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

Abstract

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.

Figure 1

Free calcium kinetics in schizonts approaching parasite egress. Cells labelled with Fluo-4 AM (5 μM), and monitored at 37°C in full medium (A) or full medium supplemented with 40 μM probenecid (B). Selected frames from the time-lapse movies on the right side of each graph show DIC and fluorescence images of pre-egress schizonts (upper set of images), pre-egress schizonts with the erythrocyte membrane permeable to external calcium (middle set of images labelled as ‘poration’ showing the highest level of fluorescence signal) and lower set of images captured erythrocyte membrane rupture and parasite egress. Bar = 5 μm.

Figure 2

Free Ca²⁺ content and free Ca²⁺ kinetics within infected cells labelled with a ratio-metric calcium probe Fura Red AM. Cells were labelled with Fura Red AM (5 μM) and monitored at 37°C in medium supplemented with 40 μM probenecid. A. Fluorescence and DIC images of labelled schizont, four images on left; parasite egress from the same schizont captured in all three channels, four images on right. Circle indicates an area of interest selected for analysis of fluorescence in the two ratio-metric channels. Bar = 5 μm. B. Free calcium content in infected cells for three different stages of parasite maturation: early trophozoites, late trophozoite and pre-egress schizonts. Cells were labelled with ratio-metric probe Fura Red AM (5 μM), and monitored at 37°C in the medium supplemented with 40 μM probebecid. Number of analysed cells: early trophozoites – 80, late trophozoites – 60, schizonts – 10. [Ca²⁺] estimation described in the Methods. C. Rate of calcium increase observed in the three different stages of parasite maturation described in B using the same labelling protocol. Number of analysed cells: early trophozoites – 25, late trophozoites – 60, schizonts – 10. Data presented as a mean ± SEM; significance evaluated using a paired t-test. A statistically significant differences (*) with P < 0.01 was found between early and late trophozoites. D. Representative time courses for the calcium increases observed in early trophozoite (filled circles) and late trophozoite (open circles). E. Time courses for the calcium increases observed in three pre-egress schizonts (circles filled with black, light gray or dark gray colour for each cell). Note that the ratio could plateau prior to egress (black circles curve).

Figure 3

Manipulating the free calcium concentration in schizonts affects parasite egress. A. Reducing the extracellular free Ca²⁺ concentration does not affect parasite egress. Synchronized cell cultures approaching the end of the cycle were pretreated with different concentrations of EDTA, pH 7.4, for 5 min at 37°C to chelate external Ca²⁺ and then observed. Drug-pretreated and control cultures were kept for two hours at 37°C to accumulate sites of egress and then egress was evaluated as described in the Methods and presented as Mean ± SEM, n = 3-4 independent experiments; 7,429 infected cells were analyzed. Concentration of free calcium was less than 10 nM according to a fluorimetric assay for free Ca²⁺. B. Reduction in intracellular Ca²⁺ concentration by BAPTA inhibits parasite egress. Synchronized cell cultures approaching the end of the cycle were pretreated outside the chambers with increasing concentrations of the cell permeant Ca²⁺ chelator BAPTA AM at 37°C for 30 min. Pretreated cultures were then injected into the chambers for microscopy and kept at 37°C for 90 min to accumulate sites of egress and then parasite egress was assessed in treated and control cultures as described above (mean ± SEM, n = 3-4 independent experiments; 9,669 infected cells were analyzed). C. The Ca²⁺ ionophore A23187 activates parasite egress only in the presence of intra-and extra-cellular Ca²⁺ and upon short (15–30 min) treatment of infected cells. Cells pretreated with EDTA (5 min at 37°C) or BAPTA AM (30 min at 37°C) or both were injected into the chambers immediately after addition of ionophore into the medium. Chambers were kept at 37°C for 15–30 min to accumulate sites of egress and then parasite egress was assessed in cultures as described above (mean ± SEM, n = 2-6 independent experiments, at least 300 infected cells were analyzed for each condition in each experiment).

Figure 4

Acceleration of parasite egress induced by inhibitors of the Plasmodium Ca²⁺ pump PfATP6. Cells were treated for 30 min at 37°C in chambers. A. ThG – Thapsigargin, mean of two independent experiments, total number of analysed infected cells, 3,300. B. CPA – cyclopiazonic acid, mean ± SEM, n = 3 independent experiments, total number of analysed cells, 3,600.

Figure 5

A23187-induced and Ca²⁺ -dependent parasite death. A. Immature schizonts from infected erythrocytes critically swelled with extruded parasitophorous vacuoles. Cultures were treated with 10 μM A23187 in chambers for two hours in the presence of fluorescent antibodies to the major erythrocyte surface protein glycophorin A (red colour in upper images) and with fluorescent phalloidin (green colour in lower images) to detect the major erythrocyte cytoskeletal protein F-actin. Note the unlabelled membranes of the parasitophorous vacuoles (black arrowheads) extruded from the labelled erythrocyte membranes; bar = 5 μm. B. Extended treatment (2 hours) of cultures with increased concentrations of ionophore inhibited parasite egress and led to accumulation of damaged schizonts (a representative experiment, mean of three measurements, total number of infected cells analysed, 1,402).