The black fly salivary protein Simukunin inhibits complement activation through the lectin pathway protease MASP-2

Salivary secretions from blood-feeding arthropods are enriched with bioactive molecules that counteract host hemostatic and immune defenses, including blood coagulation, platelet aggregation, complement activation, and vasoconstriction. Although numerous salivary components have been identified and functionally characterized across hematophagous arthropods, their specific roles in complement inhibition in black flies remain insufficiently defined. Previously, it was discovered that Simukunin, a salivary protein from Simulium vittatum, inhibits procoagulant activity. Here, using surface plasmon resonance and enzymatic assays, we found that Simukunin also acts as a specific inhibitor of the complement lectin pathway (LP). Simukunin binds with high affinity to mannose-associated serine protease 2 (MASP-2), a serine protease crucial for LP activation, preferentially blocking cleavage of C2 over C4, resulting in reduced downstream membrane attack complex deposition. No interaction with other downstream complement proteins was found. Simukunin also inhibits the procoagulant activity of MASP-2 in vitro. Notably, pretreatment with Simukunin significantly improved survival rates in a murine model of lipopolysaccharide-induced sepsis. Overall, Simukunin emerges as a novel complement inhibitor and a potential candidate for therapeutic applications.