Phenoloxidases take part in key insect physiological processes including pigmentation, cuticle tanning, wound healing, and defense responses. Some mosquito phenoloxidases generate active chemicals and eumelanin to kill and sequester malaria parasites. While reverse genetic analyses have revealed components of this enzyme system, important knowledge gaps still exist due to lack of biochemical analysis. In studies of the Manduca sexta prophenoloxidase (PPO) activation system, we accumulated wealthy knowledge on PPOs, PPO activating proteases, and their regulators. We cloned cDNAs of all nine Anopheles gambiae PPOs, expressed them as functional proteins in E. coli, crystallized three, and solved one PPO structure. Initial kinetic analysis revealed striking differences in substrate preference and catalytic efficiency. We validated the proteomic approach to semi-quantify PPOs and other proteins in the mosquito and detected PPOs in tissues using polyclonal antibodies. By injecting into female adults dsRNA encoding a conserved region in PPOs, we substantially reduced their mRNA and protein levels. Based on these advances, we propose to study the structures, functions, and expression of A. gambiae PPO1-9. Acquired knowledge and reagents will be useful for exploring how parasites evade or suppress the host immune mechanism in the future.
Mosquitoes transmit pathogens that kill approximately half a million people each year. The transmission success is in part determined by the insect immune system. This project aims at elucidating structures, functions, and expression of prophenoloxidase-1 through 9 in a major vector species, which participate in melanotic encapsulation of parasites and nematodes. Multiple phenoloxidases with different expression profiles and biochemical properties generate reactive compounds to kill and entrap the pathogens and to perform other physiological functions. Acquired knowledge and mutated genes have potentials to be applied for disrupting human disease transmission in mosquitoes.
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