Insight into the structural hierarchy of the protease cascade that regulates the mosquito melanization response

Abstract

Serine protease cascades regulate important physiological processes during infection, from complement activation and coagulation in mammals to melanization and Toll pathway activation in insects. Key components of these cascades in insects are clip domain serine proteases (CLIPs) including the catalytic, clip domain serine proteases (cSPs) and the non-catalytic, clip domain serine protease homologs (cSPHs), both of which are essential for the proper functioning of these cascades. In the context of the melanization immune response of insects, CLIPs cascades converge on the activation cleavage of the zymogen prophenoloxidase to active phenoloxidase that initiates melanin biogenesis on microbial surfaces. While we have previously provided insight into the structural hierarchy of cSPHs in these cascades in the mosquito vector of malaria Anopheles gambiae, the hierarchy of cSPs as well as their functional interactions with cSPHs remain largely unknown. Here, we define partially the structural hierarchy of An. gambiae cSPs of the CLIPB family that are key factors in the melanization response and characterize their relative contributions to the anti-bacterial melanization response and to mosquito susceptibility to bacterial infections. Based on in vivo and in vitro assays that gauge the activation cleavage profiles of candidate CLIPs, we also propose that bidirectional interactions between cSPs and cSPHs regulate signal amplification and propagation in these cascades.

Late sporogonic stages ofPlasmodiumparasites are susceptible to the melanization response inAnopheles gambiaemosquitoes

“…Membranes were blocked with 1x phosphate-buffered saline (PBS) containing 0.05% Tween 20 and 3% milk solution for 1 hour at room temperature, followed by an overnight incubation at 4°C with the corresponding primary antibodies. Primary antibodies were used according to the following dilutions: rabbit αTEP1, 1:1000 (El Moussawi et al, 2019), rabbit αCTL4, 1:1000 (Saab et al, 2024), rabbit αCLIPA14, 1:3000 (Saab et al, 2024), rabbit αCLIPA2, 1:1000 (Yassine et al, 2014), rabbit αCLIPA28, 1:1000 (El Moussawi et al, 2019), and mouse αApolipophorin II, 1:100 (Mendes et al, 2008). Next, membranes were washed with 1xPBS-Tween 20 and incubated with secondary anti-rabbit (1:14,000) or anti-mouse (1:6000) antibodies conjugated with horse-radish peroxidase for 1 hour at room temperature.…”

“…TEP1 is also essential for P. berghei and P. falciparum ookinete melanization, however, its contribution seems to depend on the refractory mosquito genetic background; it is essential for melanizing ookinetes of both species in L3-5 mosquitoes (Collins et al, 1986;Blandin et al, 2004;Eldering et al, 2016), however, it is dispensable for P. falciparum ookinete melanization in CTL null mosquitoes (Simoes et al, 2022). The clip domain serine proteases (cSPs) and their serine protease homologs (cSPHs) that are central regulators of the melanization response constitute another class of Plasmodium antagonists, as several members are critical for Plasmodium melanization (El Moussawi et al, 2019;Povelones et al, 2013;Sousa et al, 2020;Volz et al, 2006;Zhang et al, 2021), in particular the three cSPHs SPCLIP1, CLIPA8 and CLIPA28 which form a hierarchical cSPH module that is central for the activation of the protease cascades driving microbial melanization (El Moussawi et al, 2019;Saab et al, 2024;Sousa et al, 2020).…”