Malaria control and eradication strategies continue to primarily rely on vector control that is stymied by insecticide resistance in vector populations. In addition to resistance management, novel insecticides with new modes of action - particularly for which efficacy is not affected by existing resistance mechanisms - are required. The long-term goal is to develop a malaria vector control methodology based on a new insecticide target, the serine protease inhibitor serpin (SRPN)2 from the African malaria mosquito, Anopheles gambiae s.s.. SRPN2 is a key negative regulator in the extracellular proteinase cascade that controls activation of prophenoloxidase (proPO) and thus melanization - a powerful, arthropod-specific innate immune response. SRPN2 depletion from the hemolymph of adult female mosquitoes significantly reduces longevity with escalating daily mortality nine days after treatment. The objectives in this application are to identify the interactions of SRPN2 with its proteinase targets and to evaluate the malaria control potential of a SRPN2 inhibitor. The rationale for the proposed research is that detailed information on SRPN2's biological proteinase targets and mode of action will ultimately allow the design of small molecule inhibitors of SRPN2 that could act as an insecticide for mosquito vector control. Guided by our preliminary data, the following three specific aims will be pursued: (1) Determine the molecular targets of SRPN2 inhibitory function;(2) Determine the molecular interface between SRPN2 and its target proteinase(s);and (3) Model the impact of SRPN2 depletion on disease transmission. Under the first aim, a combination of biochemical and genetic approaches that have been used successfully in the applicant's laboratory will be used to identify serine proteinase targets of SRPN2 based on their ability to (a) form covalent complexes with the serpin and (b) revert the serpin's depletion phenotype. Under the second aim, the molecular interactions between SRPN2 and one of its target proteinases, CLIPB9, will be identified by protein crystallography and mutational analysis. Under the third aim, the potential effect of SRPN2 inhibition on malaria transmission and resistance development against a potential SRPN2 inhibitor will be assessed by mathematical modeling. The proposed research is innovative, as it will for the first time evaluate the mosquito immune system as a physiological target for novel insecticides. Additionally, this project will use microfluidics as a highly innovative approach to the study of mosquito innate immunity, which if successful will be transformative to the field of insect biochemistry. This project is significant as it will provide fundamental knowledge of the nature of serine proteinase cascades that regulate melanization in mosquitoes. Ultimately, it has the potential to advance the development of new a generation of insecticide targets for malaria control.

Public Health Relevance

Vector-borne diseases, especially malaria, continue to be a major public health threat world-wide, with roughly half of the world population at risk of malaria. The proposed project is relevant to public health because the identification of novel insecticide targets in mosquitoes will allow the development of new vector control measures for malaria control. Therefore, the proposed project is relevant to the NIH-NIAID's mission in relation to the understanding and prevention of re-emerging infectious diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI095842-01
Application #
8163820
Study Section
Vector Biology Study Section (VB)
Program Officer
Costero, Adriana
Project Start
2011-05-01
Project End
2015-04-30
Budget Start
2011-05-01
Budget End
2012-04-30
Support Year
1
Fiscal Year
2011
Total Cost
$381,807
Indirect Cost
Name
Kansas State University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
929773554
City
Manhattan
State
KS
Country
United States
Zip Code
66506
Rhodes, Victoria L; Thomas, Matthew B; Michel, Kristin (2018) The interplay between dose and immune system activation determines fungal infection outcome in the African malaria mosquito, Anopheles gambiae. Dev Comp Immunol 85:125-133
Mills, M K; Nayduch, D; McVey, D S et al. (2017) Functional Validation of Apoptosis Genes IAP1 and DRONC in Midgut Tissue of the Biting Midge Culicoides sonorensis (Diptera: Ceratopogonidae) by RNAi. J Med Entomol 54:559-567
Meekins, David A; Kanost, Michael R; Michel, Kristin (2017) Serpins in arthropod biology. Semin Cell Dev Biol 62:105-119
Zhang, Xin; An, Chunju; Sprigg, KaraJo et al. (2016) CLIPB8 is part of the prophenoloxidase activation system in Anopheles gambiae mosquitoes. Insect Biochem Mol Biol 71:106-15
Bryant, William B; Michel, Kristin (2016) Anopheles gambiae hemocytes exhibit transient states of activation. Dev Comp Immunol 55:119-29
Regna, Kimberly; Harrison, Rachel M; Heyse, Shannon A et al. (2016) RNAi Trigger Delivery into Anopheles gambiae Pupae. J Vis Exp :
Meekins, David A; Zhang, Xin; Battaile, Kevin P et al. (2016) 1.45?Å resolution structure of SRPN18 from the malaria vector Anopheles gambiae. Acta Crystallogr F Struct Biol Commun 72:853-862
Zhang, Xin; Meekins, David A; An, Chunju et al. (2015) Structural and inhibitory effects of hinge loop mutagenesis in serpin-2 from the malaria vector Anopheles gambiae. J Biol Chem 290:2946-56
Mills, M K; Nayduch, D; Michel, K (2015) Inducing RNA interference in the arbovirus vector, Culicoides sonorensis. Insect Mol Biol 24:105-14
Zhang, Xin; Mysore, Keshava; Flannery, Ellen et al. (2015) Chitosan/interfering RNA nanoparticle mediated gene silencing in disease vector mosquito larvae. J Vis Exp :

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