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. However, most in vitro growth assays are carried out in static conditions, which are a poor mimic for growth in the body. Previously, more dynamic growth conditions were created using orbital shaking platforms, but exploration of the impact of shaking has been limited, and no attempt has been made to understand the forces generated by the resultant fluid motion. For several strains of the deadliest malaria species, Plasmodium falciparum , we show that growth under shaking is strongly impacted by shaking speed, vessel geometry and haematocrit. 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, at which point the forces that RBCs and parasites are exposed to are comparable to the forces in the microvasculature. By contrast, the growth rate is increased in more turbulent conditions generated by higher shaking speeds. Using a panel of P. falciparum lines in which known invasion proteins were disrupted, we show that the critical shaking speed can reveal new growth phenotypes, showing several ligands have greater importance in high wall shear stress conditions.