The CLIP-domain serine protease CLIPC9 regulates melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vector Anopheles 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 microbial 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 melanotic mosquito 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 microbial melanization in An. gambiae and shed light on how the CLIP-SPH cascade regulates this potent immune response.

The Route of Infection Influences the Contribution of Key Immunity Genes to Antibacterial Defense in <b><i>Anopheles gambiae</i></b>

“…CLIPs are key components of serine protease cascades that regulate important insect immune responses specifically melanization and Toll pathway activation [3, 65, 66]. Among the enriched CLIPs, CLIPB4 and CLIPC9, both catalytic clips, are involved in the melanization of P. berghei ookinetes in refractory mosquito backgrounds [67, 68], while CLIPA8 and CLIPA7 are noncatalytic CLIPs that act as positive and negative regulators of Plasmodium melanization, respectively [67]. The melanization response to fungal infections requires CLIPA8 [69], while both CLIPA8 and CLIPC9 play an essential role in the melanization response to bacterial infections [68, 70, 71].…”

“…Among the enriched CLIPs, CLIPB4 and CLIPC9, both catalytic clips, are involved in the melanization of P. berghei ookinetes in refractory mosquito backgrounds [67, 68], while CLIPA8 and CLIPA7 are noncatalytic CLIPs that act as positive and negative regulators of Plasmodium melanization, respectively [67]. The melanization response to fungal infections requires CLIPA8 [69], while both CLIPA8 and CLIPC9 play an essential role in the melanization response to bacterial infections [68, 70, 71]. CLIPA1, CLIPA4, CLIPA6, and CLIPB1 do not seem to be involved in Plasmodium melanization [67], whereas the roles of CLIPC4 and CLIPB13 in the melanization response remain to be elucidated.…”

CLIPB10 is a Terminal Protease in the Regulatory Network That Controls Melanization in the African Malaria Mosquito Anopheles gambiae

“…gambiae , and a potential activator of CLIPB10 during infection-induced melanization. The data provided by Sousa and co-authors also suggest that CLIPC9 undergoes localized activation cleavage, binding to microbial surfaces in a cleaved form ( Sousa et al, 2020 ). The cleavage of CLIPC9 depends on components of the complement-like pathway, but the nascent protease mediating CLIPC9 cleavage awaits identification.…”

“…Examples of such proPO activation cascades have been described in several model organisms, including M. sexta ( Gorman et al, 2007 ; Wang and Jiang, 2007 ), Tenebrio molitor ( Kan et al, 2008 ; Jiang et al, 2009 ), and most recently Helicoverpa armigera ( Wang Q. et al, 2020 ). While currently no data exist that show activation cleavage of either CLIPB9 or CLIPB10 by an endogenous CLIPC, recent genetic evidence suggests that CLIPC9 is required for humoral melanization of parasites as well as tissue melanization ( Sousa et al, 2020 ). Concomitant knockdown of CTL4 and CLIPC9 reversed ookinete melanization without rescuing parasite killing, thus phenocopying the CTL4 / CLIPB10 double-kd.…”

Trained Immunity in Anopheles gambiae: Antibacterial Immunity Is Enhanced by Priming via Sugar Meal Supplemented With a Single Gut Symbiotic Bacterial Strain

“…This process requires complex interactions of different members in the CLIPs B, C, and A as well as serpins (An et al, 2011;Gulley et al, 2013;Povelones et al, 2013;Zhang et al, 2015;Cao et al, 2017;He et al, 2017;Meekins et al, 2017;Nakhleh et al, 2017;El Moussawi et al, 2019;Sousa et al, 2020). It has been shown recently that microbial melanization can be triggered by E. coli infection (Sousa et al, 2020). The genes that participate in modulating melanization were enriched in the transcriptomes responsive to the priming regimes.…”