The CLIP-domain serine protease CLIPC9 regulates melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vectorAnopheles gambiae

Abstract

The arthropod melanization immune response is activated by extracellular protease cascades predominantly comprised of CLIP-domain serine proteases (CLIP-SPs) and serine protease homologs (CLIP-SPHs). In the malaria vector, Anopheles gambiae, the CLIP-SPHs SPCLIP1, CLIPA8, and CLIPA28 form the core of a hierarchical cascade downstream of mosquito complement that is required for melanization. However, our understanding of the regulatory relationship of the CLIP-SPH cascade with the catalytic CLIP-SPs driving melanization is incomplete. Here, we report on the development of a novel screen to identify melanization pathway components based on the quantitation of infection-induced excreta, eliminating the need for microdissections or hemolymph enzymatic assays. Using this screen, we identified CLIPC9 and subsequent functional analyses established that this protease is essential for the melanization of both Escherichia coli and the rodent malaria parasite Plasmodium berghei. Mechanistically, septic infection with E. coli promotes CLIPC9 cleavage and both full-length and cleaved CLIPC9 localize to this bacterium in a CLIPA8-dependent manner. The steady state level of CLIPC9 in the hemolymph is regulated by thioester-containing protein 1 (TEP1), suggesting it functions downstream of mosquito complement. In support, CLIPC9 cleavage is inhibited following SPCLIP1, CLIPA8, and CLIPA28 knockdown positioning it downstream of the CLIP-SPH cascade. Moreover, like CLIPA8 and CLIPA28, CLIPC9 processing is negatively regulated by serine protease inhibitor 2 (SRPN2). This report demonstrates how our novel excretion-based approach can be utilized to dissect the complex protease networks regulating mosquito melanization. Collectively, our findings establish that CLIPC9 is required for An. gambiae melanization and shed light on how the CLIP-SPH cascade regulates this potent immune response.

Transcriptome Profiles of Anopheles Gambiae Harboring Natural Low-Level Plasmodium Infection Reveal Adaptive Advantages For The Mosquito

“…Serine proteases constitute the majority of mosquito immune system moderators that are activated in response to pathogen recognition (Kumar et al, 2018). While there are hundreds of serine proteases functioning in a variety of mosquito molecular processes, eight CLIP-domain serine proteases (CLIPs)-SPCLIP1, CLIPA2, CLIPA7, CLIPA8, CLIPA14, CLIPA28, CLIPB14, and CLIPB15mediate P. berghei melanization (Nakhleh et al, 2017;Kumar et al, 2018;Sousa et al, 2020). No AgCLIP transcripts were significantly more abundant in heads of sporozoite-infected mosquitoes, although eight were less abundant in treatment groups for which mRNAs of AgCLIPA7 (-1.34-fold: padj = <0.0001; AGAP011792), AgCLIPA14 (-1.2-fold; p-adj = <0.0001; AGAP011788) and AgCLIPA28 (-0.92-fold: p-adj = 0.02; AGAP010968) were significantly under-represented relative to uninfected controls (Figure 10C).…”