Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor

Malaria parasite egress from host erythrocytes (RBCs) is regulated by discharge of a parasite serine protease called SUB1 into the parasitophorous vacuole (PV). There, SUB1 activates a PV‐resident cysteine protease called SERA6, enabling host RBC rupture through SERA6‐mediated degradation of the RBC cytoskeleton protein β‐spectrin. Here, we show that the activation of Plasmodium falciparum SERA6 involves a second, autocatalytic step that is triggered by SUB1 cleavage. Unexpectedly, autoproteolytic maturation of SERA6 requires interaction in multimolecular complexes with a distinct PV‐located protein cofactor, MSA180, that is itself a SUB1 substrate. Genetic ablation of MSA180 mimics SERA6 disruption, producing a fatal block in β‐spectrin cleavage and RBC rupture. Drug‐like inhibitors of SERA6 autoprocessing similarly prevent β‐spectrin cleavage and egress in both P. falciparum and the emerging zoonotic pathogen P. knowlesi. Our results elucidate the egress pathway and identify SERA6 as a target for a new class of antimalarial drugs designed to prevent disease progression.