Evidence against a Role of Elevated Intracellular Ca²⁺ during Plasmodium falciparum Preinvasion.

Abstract

Severe malaria is primarily caused by Plasmodium falciparum parasites during their asexual reproduction cycle within red blood cells. One of the least understood stages in this cycle is the brief pre-invasion period during which merozoite-red cell contacts lead to apical alignment of the merozoite in readiness for penetration, a stage of major relevance in the control of invasion efficiency. Red blood cell deformations associated with this process were suggested to be active plasma membrane responses mediated by transients of elevated intracellular calcium. Few studies have addressed this hypothesis due to technical challenges, and the results remained inconclusive. Here, Fluo-4 was used as a fluorescent calcium indicator with optimized protocols to investigate the distribution of the dye in red blood cell populations used as falciparum invasion targets in egress-invasion assays. Pre-invasion dynamics was observed simultaneously under bright-field and fluorescence microscopy by recording egress-invasion events. All the egress-invasion sequences showed red blood cell deformations of varied intensities during the pre-invasion period and the echinocytic changes that follow during invasion. Intra-erythrocytic calcium signals were absent throughout this interval in over half the records, and totally absent during the pre-invasion period regardless of deformation strength. When present, calcium signals were of a punctate modality, initiated within merozoites already poised for invasion. These results argue against a role of elevated intracellular calcium during the pre-invasion stage. We suggest an alternative mechanism of merozoite-induced pre-invasion deformations based on passive red cell responses to transient agonist-receptor interactions associated with the formation of adhesive coat filaments.