Cytoplasmic free Ca2+ is essential for multiple steps in malaria parasite egress from infected erythrocytes.

Background: Egress of Plasmodium falciparum, from erythrocytes at the end of its asexual cycle and subsequent parasite invasion into new host cells, is responsible for parasite dissemination in the human body. The egress pathway is emerging as a coordinated multistep programme that extends in time for tens of minutes, ending with rapid parasite extrusion from erythrocytes. While the Ca²⁺ regulation of the invasion of P. falciparum in erythrocytes is well established, the role of Ca²⁺ in parasite egress is poorly understood. This study analysed the involvement of cytoplasmic free Ca²⁺ in infected erythrocytes during the multistep egress programme of malaria parasites. Methods: Live-cell fluorescence microscopy was used to image parasite egress from infected erythrocytes, assessing the effect of drugs modulating Ca²⁺ homeostasis on the egress programme. Results: A steady increase in cytoplasmic free Ca²⁺ is found to precede parasite egress. This increase is independent of extracellular Ca²⁺ for at least the last two hours of the cycle, but is dependent upon Ca²⁺ release from internal stores. Intracellular BAPTA chelation of Ca²⁺ within the last 45 minutes of the cycle inhibits egress prior to parasitophorous vacuole swelling and erythrocyte membrane poration, two characteristic morphological transformations preceding parasite egress. Inhibitors of the parasite endoplasmic reticulum (ER) Ca²⁺-ATPase accelerate parasite egress, indicating that Ca²⁺ stores within the ER are sufficient in supporting egress. Markedly accelerated egress of apparently viable parasites was achieved in mature schizonts using Ca²⁺ ionophore A23187. Ionophore treatment overcomes the BAPTA-induced block of parasite egress, confirming that free Ca²⁺ is essential in egress initiation. Ionophore treatment of immature schizonts had an adverse effect inducing parasitophorous vacuole swelling and killing the parasites within the host cell. Conclusions: The parasite egress programme requires intracellular free Ca²⁺ for egress initiation, vacuole swelling, and host cell cytoskeleton digestion. The evidence that parasitophorous vacuole swelling, a stage of unaffected egress, is dependent upon a rise in intracellular Ca²⁺ suggests a mechanism for ionophore-inducible egress and a new target for Ca²⁺ in the programme liberating parasites from the host cell. A regulatory pathway for egress that depends upon increases in intracellular free Ca²⁺ is proposed. [ABSTRACT FROM AUTHOR]