MMV687794 blocks Plasmodium falciparum invasion of red blood cells by targeting a Surface-associated Lipid-Interacting Rhoptry Protein, PfSLIRP

Invasion of red blood cells (RBCs) by the human malaria parasite, Plasmodium falciparum, drives disease. During invasion, the parasite pushes its way into the RBC while wrapping the RBC membrane around itself to establish the parasitophorous vacuole, a stable niche within the RBC where the parasite grows. To better understand invasion, we investigated the mechanism of action of an invasion-inhibitory compound, MMV687794 (MMV794). Lattice light-sheet microscopy revealed that MMV794 blocks parasite entry by preventing parasitophorous vacuole formation. In vitro drug resistance selection of parasites with MMV794 found mutations to the /{beta} hydrolase, PF3D7_0403800, and engineering one of these mutations (C36W) into parasites by CRISPR/Cas9 recapitulated the resistance phenotype. Expansion microscopy demonstrated that this protein is expressed in schizonts, localising to the surface of rhoptries, which are specialised apical secretory organelles that function during invasion. Lipidomics and proteomics analyses of C36W parasites uncovered widespread changes to lipid composition/homeostasis and altered abundance of proteins involved in invasion, indicating a role for PF3D7_0403800 in invasion-associated lipid metabolism. Finally, we used solvent-induced proteome profiling and reactivity assays to confirm drug-target engagement. Together, our findings identify a novel Surface-associated Lipid-Interacting Rhoptry Protein (PfSLIRP) that coordinates lipid metabolism at the rhoptries to enable RBC invasion.