An extracellular serine protease network coordinates key mechanisms of insect defense against microbial infection. Proteolytic activation of prophenoloxidase (PPO) generates phenoloxidase that catalyzes the production of reactive compounds to sequester and kill invading pathogens and parasites. Proteolytic processing of sp?tzle precursor triggers the Toll pathway, which induces the synthesis of antimicrobial peptides and other defense proteins. In insect vectors of human diseases, initiation and regulation of these protease systems may be disrupted by proteins from the intruders. Knowledge of such protein interactions from biochemical model insects such as Manduca sexta will be useful for understanding and manipulating similar systems in arthropod vectors to interfere with disease transmission. We have discovered a branch of the M. sexta protease network that detects Gram-positive bacteria and fungi and mediates PPO activation. In this network, pathogen recognition receptors bind to microbes and initiate protease pathways for PPO and Toll activation. We have identified a new Sushi-domain protease that may participate in a branch of the pathway responsive to Gram-negative bacterial infection. A positive feedback loop in the protease system controls locality and potency of the defense responses. We plan to extend our research on the PPO activation reaction by examining the initiation and regulation of immune protease pathways.
The specific aims of this project are: 1) Characterization of the pathway initiation by Gram-negative bacteria;2) Elucidation of the positive regulatory mechanisms in the protease system;3) Investigation of the structure, function, and activation of M. sexta PPO.

Public Health Relevance

This research will continue to elucidate the function and regulation of a serine protease network during innate immune responses, which produces phenoloxidase and antimicrobial peptides to entrap and kill parasites that cause malaria and filariasis. The acquired knowledge will be useful for disrupting the transmission of human diseases in vector species.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Vector Biology Study Section (VB)
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Gindhart, Joseph G
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Oklahoma State University Stillwater
Other Basic Sciences
Schools of Earth Sciences/Natur
United States
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Zhang, Xiufeng; Zheng, Yun; Cao, Xiaolong et al. (2015) Identification and profiling of Manduca sexta microRNAs and their possible roles in regulating specific transcripts in fat body, hemocytes, and midgut. Insect Biochem Mol Biol 62:22-Nov
Wang, Yang; Lu, Zhiqiang; Jiang, Haobo (2014) Manduca sexta proprophenoloxidase activating proteinase-3 (PAP3) stimulates melanization by activating proPAP3, proSPHs, and proPOs. Insect Biochem Mol Biol 50:82-91
Zhang, Xiufeng; Zheng, Yun; Jagadeeswaran, Guru et al. (2014) Identification of conserved and novel microRNAs in Manduca sexta and their possible roles in the expression regulation of immunity-related genes. Insect Biochem Mol Biol 47:12-22
Zhang, Shuguang; Cao, Xiaolong; He, Yan et al. (2014) Semi-quantitative analysis of changes in the plasma peptidome of Manduca sexta larvae and their correlation with the transcriptome variations upon immune challenge. Insect Biochem Mol Biol 47:46-54
Gunaratna, Ramesh T; Jiang, Haobo (2013) A comprehensive analysis of the Manduca sexta immunotranscriptome. Dev Comp Immunol 39:388-98
Zhao, Picheng; Wang, Yang; Jiang, Haobo (2013) Biochemical properties, expression profiles, and tissue localization of orthologous acetylcholinesterase-2 in the mosquito, Anopheles gambiae. Insect Biochem Mol Biol 43:260-71
Zhang, Xiufeng; Zheng, Yun; Jagadeeswaran, Guru et al. (2012) Identification and developmental profiling of conserved and novel microRNAs in Manduca sexta. Insect Biochem Mol Biol 42:381-95
Wang, Yang; Sumathipala, Niranji; Rayaprolu, Subrahmanyam et al. (2011) Recognition of microbial molecular patterns and stimulation of prophenoloxidase activation by a ?-1,3-glucanase-related protein in Manduca sexta larval plasma. Insect Biochem Mol Biol 41:322-31
Zhao, Picheng; Lu, Zhiqiang; Strand, Michael R et al. (2011) Antiviral, anti-parasitic, and cytotoxic effects of 5,6-dihydroxyindole (DHI), a reactive compound generated by phenoloxidase during insect immune response. Insect Biochem Mol Biol 41:645-52
An, Chunju; Jiang, Haobo; Kanost, Michael R (2010) Proteolytic activation and function of the cytokine Spatzle in the innate immune response of a lepidopteran insect, Manduca sexta. FEBS J 277:148-62

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