Functional and molecular characterization of the serine protease homologue CLIPA28 in the melanization response of Anopheles gambiae mosquitoes

Abstract

Anopheles gambiae mosquitoes are the main vectors of the human malaria parasite Plasmodium falciparum in sub-Saharan Africa. The innate immune system of mosquitoes is a major determinant of their vectorial capacity. Catalytic clip domain serine proteases (cSP) and their non-catalytic homologs (referred to as clip-domain serine protease homologs, cSPH) are key components of extracellular enzymatic cascades that control diverse immune responses in insects including melanization, antimicrobial peptide synthesis, and complement-mediated responses. Despite being non-catalytic, the activation of cSPHs, requires their proteolytic cleavage, yet factors that control their activation and the complexity of their interactions within these cascades remain unclear. Here, we describe the functional and molecular characterization of a novel cSPH (CLIPA28), identified based on its co-regulated expression profile with CLIPA2, a key negative regulator of mosquito complement and melanization immune responses, in a transcriptomic database using Pearson correlation analysis. We show that CLIPA28 is required for the mosquito melanization response. Its knockdown (kd) abolished Plasmodium berghei ookinete melanization in refractory mosquito genotypes and significantly reduced hemolymph phenoloxidase activity after systemic bacterial infections. CLIPA28 kd didn’t affect mosquito tolerance nor resistance to bacterial infections, but significantly compromised both in response to fungal challenge, which supports our previous published observations that melanization is required for anti-fungal defense. Biochemical analysis revealed that CLIPA28 is rapidly cleaved in the hemolymph following systemic infections. Using an RNAi-mediated gene silencing and western blot analysis, we show that the mosquito complement-like protein TEP1 and two other cSPs known to be required for melanization, SPCLIP1 and CLIPA8, are required factors for CLIPA28 cleavage, and that these three positive regulatory cSPHs are activated in a hierarchically ordered