Breakdown in membrane asymmetry regulation leads to monocyte recognition of P. falciparum-infected red blood cells

Author summary Malaria remains the deadliest parasitic disease in the world despite years of sustained effort, new drug development, and a greater understanding of the parasite and its interactions with its host. We examined a series of interconnected processes, from lipid and calcium sequestration through to recognition by immune cells. We show here that the uptake of cholesterol and calcium ions induce changes to the parasitised host red blood cell. These changes affect the activity of lipid-transporting enzymes in the host cell, which keep certain phospholipids on specific sides of the membrane: a concept called membrane asymmetry. We show that the parasite causes activation of a calcium- and cholesterol-sensitive enzyme, scramblase, so phospholipids are scrambled back and forth. Therefore, the lipid phosphatidylserine becomes exposed in the membrane outer layer, acting as an 'eat me' signal to phagocytes including monocytes. Throughout our study, we show that the infected cell is expending energy to compensate for the processes which ultimately lead to phagocytosis. But despite these compensatory processes, a portion of parasites are ingested by monocytes. Our systematic exploration of this pathway addresses contradictory findings from past reports, and exposes vulnerabilities in the parasite's metabolism that could be used for targeted drug design. The human malaria parasite Plasmodium falciparum relies on lipids to survive; this makes its lipid metabolism an attractive drug target. The lipid phosphatidylserine (PS) is usually confined to the inner leaflet of the red blood cell membrane (RBC) bilayer; however, some studies suggest that infection with the intracellular parasite results in the presence of this lipid in the RBC membrane outer leaflet, where it could act as a recognition signal to phagocytes. Here, we used fluorescent lipid analogues and probes to investigate the enzymatic reactions responsible for maintaining asymmetry between membrane leaflets, and found that in parasitised RBCs the maintenance of membrane asymmetry was partly disrupted, and PS was increased in the outer leaflet. We examined the underlying causes for the differences between uninfected and infected RBCs using fluorescent dyes and probes, and found that calcium levels increased in the infected RBC cytoplasm, whereas membrane cholesterol was depleted from the erythrocyte plasma membrane. We explored the resulting effect of PS exposure on enhanced phagocytosis by monocytes, and show that infected RBCs must expend energy to limit phagocyte recognition, and provide experimental evidence that PS exposure contributes to phagocytic recognition of P. falciparum-infected RBCs. Together, these findings underscore the pivotal role for PS exposure on the surface of Plasmodium falciparum-infected erythrocytes for in vivo interactions with the host immune system, and provide a rationale for targeted antimalarial drug design.

Automated summary

The study reveals that infection by the malaria parasite disrupts the symmetry of red blood cell membranes, leading to the exposure of phosphatidylserine and increased phagocytosis. Infected cells need to expend energy to resist phagocyte recognition, and the exposure of phosphatidylserine contributes to the recognition of Plasmodium falciparum-infected cells by the immune system.