Dynamics and Topology of Heparin Interaction with Malaria Parasite-Infected Erythrocytes Revealed by Live Cell Time Lapse Microscopy

Abstract

Heparin strongly inhibits malaria parasite invasion of human erythrocytes, but the mechanism is poorly defined. Using live cell microscopy we show that egress of Plasmodium falciparum from erythrocytes is also inhibited by heparin in a dose-dependent manner. Heparin blocks host erythrocyte membrane rupture (but not parasite vacuolar membrane rupture) and merozoite dispersion. Fluorescent live-cell microscopy of infected erythrocytes releasing mature merozoites showed that heparin binds different targets independently and sequentially, consistent with the inhibition of both parasite egress and invasion. Heparin enters infected erythrocytes through pores, which we previously showed to form in the erythrocyte membrane right before egress; it binds two targets, the inner surface of the erythrocyte membrane and the surface of mature merozoites. The transient nature of heparin presence on the merozoite surface prompted us to explore the hypothesis that heparin binds the major surface antigen, MSP1 that shed by PfSub2 protease upon merozoite natural invasion of new erythrocytes. Indeed, by blocking protease activity we prevented shedding of heparin complex from merozoites. Binding of heparin with MSP1 can explain also defective merozoite dispersion prior to egress, when merozoites topologically are close and can be glued by heparin. A third heparin target was observed shortly after merozoite egress. It was co-localized with the proteins expelled from the merozoite apical prominence, likely rhoptry material needed for merozoite invasion. Thus, heparin attacks the most vulnerable stage of parasite asexual cycle, i.e. merozoite transition from cell to cell, mimicking the effect by which human antibodies neutralize malaria parasites.