Extracellular serine proteases and their non-catalytic homologs coordinate key defense mechanisms in insects. This includes responses of mosquitoes against pathogens and parasites that cause serious human diseases. Proteolytic cleavage generates active phenoloxidases (POs), thioester proteins (TEPs), Sptzle and other cytokines. PO catalyzes the production of reactive chemicals to sequester and kill the invading organisms. TEPs opsonize parasites, targeting them for destruction. Sptzle and other cytokines trigger intracellular signaling pathways to induce the expression of antimicrobial peptides and other defense proteins. In insect vectors of human diseases, the protease networks may be evaded or disrupted by proteins from the intruders. Knowledge of the system components and their interactions from biochemical model insects such as Manduca sexta is useful for gaining detailed molecular understanding of fundamental aspects of insect innate immunity, including these protease cascades and provides basic knowledge that can guide studies of similar systems in insect disease vectors. We have elucidated a part of the M. sexta protease network that activates proPO in response to bacteria and fungi. In this network, recognition proteins bind to microbes and activate the proteases in a cascade mode. We have annotated 193 genes encoding serine proteases and their homologs, identified 36 of the proteins in larval hemolymph, and acquired data indicating that some of them play critical roles in immune signal transduction. Based on the molecular probes, purified proteins and working experience, we propose to investigate two critical steps of the protease network, which are conserved in dipteran species, by combining hemolymph fractionation, recombinant protein production and processing, sequence and expression information, and state-of-the-art mass spectrometry.
The specific aims of this project are to: 1) characterize the system initiation by examining interactions of peptidoglycans, two recognition proteins, and hemolymph protease-14 precursor (proHP14) at the domain level; 2) identify 1?2 proHP6-activating proteases and elucidate their activation mechanisms. New knowledge gained in this project will improve understanding of insect immunity at a biochemical level and stimulate related research in vector species.

Public Health Relevance

This research will further elucidate the constitution, function and regulation of an extracellular network of extracellular serine proteases and their homologs in a model insect, which mediates immune responses by producing active phenoloxidases, cytokines and antimicrobial peptides to kill pathogens that cause serious human diseases such as malaria. The acquired knowledge will be useful for understanding similar systems and disrupting parasite transmission in vector species.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058634-16
Application #
9787522
Study Section
Vector Biology Study Section (VB)
Program Officer
Barski, Oleg
Project Start
1999-03-01
Project End
2022-08-31
Budget Start
2019-09-01
Budget End
2020-08-31
Support Year
16
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Oklahoma State University Stillwater
Department
Other Basic Sciences
Type
Earth Sciences/Resources
DUNS #
049987720
City
Stillwater
State
OK
Country
United States
Zip Code
74078
He, Yan; Wang, Yang; Hu, Yingxia et al. (2018) Manduca sexta hemolymph protease-2 (HP2) activated by HP14 generates prophenoloxidase-activating protease-2 (PAP2) in wandering larvae and pupae. Insect Biochem Mol Biol 101:57-65
Cao, Xiaolong; Jiang, Haobo (2018) Building a platform for predicting functions of serine protease-related proteins in Drosophila melanogaster and other insects. Insect Biochem Mol Biol 103:53-69
Yang, Fan; Wang, Yang; Sumathipala, Niranji et al. (2018) Manduca sexta serpin-12 controls the prophenoloxidase activation system in larval hemolymph. Insect Biochem Mol Biol 99:27-36
Feng, Congjing; Zhao, Ya; Chen, Kangkang et al. (2018) Clip domain prophenoloxidase activating protease is required for Ostrinia furnacalis Guenée to defend against bacterial infection. Dev Comp Immunol 87:204-215
Li, Miao; Christen, Jayne M; Dittmer, Neal T et al. (2018) The Manduca sexta serpinome: Analysis of serpin genes and proteins in the tobacco hornworm. Insect Biochem Mol Biol 102:21-30
Wang, Yang; Jiang, Haobo (2017) Prophenoloxidase activation and antimicrobial peptide expression induced by the recombinant microbe binding protein of Manduca sexta. Insect Biochem Mol Biol 83:35-43
Schrag, Lynn G; Cao, Xiaolong; Herrera, Alvaro I et al. (2017) Solution Structure and Expression Profile of an Insect Cytokine: Manduca sexta Stress Response Peptide-2. Protein Pept Lett 24:3-11
He, Yan; Wang, Yang; Yang, Fan et al. (2017) Manduca sexta hemolymph protease-1, activated by an unconventional non-proteolytic mechanism, mediates immune responses. Insect Biochem Mol Biol 84:23-31
Cao, Xiaolong; Gulati, Mansi; Jiang, Haobo (2017) Serine protease-related proteins in the malaria mosquito, Anopheles gambiae. Insect Biochem Mol Biol 88:48-62
He, Xuesong; Cao, Xiaolong; He, Yan et al. (2017) Hemolymph proteins of Anopheles gambiae larvae infected by Escherichia coli. Dev Comp Immunol 74:110-124

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