The serine protease homolog Skanda modulates Toll-Phenoloxidase-mediated immunity in Drosophila

Extracellular serine protease (SP) cascades are central regulators of insect innate immunity. These cascades are negatively controlled by serine protease inhibitors (serpins) and fine-tuned by serine protease homologs (SPHs), which resemble SPs but lack catalytic activity. In Drosophila , a key SP cascade—the Toll–phenoloxidase (PO) pathway—governs both the melanization response and Toll-dependent antimicrobial peptide production. This cascade is triggered by secreted pattern-recognition receptors or microbial proteases and converges on two clip-domain SPs, Persephone and Hayan, which activate the Toll ligand Spätzle via the Spätzle-Processing Enzyme (SPE) and process prophenoloxidases. Here, we characterize the SPH Skanda and uncover its role in the Toll–PO cascade. skanda is genomically clustered with hayan and persephone and is transcriptionally induced upon infection. Skanda is unusual among SPHs, containing a long serine/threonine-rich region, two clip domains, and atypical disulfide bonds. Skanda is unstable and subject to cleavage by Grass. Functional assays show that Skanda dampens activation of Hayan, and to a lesser extent Persephone, within the Toll–PO cascade. Notably, skanda -deficient flies are highly susceptible to Staphylococcus aureus despite displaying normal Toll signaling and cuticular melanization. Moreover, compound mutants lacking two members of the hayan–psh–skanda cluster reveal a hidden contribution of Skanda to Toll activation in the absence of Persephone. Together, our results identify Skanda as a modulatory SPH that fine-tunes Toll pathway activity in concert with Persephone and Hayan.