Actomyosin forces and the energetics of red blood cell invasion by the malaria parasite Plasmodium falciparum

Author summary Malaria is a widespread infectious disease carried by mosquitoes, caused by several species of single-celled parasites of which the deadliest is Plasmodium falciparum. After a bite from an infected mosquito and a silent symptomatic livers stage, symptomatic disease ensues, caused by cyclical invasion of red blood cells and replication within over a 48-hour cycle. The force required for invasion is thought to depend on a parasite molecular motor known as myosin A (PfMyoA), but it is unclear which steps of invasion need force from this motor and what roles are played by PfMyoA and related motor proteins. Here, we generated a series of modified parasites with mutated motor proteins, resulting in a range of invasion defects from mild to completely blocked. We analysed these parasites during invasion by video microscopy, identifying mutants that stalled at different stages or took longer to invade. Together, our analysis reveals three distinct energetic steps during invasion when PfMyoA is required and sheds light on the contribution of other motor proteins. Since parasite myosins are only distantly related to those of humans, understanding their roles during invasion could unearth effective and specific targets for future drug development. All symptoms of malaria disease are associated with the asexual blood stages of development, involving cycles of red blood cell (RBC) invasion and egress by the Plasmodium spp. merozoite. Merozoite invasion is rapid and is actively powered by a parasite actomyosin motor. The current accepted model for actomyosin force generation envisages arrays of parasite myosins, pushing against short actin filaments connected to the external milieu that drive the merozoite forwards into the RBC. In Plasmodium falciparum, the most virulent human malaria species, Myosin A (PfMyoA) is critical for parasite replication. However, the precise function of PfMyoA in invasion, its regulation, the role of other myosins and overall energetics of invasion remain unclear. Here, we developed a conditional mutagenesis strategy combined with live video microscopy to probe PfMyoA function and that of the auxiliary motor PfMyoB in invasion. By imaging conditional mutants with increasing defects in force production, based on disruption to a key PfMyoA phospho-regulation site, the absence of the PfMyoA essential light chain, or complete motor absence, we define three distinct stages of incomplete RBC invasion. These three defects reveal three energetic barriers to successful entry: RBC deformation (pre-entry), mid-invasion initiation, and completion of internalisation, each requiring an active parasite motor. In defining distinct energetic barriers to invasion, these data illuminate the mechanical challenges faced in this remarkable process of protozoan parasitism, highlighting distinct myosin functions and identifying potential targets for preventing malaria pathogenesis.