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Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem.

Gazarini ML, Thomas AP, Pozzan T, Garcia CR - J. Cell Biol. (2003)

Bottom Line: This allowed selective loading of the Ca2+ probes within the PV.The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism.We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Institute of Biosciences, University of São Paulo, São Paulo 05508-900, Brazil.

ABSTRACT
Malaria parasites, Plasmodia, spend most of their asexual life cycle within red blood cells, where they proliferate and mature. The erythrocyte cytoplasm has very low [Ca2+] (<100 nM), which is very different from the extracellular environment encountered by most eukaryotic cells. The absence of extracellular Ca2+ is usually incompatible with normal cell functions and survival. In the present work, we have tested the possibility that Plasmodia overcome the limitation posed by the erythrocyte intracellular environment through the maintenance of a high [Ca2+] within the parasitophorous vacuole (PV), the compartment formed during invasion and within which the parasites grow and divide. Thus, Plasmodia were allowed to invade erythrocytes in the presence of Ca2+ indicator dyes. This allowed selective loading of the Ca2+ probes within the PV. The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism. We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV. A transient drop of [Ca2+] in the PV for a period as short as 2 h affects the maturation process of the parasites within the erythrocytes, with a major reduction 48 h later in the percentage of schizonts, the form that re-invades the red blood cells.

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Simultaneous imaging of the [Ca2+] in the PV and cytosol. 10 μM Mag-Fura-2 acid was present during invasion with P. falciparum, and the cells were then loaded with Fluo-3/AM after invasion. The buffer contained 1 mM CaCl2 and 2 mM Probenecid to avoid release of loaded Fluo-3. (A) Changes in parasite cytosolic [Ca2+] monitored by Fluo-3 fluorescence (arbitrary units) on addition of 10 μM THG and 10 μM ionomycin. (B) Changes of PV [Ca2+] monitored as the ratio of Mag-Fura-2 fluorescence excited at 351 and 375 nm in the same cell. (C) Phase-contrast image of the invaded RBC. (D) Fluo-3 fluorescence signal (in the parasite cytoplasm). (E) Mag-Fura-2 (in the PV) fluorescence. The small reversible drop in fluorescence observed in A and B is an addition artifact (a slight change in focus). In fact, we have also observed it on addition of medium only. (F) Mouse RBCs were infected with P. chabaudi in the presence of 10 μM Mag-Fura-2 acid in the standard medium containing CaCl2. After washing the excess dye, the infected cells (107 cells) were resuspended in the fluorimeter cuvette, equipped with magnetic stirring, in medium without added CaCl2. The arrow shows the time of addition of 15 μM ionomycin. The Ca2+ concentration in the PV was calculated, after lysing the cells, using the F-4500 intracellular cation measurement system software (Hitachi). The [Ca2+] of the PV, measured 30 min after completion of invasion, was found to be 41 ± 1 μM (n = 5).
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fig4: Simultaneous imaging of the [Ca2+] in the PV and cytosol. 10 μM Mag-Fura-2 acid was present during invasion with P. falciparum, and the cells were then loaded with Fluo-3/AM after invasion. The buffer contained 1 mM CaCl2 and 2 mM Probenecid to avoid release of loaded Fluo-3. (A) Changes in parasite cytosolic [Ca2+] monitored by Fluo-3 fluorescence (arbitrary units) on addition of 10 μM THG and 10 μM ionomycin. (B) Changes of PV [Ca2+] monitored as the ratio of Mag-Fura-2 fluorescence excited at 351 and 375 nm in the same cell. (C) Phase-contrast image of the invaded RBC. (D) Fluo-3 fluorescence signal (in the parasite cytoplasm). (E) Mag-Fura-2 (in the PV) fluorescence. The small reversible drop in fluorescence observed in A and B is an addition artifact (a slight change in focus). In fact, we have also observed it on addition of medium only. (F) Mouse RBCs were infected with P. chabaudi in the presence of 10 μM Mag-Fura-2 acid in the standard medium containing CaCl2. After washing the excess dye, the infected cells (107 cells) were resuspended in the fluorimeter cuvette, equipped with magnetic stirring, in medium without added CaCl2. The arrow shows the time of addition of 15 μM ionomycin. The Ca2+ concentration in the PV was calculated, after lysing the cells, using the F-4500 intracellular cation measurement system software (Hitachi). The [Ca2+] of the PV, measured 30 min after completion of invasion, was found to be 41 ± 1 μM (n = 5).

Mentions: The different behavior of the PV with respect to the parasite cytoplasm is even more evident in the experiment presented in Fig. 4 for P. falciparum, and identical results were obtained with P. chabaudi. In this experiment, invasion was carried out in medium containing the free acid form of another Ca2+ indicator, Mag-Fura-2, to monitor [Ca2+] within the PV. After invasion, the infected RBCs were incubated with Fluo-3/AM to load the parasite cytoplasm. The spectral characteristics of Mag-Fura-2 are sufficiently different from those of Fluo-3 that the signals from the two dyes are easily distinguished. Fig. 4 shows the two confocal images of a cell doubly loaded with Mag-Fura-2 (acid) in the PV and Fluo-3 (AM form) in the cytoplasm (Fig. 4, E and D, respectively). The phase image is also presented in C. The different localization of the two dyes appears quite clear; Fluo-3 within the parasite and Mag-Fura-2 around it. Fig. 4 shows the simultaneous dynamic measurements of [Ca2+] in the cytosol (Fig. 4 A, Fluo-3 AM) and PV (Fig. 4 B, Mag-Fura-2 acid) in the same cell. Mag-Fura-2 is a “ratiometric” dye, i.e., Ca2+ binding has opposite effects on the fluorescence emitted on excitation at 340 and 380 nm. Thus, an increase in [Ca2+], as revealed by Fluo-3, results in an increase of fluorescence, whereas an increase of the 340/380 nm ratio with Mag-Fura-2 is more directly related to the absolute increase of [Ca2+] (Grynkiewicz et al., 1985). In Fig. 4, the cells were first treated with the inhibitor of the sarco-ER Ca2+ ATPase (SERCA) thapsigargin (THG), a drug known to cause the release of Ca2+ from internal stores. Addition of THG resulted in elevation of [Ca2+], measured with both Fluo-3 and Mag-Fura-2. However, the subsequent addition of ionomycin had opposite effects on the [Ca2+] monitored by the two indicators; a further increase in [Ca2+] was monitored with cytoplasmic Fluo-3, whereas a large decrease was revealed by Mag-Fura-2 trapped in the PV. The interpretation of this experiment appears straightforward: THG mobilizes Ca2+ from Plasmodium intracellular stores, thus increasing cytoplasmic [Ca2+]; Ca2+ is then pumped out into the PV and revealed by Mag-Fura-2. Indeed, a clear delay is observed in the peak of the Mag-Fura-2 signal, as compared with that of Fluo-3. Ionomycin further releases Ca2+ from stores and causes an additional small increase in the [Ca2+] of the cytoplasm, but, given that the [Ca2+] of the PV is much higher than in the cytosol of the RBC, the ionophore transports Ca2+ out of the PV, leading to a major decrease of the Mag-Fura-2 signal.


Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem.

Gazarini ML, Thomas AP, Pozzan T, Garcia CR - J. Cell Biol. (2003)

Simultaneous imaging of the [Ca2+] in the PV and cytosol. 10 μM Mag-Fura-2 acid was present during invasion with P. falciparum, and the cells were then loaded with Fluo-3/AM after invasion. The buffer contained 1 mM CaCl2 and 2 mM Probenecid to avoid release of loaded Fluo-3. (A) Changes in parasite cytosolic [Ca2+] monitored by Fluo-3 fluorescence (arbitrary units) on addition of 10 μM THG and 10 μM ionomycin. (B) Changes of PV [Ca2+] monitored as the ratio of Mag-Fura-2 fluorescence excited at 351 and 375 nm in the same cell. (C) Phase-contrast image of the invaded RBC. (D) Fluo-3 fluorescence signal (in the parasite cytoplasm). (E) Mag-Fura-2 (in the PV) fluorescence. The small reversible drop in fluorescence observed in A and B is an addition artifact (a slight change in focus). In fact, we have also observed it on addition of medium only. (F) Mouse RBCs were infected with P. chabaudi in the presence of 10 μM Mag-Fura-2 acid in the standard medium containing CaCl2. After washing the excess dye, the infected cells (107 cells) were resuspended in the fluorimeter cuvette, equipped with magnetic stirring, in medium without added CaCl2. The arrow shows the time of addition of 15 μM ionomycin. The Ca2+ concentration in the PV was calculated, after lysing the cells, using the F-4500 intracellular cation measurement system software (Hitachi). The [Ca2+] of the PV, measured 30 min after completion of invasion, was found to be 41 ± 1 μM (n = 5).
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fig4: Simultaneous imaging of the [Ca2+] in the PV and cytosol. 10 μM Mag-Fura-2 acid was present during invasion with P. falciparum, and the cells were then loaded with Fluo-3/AM after invasion. The buffer contained 1 mM CaCl2 and 2 mM Probenecid to avoid release of loaded Fluo-3. (A) Changes in parasite cytosolic [Ca2+] monitored by Fluo-3 fluorescence (arbitrary units) on addition of 10 μM THG and 10 μM ionomycin. (B) Changes of PV [Ca2+] monitored as the ratio of Mag-Fura-2 fluorescence excited at 351 and 375 nm in the same cell. (C) Phase-contrast image of the invaded RBC. (D) Fluo-3 fluorescence signal (in the parasite cytoplasm). (E) Mag-Fura-2 (in the PV) fluorescence. The small reversible drop in fluorescence observed in A and B is an addition artifact (a slight change in focus). In fact, we have also observed it on addition of medium only. (F) Mouse RBCs were infected with P. chabaudi in the presence of 10 μM Mag-Fura-2 acid in the standard medium containing CaCl2. After washing the excess dye, the infected cells (107 cells) were resuspended in the fluorimeter cuvette, equipped with magnetic stirring, in medium without added CaCl2. The arrow shows the time of addition of 15 μM ionomycin. The Ca2+ concentration in the PV was calculated, after lysing the cells, using the F-4500 intracellular cation measurement system software (Hitachi). The [Ca2+] of the PV, measured 30 min after completion of invasion, was found to be 41 ± 1 μM (n = 5).
Mentions: The different behavior of the PV with respect to the parasite cytoplasm is even more evident in the experiment presented in Fig. 4 for P. falciparum, and identical results were obtained with P. chabaudi. In this experiment, invasion was carried out in medium containing the free acid form of another Ca2+ indicator, Mag-Fura-2, to monitor [Ca2+] within the PV. After invasion, the infected RBCs were incubated with Fluo-3/AM to load the parasite cytoplasm. The spectral characteristics of Mag-Fura-2 are sufficiently different from those of Fluo-3 that the signals from the two dyes are easily distinguished. Fig. 4 shows the two confocal images of a cell doubly loaded with Mag-Fura-2 (acid) in the PV and Fluo-3 (AM form) in the cytoplasm (Fig. 4, E and D, respectively). The phase image is also presented in C. The different localization of the two dyes appears quite clear; Fluo-3 within the parasite and Mag-Fura-2 around it. Fig. 4 shows the simultaneous dynamic measurements of [Ca2+] in the cytosol (Fig. 4 A, Fluo-3 AM) and PV (Fig. 4 B, Mag-Fura-2 acid) in the same cell. Mag-Fura-2 is a “ratiometric” dye, i.e., Ca2+ binding has opposite effects on the fluorescence emitted on excitation at 340 and 380 nm. Thus, an increase in [Ca2+], as revealed by Fluo-3, results in an increase of fluorescence, whereas an increase of the 340/380 nm ratio with Mag-Fura-2 is more directly related to the absolute increase of [Ca2+] (Grynkiewicz et al., 1985). In Fig. 4, the cells were first treated with the inhibitor of the sarco-ER Ca2+ ATPase (SERCA) thapsigargin (THG), a drug known to cause the release of Ca2+ from internal stores. Addition of THG resulted in elevation of [Ca2+], measured with both Fluo-3 and Mag-Fura-2. However, the subsequent addition of ionomycin had opposite effects on the [Ca2+] monitored by the two indicators; a further increase in [Ca2+] was monitored with cytoplasmic Fluo-3, whereas a large decrease was revealed by Mag-Fura-2 trapped in the PV. The interpretation of this experiment appears straightforward: THG mobilizes Ca2+ from Plasmodium intracellular stores, thus increasing cytoplasmic [Ca2+]; Ca2+ is then pumped out into the PV and revealed by Mag-Fura-2. Indeed, a clear delay is observed in the peak of the Mag-Fura-2 signal, as compared with that of Fluo-3. Ionomycin further releases Ca2+ from stores and causes an additional small increase in the [Ca2+] of the cytoplasm, but, given that the [Ca2+] of the PV is much higher than in the cytosol of the RBC, the ionophore transports Ca2+ out of the PV, leading to a major decrease of the Mag-Fura-2 signal.

Bottom Line: This allowed selective loading of the Ca2+ probes within the PV.The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism.We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Institute of Biosciences, University of São Paulo, São Paulo 05508-900, Brazil.

ABSTRACT
Malaria parasites, Plasmodia, spend most of their asexual life cycle within red blood cells, where they proliferate and mature. The erythrocyte cytoplasm has very low [Ca2+] (<100 nM), which is very different from the extracellular environment encountered by most eukaryotic cells. The absence of extracellular Ca2+ is usually incompatible with normal cell functions and survival. In the present work, we have tested the possibility that Plasmodia overcome the limitation posed by the erythrocyte intracellular environment through the maintenance of a high [Ca2+] within the parasitophorous vacuole (PV), the compartment formed during invasion and within which the parasites grow and divide. Thus, Plasmodia were allowed to invade erythrocytes in the presence of Ca2+ indicator dyes. This allowed selective loading of the Ca2+ probes within the PV. The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism. We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV. A transient drop of [Ca2+] in the PV for a period as short as 2 h affects the maturation process of the parasites within the erythrocytes, with a major reduction 48 h later in the percentage of schizonts, the form that re-invades the red blood cells.

Show MeSH
Related in: MedlinePlus