Abstract
Serine protease cascades regulate important insect immune responses namely melanization and Toll pathway activation. An important component of these cascades are clip-domain serine protease homologs (cSPHs), which are non-catalytic, but essential for activating the enzyme prophenoloxidase (PPO) in the melanization response during septic infections. 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 report the identification of CLIPA28 as a novel immune-related cSPH in the malaria vector Anopheles gambiae. Functional genetic analysis using RNA interference (RNAi) revealed that CLIPA28 is essential for the melanization of Plasmodium berghei parasites in refractory mosquitoes, and for mosquito resistance to fungal infections. We further show, using combined biochemical and genetic approaches, that CLIPA28 is member of a network of at least four cSPHs, whereby members are activated in a hierarchical manner following septic infections. Depletion of the complement-like protein TEP1 abolished the activation of this network after septic infections, whereas, depletion of the serine protease inhibitor 2 (SRPN2) triggered enhanced network activation, even in naïve mosquitoes, culminating in a dramatic reduction in cSPHs hemolymph levels, which paralleled that of PPO. Our data suggest that cSPHs are engaged in complex and multilayered interactions within serine protease cascades that regulate melanization, and identify TEP1 and SRPN2 as two master regulators of the cSPH network.
Highlights
In insects, key humoral immune responses such as antimicrobial peptide synthesis by the Toll pathway [1], melanization [2,3,4], and complement-mediated attack [5] are regulated by serine protease cascades composed mainly of clip-domain serine proteases that are present in the hemolymph as zymogens
Functional studies in wildtype and refractory A. gambiae genetic backgrounds allowed the identification of several containing serine protease homologs (cSPHs) that regulate the melanization of P. berghei ookinetes, as they egress from midgut epithelial cells into the basal labyrinth
To identify novel cSPHs involved in the mosquito melanization response we searched for genes that are significantly co-regulated with CLIPA2 in a developmental transcriptome data set of Expressed Sequence Tags [34]
Summary
Key humoral immune responses such as antimicrobial peptide synthesis by the Toll pathway [1], melanization [2,3,4], and complement-mediated attack [5] are regulated by serine protease cascades composed mainly of clip-domain serine proteases (cSP) that are present in the hemolymph as zymogens. Not all cSPs are catalytic, those that lack one or more of the three residues (His, Asp, Ser) that form the catalytic triad are non-catalytic [ known as clip-domain containing serine protease homologs (cSPHs)] These cascades have been mostly studied in the context of melanization, an immune effector response that is triggered locally in response to cuticle injury, or systemically following microbial invasion of the hemocoel [6,7]. It is characterized by the synthesis of melanin and its cross-linking with molecules on microbial surfaces, or in injured areas resulting in the killing of the invader, and hardening of the wound clot. Systemic infections rapidly activate the melanization response by triggering the proteolytic processing of cSPs and cSPHs, leading to prophenoloxidase (PPO) cleavage into active PO by a terminal cSP in the cascade, known as a prophenoloxidase activating proteinase (PAP)
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