Growing Malaria Parasites at a Critical Shaking Speed Mimicking Physiological Flow Reveals New Phenotypes for Invasion Ligands

Malaria kills over 600,000 people annually, with all the clinical symptoms being caused by the blood stage of the infection. Malaria parasites invade red blood cells (RBCs), where they grow and multiply until daughter parasites egress to invade new RBCs. This cycle happens primarily in the blood circulation, bone marrow, and spleen, where parasites and RBCs are exposed to flow-generated forces. Despite this, almost all in vitro growth assays are carried out in static conditions, which are a poor mimic for the conditions that malaria parasites encounter in the body. Therefore, we systematically tested the impact of dynamic conditions created by orbital shaking platforms on parasite growth and explored the link between growth and the wall shear stress forces generated by fluid motion. For several strains of the deadliest malaria species, Plasmodium falciparum, we showed that strikingly, for any given vessel, there is a critical shaking speed at which growth rates are reduced, corresponding to when the RBCs start to aggregate in the centre of the well, before growth increases again at higher shaking speeds. The force that parasites are exposed to at this critical shaking speed corresponds cloesly with previous measurements for the forces that RBCs and parasites are exposed to in the microvasculature. During invasion, the early attachment of the parasites to RBCs is dependent on two families of attachment proteins, the Erythrocyte Binding Antigen (PfEBA) family and the Reticulocyte Binding Protein Homologue (PfRH) family, which are thought to be largely redundant in function. We used a panel of PfEBA and PfRH knock-out lines to show for the first time that several of these ligands have greater importance in high wall shear stress conditions. This both adds new understanding to the function of these ligand families, and indicates that the concept of the critical shaking speed can reveal new parasite growth phenotypes.