bacteria. J. Biol. Chem. 275: 37373-37381.


Progress 10/01/99 to 12/31/99

Outputs
Infection in insects provokes defensive responses by hemocytes, synthesis of antimicrobial proteins by fat body, and activation of pro-phenol oxidase. Our goal is to understand how recognition of microorganisms triggers pro-phenol oxidase activation or produces a signal that is transmitted to fat body cells and hemocytes to stimulate their responses. Our hypothesis is that binding of polysaccharides from microbial cell walls by lectins in hemolymph is a mechanism for recognition of infection that results in activation of serine proteases in insect hemolymph; proteolytic processing of specific plasma proteins then produces signaling molecules, which stimulate biochemical and cellular protective responses. Parasites and pathogens adapted for using specific insects as hosts might have evolved mechanisms to avoid activating such protease response systems. Activation of proteases also leads to activation of hemolymph phenoloxidase and the subsequent production of melanin that encapsulates parasites and coats wound sites. Our objectives are to identify and characterize lectins and proteases in hemolymph of Manduca sexta that participate in responses to wounding and infection and to gain an understanding of how the proteases are activated and regulated. We have identified a family of C-type lectins in Manduca hemolymph. These proteins bind to the surface of bacteria and stimulate activation of the prophenoloxidase pathway. We have purified four such proteins and cloned cDNAs which encode them. Their synthesis is induced upon bacterial infection. We have also purified and cloned a cDNA for a beta-1,3-glucan binding protein from Manduca hemolymph. This protein binds to the surface of fungi. The interaction of the beta-1,3-glucan binding protein with fungal cells walls also triggers activation of the prophenoloxidase pathway. Thus, we have identified two types of proteins in insect hemolymph, which function in recognition of microbial infection.

Impacts
Insects respond to infections with protective responses, but the molecular mechanisms by which they detect microbial invasion have been unclear. We are beginning to characterize insect proteins which bind specifically to polysaccharides in bacterial and fungal cell walls and that are involved triggering antimicrobial responses. Understanding of the biochemistry of these proteins may lead to new strategies for microbial control of insect pests.

Publications

  • Yu, X.-Q., Gan, H., and Kanost, M.R.. 1999. An inducible C-type lectin from an insect, Manduca sexta, stimulates activation of plasma prophenol oxidase. Insect Bioch. Molec. Biol. 29: 585-597.
  • Yu, X.-Q. and Kanost, M.R. 1999. Developmental expression of Manduca sexta hemolin. Arch. Insect. Biochem. Physiol. 42: 198-212.
  • Kanost, M.R., Jiang, H., Wang, Y., Yu, X.-Q., Ma, C.C., and Zhu, Y. 1999. Hemolymph proteinases in immune responses of Manduca sexta. In "Phylogenetic Perspectives on the Vertebrate Immune System." (G. Beck and M. Sugumaran, eds.) Plenum, New York, in press.
  • Yu, X-Q. and Kanost, M.R. 1999. A family of C-type lectins in Manduca sexta. In "Phylogenetic Perspectives on the Vertebrate Immune System." (G. Beck and M. Sugumaran, eds.) Plenum, New York, in press.
  • Ma, C. and Kanost, M.R. 1999. cDNA cloning and characterization of a beta-1,3-glucan-recognition protein from the tobacco hornworm, Manduca sexta. In "Phylogenetic Perspectives on the Vertebrate Immune System." (G. Beck and M. Sugumaran, eds.) Plenum, New York, in press.
  • Ma, C. and Kanost, M.R. 1999. A beta,1-3-glucan-recognition protein from an insect, Manduca sexta, agglutinates microorganisms and activates the phenoloxidase cascade. J. Biol. Chem. In press.