Distinct external signals trigger sequential release of apical organelles during erythrocyte invasion by malaria parasites

Abstract

The invasion of erythrocytes by Plasmodium merozoites requires specific interactions between host receptors and parasite ligands. Parasite proteins that bind erythrocyte receptors during invasion are localized in apical organelles called micronemes and rhoptries. The regulated secretion of microneme and rhoptry proteins to the merozoite surface to enable receptor binding is a critical step in the invasion process. The sequence of these secretion events and the external signals that trigger release are not known. We have used time-lapse video microscopy to study changes in intracellular calcium levels in Plasmodium falciparum merozoites during erythrocyte invasion. In addition, we have developed flow cytometry based methods to measure relative levels of cytosolic calcium and study surface expression of apical organelle proteins in P. falciparum merozoites in response to different external signals. We demonstrate that exposure of P. falciparum merozoites to low potassium ion concentrations as found in blood plasma leads to a rise in cytosolic calcium levels through a phospholipase C mediated pathway. Rise in cytosolic calcium triggers secretion of microneme proteins such as the 175 kD erythrocyte binding antigen (EBA175) and apical membrane antigen-1 (AMA-1) to the merozoite surface. Subsequently, interaction of EBA175 with glycophorin A (glyA), its receptor on erythrocytes, restores basal cytosolic calcium levels and triggers release of rhoptry proteins. Our results identify for the first time the external signals responsible for the sequential release of microneme and rhoptry proteins during erythrocyte invasion and provide a starting point for the dissection of signal transduction pathways involved in regulated exocytosis of these key apical organelles. Signaling pathway components involved in apical organelle discharge may serve as novel targets for drug development since inhibition of microneme and rhoptry secretion can block invasion and limit blood-stage parasite growth.

Figure 1. Cytosolic calcium levels in P. falciparum merozoites during erythrocyte invasion.

Late stage P. falciparum schizonts were labeled with Fluo-4AM and added to uninfected erythrocytes to allow re-invasion. Calcium levels were monitored in released merozoites during invasion by time-lapse video microscopy. Both DIC and fluorescence images were acquired at 2 second intervals. Selected pairs of DIC and fluorescence images with time elapsed between frames in seconds (Sec) are shown. The mean fluorescence intensity (MFI) after subtraction of background in an individual merozoite that successfully completed invasion (marked by arrows) is indicated. White bar indicates 5 µm. Also see Supplementary Information, Videos S1, S2 and S3.

Figure 2. Cytosolic calcium levels and expression of EBA175 on merozoite surface detected by flow cytometry following treatment with calcium ionophore A23187.

(A–B) Cytosolic calcium levels in merozoites upon treatment with ionophore A23187. Changes in cytosolic calcium levels were detected in P. falciparum merozoites labeled with Fluo-4AM by flow cytometry after treatment with either A23187 (A) or BAPTA-AM followed by A23187 (BA + A23187) (B). Mean fluorescence intensities (MFI), which reflect relative levels of free cytosolic calcium [Ca⁺²], measured by flow cytometry are shown as a function of time in seconds. Arrow indicates time-point at which A23187 was added. (C–F) Surface expression of microneme, rhoptry and merozoite surface proteins upon treatment with A23187. Expression of microneme protein EBA175 (C), rhoptry protein CLAG3.1 (E) and merozoite surface protein MSP4 (F) was detected on surface of P. falciparum merozoites isolated in RPMI1640 medium (RPMI, red) or following treatment with either A23187 (A23187, blue) or treatment with BAPTA-AM followed by A23187 (BA + A23187, green) using specific sera by flow cytometry. Microneme protein EBA175 was also detected in merozoites permeabilized with 0.05% saponin using specific sera (D). Untreated merozoites stained with pre-immune serum (PIS, black) were used as controls (C–F).

Figure 3. Expression of EBA175 on merozoite surface detected by immunofluorescence assay (IFA) following treatment with calcium ionophore A23187.

Expression of microneme protein EBA175 on surface of P. falciparum merozoites isolated in RPMI1640 medium (RPMI) (A) or following treatment with either A23187 (RPMI + A23187) (B) or BAPTA-AM followed by A23187 (RPMI + BA + A23187) (C) was detected by IFA using anti-EBA175 rabbit sera followed by FITC-conjugated anti-rabbit IgG goat sera (green). Nuclear DNA was counterstained with DAPI (blue). Bright field, bright field merged with DAPI staining and merged fluorescence images (DAPI and FITC) are shown. Black bar indicates 2 µm.

Figure 4. Cytosolic calcium levels and expression of EBA175 on merozoite surface in response to changes in ionic conditions.

(A) Cytosolic calcium levels in merozoites under different ionic conditions. P. falciparum merozoites were isolated in buffer mimicking intracellular conditions (IC – 5 mM NaCl, 140 mM KCl, 1 mM EGTA). Merozoites were labeled with Fluo-4AM and cytosolic calcium levels [Ca⁺²] were measured by flow cytometry before and after transfer from IC buffer to buffer mimicking extracellular conditions (EC – 140 mM NaCl, 5 mM KCl, 1 mM CaCl₂ (IC-EC, brown)) or IC K_low buffer (IC K_low - 5 mM NaCl, 5 mM KCl, 135 mM choline-Cl, 1 mM EGTA (IC-IC K_low, green)). Merozoites isolated in IC buffer were transferred to EC buffer after prior treatment with either BAPTA-AM (IC+BA-EC+BA, blue) or U73122 (IC+U73122-EC+U73122, red) or its inactive analog U73343 (IC+U73343-EC+U73343, purple). Mean fluorescence intensities (MFI), which reflect relative levels of free cytosolic calcium measured by flow cytometry, are shown as a function of time in seconds. Arrow indicates time-point at which merozoites were transferred from IC buffer. (B) Surface expression of EBA175 under different ionic conditions. Expression of EBA-175 was detected on surface of merozoites isolated in IC buffer (IC, red), following transfer from IC to EC buffer (EC, blue) or following transfer from IC to EC buffer after prior treatment with BAPTA-AM (EC + BA, green) using specific sera by flow cytometry. Merozoites isolated in IC buffer and stained with pre-immune serum (PIS, black) were used as controls. (C) Surface expression of EBA175 in response to change in [K⁺]. Expression of EBA-175 was detected on surface of merozoites isolated in IC buffer (IC, red), after transfer from IC to EC buffer (EC, blue), IC K_low buffer (IC K_low, pink) or EC w/o Ca⁺² buffer (140 mM NaCl, 5 mM KCl, 1 mM EGTA (EC w/o Ca⁺², green) using specific sera by flow cytometry. (D) Effect of PLC inhibitor U73122 on surface expression of EBA175. Expression of EBA175 was detected on surface of merozoites isolated in IC buffer (IC, red), following transfer from IC to EC buffer (EC, blue) or from IC to EC buffer following pre-treatment with either U73122 (EC+U73122, green) or its inactive analog U73343 (EC+U73343, pink) using specific sera by flow cytometry. (E) Invasion rates in presence of calcium signaling pathway inhibitors. Merozoites were allowed to invade erythrocytes in presence of calcium chelator BAPTA-AM (EC + BA), PLC inhibitor U73122 (EC+U73122) or its inactive analog U73343 (EC+U73343). Newly invaded rings were scored by Giemsa staining. Invasion rates in RPMI1640 medium in absence of any calcium modulating agents were used as controls to determine invasion inhibition rates in presence of BAPTA-AM, U73122 and U73343.

Figure 5. Expression of EBA175 on merozoite surface detected by immunofluorescence assay (IFA) in response to changes in ionic conditions.

Expression of microneme protein EBA175 on surface of P. falciparum merozoites isolated in IC buffer (IC) (A) or following transfer to EC buffer (EC) (B) or following transfer to EC buffer after pre-treatment with BAPTA-AM (EC + BA) (C) was detected by IFA using anti-EBA175 rabbit sera followed by FITC-conjugated anti-rabbit IgG goat sera (green). Nuclear DNA was counterstained with DAPI (blue). Bright field, bright field merged with DAPI staining and merged fluorescence images (DAPI and FITC) are shown. Black bar indicates 2 µm.

Figure 6. Cytosolic calcium levels and translocation of rhoptry protein CLAG3.1 to merozoite surface in response to interaction with glyA.

(A) Expression of CLAG3.1 on merozoite surface following interaction with glyA. Expression of CLAG3.1 was detected on surface of P. falciparum merozoites isolated in buffer mimicking intracellular conditions (IC – 5 mM NaCl, 140 mM KCl, 1 mM EGTA, red), or after transfer from IC buffer to buffer mimicking extracellular conditions (EC - 140 mM NaCl, 5 mM KCl, 1 mM CaCl₂, blue) or after transfer from IC buffer to EC buffer containing glyA (EC + GlyA, green) using specific sera by flow cytometry. Merozoites isolated in IC buffer and stained with pre-immune sera (PIS, black) were used as controls. (B) Changes in cytosolic calcium levels in P. falciparum 3D7 merozoites following transfer from intracellular to extracellular ionic conditions and following engagement with glyA. P. falciparum 3D7 merozoites were isolated in IC buffer. Merozoites were labeled with Fluo-4AM and cytosolic calcium levels [Ca⁺²] were measured by flow cytometry before and after transfer from IC to EC buffer (IC-EC) and from EC buffer to EC buffer containing glyA (EC + GlyA). (C) Translocation of CLAG3.1 to surface of P. falciparum 3D7Δ175 merozoites following receptor engagement. Expression of CLAG3.1 was detected on surface of P. falciparum 3D7Δ175 merozoites isolated in IC buffer (IC, red), after transfer from IC buffer to EC buffer (EC, blue), after transfer from IC buffer to EC buffer containing glyA (EC + GlyA, green) or after transfer from IC buffer to EC buffer containing red blood cell (RBC) ghosts (EC + RBC ghosts, pink) using specific sera by flow cytometry. P. falciparum 3D7Δ175 merozoites in IC buffer stained with pre-immune sera (PIS, black) were used as control. (D) Changes in cytosolic calcium levels in P. falciparum 3D7Δ175 merozoites after transfer from intracellular to extracellular conditions and following binding to erythrocyte ghosts. P. falciparum 3D7Δ175 merozoites were isolated in IC buffer. Merozoites were labeled with Fluo-4AM and cytosolic calcium levels were measured by flow cytometry. Merozoites were transferred first from IC buffer to EC buffer (IC-EC) and from EC buffer to EC buffer containing red blood cell ghosts (EC + RBC ghosts). Mean fluorescence intensities (MFI), which reflect relative levels of free cytosolic calcium measured by flow cytometry are shown as a function of time in seconds (B, D). Arrows indicate time-points at which merozoites were transferred from IC buffer to EC buffer (IC-EC) (B, D) or from EC buffer to EC buffer containing gly A (EC + Gly A) (B) or from EC buffer to EC buffer containing RBC ghosts (EC + RBC ghosts) (D).

Figure 7. Expression of CLAG3.1 on merozoite surface detected by immunofluorescence assay (IFA) in response to interaction with glyA.

Expression of rhoptry protein CLAG3.1 on surface of P. falciparum merozoites isolated in IC buffer (IC) (A) or following transfer to EC buffer (EC) (B) or following transfer to EC buffer containing glyA (EC + GlyA) (C) detected by IFA using anti-CLAG3.1 rabbit sera followed by FITC-conjugated (green) anti-rabbit IgG goat sera. Nuclear DNA was counterstained with DAPI (blue). Bright field, bright field merged with DAPI staining and merged fluorescence images (DAPI and FITC) are shown. Black bar indicates 2 µm.