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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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Effects of extracellular Ca2+ on the [Ca2+] in the PV and on parasite development. P. falciparum was allowed to invade RBC in Ca2+ medium and Fluo-3 acid followed by 30 min incubation either in medium containing 1 mM CaCl2 or in [Ca2+]-free medium supplemented with 10 mM EGTA. Other conditions were as in Fig. 2. (A) Kinetics of the Fluo-3 signal in four typical cells. The black traces refer to three cells incubated initially in Ca2+ medium followed by [Ca2+]-free medium and EGTA (where indicated). The red trace refers to a single typical control cell continuously incubated in normal calcium-containing medium. The values have been normalized to the fluorescence measured at the beginning of the experiment. For clarity, the three black traces have been offset vertically by 5%. (B) Effect of a 2-h exposure to 10 mM EGTA on the proportions of P. falciparum forms measured 48 h after invasion. P. falciparum were allowed to invade the RBC in normal medium and were then incubated in medium supplemented with 10 mM EGTA for 2 h, and finally transferred to the normal Ca2+-containing medium. (C) Parasitemia (number of parasites/1,000 cells) in both conditions. Results in B and C are means ± SEM from three independent experiments; data were compared by one-way ANOVA and then by a Newman-Keuls test. Asterisk denotes statistical significance with respect to control values (P < 0.05).
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fig6: Effects of extracellular Ca2+ on the [Ca2+] in the PV and on parasite development. P. falciparum was allowed to invade RBC in Ca2+ medium and Fluo-3 acid followed by 30 min incubation either in medium containing 1 mM CaCl2 or in [Ca2+]-free medium supplemented with 10 mM EGTA. Other conditions were as in Fig. 2. (A) Kinetics of the Fluo-3 signal in four typical cells. The black traces refer to three cells incubated initially in Ca2+ medium followed by [Ca2+]-free medium and EGTA (where indicated). The red trace refers to a single typical control cell continuously incubated in normal calcium-containing medium. The values have been normalized to the fluorescence measured at the beginning of the experiment. For clarity, the three black traces have been offset vertically by 5%. (B) Effect of a 2-h exposure to 10 mM EGTA on the proportions of P. falciparum forms measured 48 h after invasion. P. falciparum were allowed to invade the RBC in normal medium and were then incubated in medium supplemented with 10 mM EGTA for 2 h, and finally transferred to the normal Ca2+-containing medium. (C) Parasitemia (number of parasites/1,000 cells) in both conditions. Results in B and C are means ± SEM from three independent experiments; data were compared by one-way ANOVA and then by a Newman-Keuls test. Asterisk denotes statistical significance with respect to control values (P < 0.05).

Mentions: The final question is whether the relatively high level of Ca2+ in the PVM is just accidental or whether it is essential for the proper development of the parasites within the RBC. The simplest direct test to answer this question is to artificially decrease the [Ca2+] in the PV and monitor the effect on parasite development. To this end, P. chabaudi or P. falciparum were allowed to invade RBCs in normal [Ca2+]-containing medium containing Fluo-3 (free acid, to monitor the [Ca2+] within the PV), and then they were incubated in Ca2+-free medium. As shown in Fig. 6 A, the Fluo-3 signal representing PV [Ca2+] for P. falciparum-infected RBC remained constant for a few minutes and then slowly decreased (similar results were obtained with P. chabaudi; not depicted). After 30 min under these conditions, the fluorescence of Fluo-3 was about 50% of the initial value. Continuous incubation for 20 h in EGTA completely prevented the maturation of the parasites, but many RBCs appeared damaged under these conditions. A less drastic protocol was thus adopted; the invaded RBCs were incubated for 2 h in an EGTA-containing medium and then were returned to the normal [Ca2+]-containing medium. Fig. 6 (B and C) shows that after 48 h, the number of infected RBCs (parasitemia) is identical in controls and in cells that have been treated for 2 h in EGTA (Fig. 6 C), indicating that the Ca2+ removal protocol did not damage the RBC. However, the percentage of immature forms was significantly increased in the Ca2+-depleted condition, and the mature form, the schizonts, was reduced by about 30%. This experiment demonstrates that maintaining a high [Ca2+] in the PV is necessary for a normal maturation of Plasmodia within the RBC, and that even a short depletion of [Ca2+] in the PV results in a substantial alteration in the maturation process.


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)

Effects of extracellular Ca2+ on the [Ca2+] in the PV and on parasite development. P. falciparum was allowed to invade RBC in Ca2+ medium and Fluo-3 acid followed by 30 min incubation either in medium containing 1 mM CaCl2 or in [Ca2+]-free medium supplemented with 10 mM EGTA. Other conditions were as in Fig. 2. (A) Kinetics of the Fluo-3 signal in four typical cells. The black traces refer to three cells incubated initially in Ca2+ medium followed by [Ca2+]-free medium and EGTA (where indicated). The red trace refers to a single typical control cell continuously incubated in normal calcium-containing medium. The values have been normalized to the fluorescence measured at the beginning of the experiment. For clarity, the three black traces have been offset vertically by 5%. (B) Effect of a 2-h exposure to 10 mM EGTA on the proportions of P. falciparum forms measured 48 h after invasion. P. falciparum were allowed to invade the RBC in normal medium and were then incubated in medium supplemented with 10 mM EGTA for 2 h, and finally transferred to the normal Ca2+-containing medium. (C) Parasitemia (number of parasites/1,000 cells) in both conditions. Results in B and C are means ± SEM from three independent experiments; data were compared by one-way ANOVA and then by a Newman-Keuls test. Asterisk denotes statistical significance with respect to control values (P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172890&req=5

fig6: Effects of extracellular Ca2+ on the [Ca2+] in the PV and on parasite development. P. falciparum was allowed to invade RBC in Ca2+ medium and Fluo-3 acid followed by 30 min incubation either in medium containing 1 mM CaCl2 or in [Ca2+]-free medium supplemented with 10 mM EGTA. Other conditions were as in Fig. 2. (A) Kinetics of the Fluo-3 signal in four typical cells. The black traces refer to three cells incubated initially in Ca2+ medium followed by [Ca2+]-free medium and EGTA (where indicated). The red trace refers to a single typical control cell continuously incubated in normal calcium-containing medium. The values have been normalized to the fluorescence measured at the beginning of the experiment. For clarity, the three black traces have been offset vertically by 5%. (B) Effect of a 2-h exposure to 10 mM EGTA on the proportions of P. falciparum forms measured 48 h after invasion. P. falciparum were allowed to invade the RBC in normal medium and were then incubated in medium supplemented with 10 mM EGTA for 2 h, and finally transferred to the normal Ca2+-containing medium. (C) Parasitemia (number of parasites/1,000 cells) in both conditions. Results in B and C are means ± SEM from three independent experiments; data were compared by one-way ANOVA and then by a Newman-Keuls test. Asterisk denotes statistical significance with respect to control values (P < 0.05).
Mentions: The final question is whether the relatively high level of Ca2+ in the PVM is just accidental or whether it is essential for the proper development of the parasites within the RBC. The simplest direct test to answer this question is to artificially decrease the [Ca2+] in the PV and monitor the effect on parasite development. To this end, P. chabaudi or P. falciparum were allowed to invade RBCs in normal [Ca2+]-containing medium containing Fluo-3 (free acid, to monitor the [Ca2+] within the PV), and then they were incubated in Ca2+-free medium. As shown in Fig. 6 A, the Fluo-3 signal representing PV [Ca2+] for P. falciparum-infected RBC remained constant for a few minutes and then slowly decreased (similar results were obtained with P. chabaudi; not depicted). After 30 min under these conditions, the fluorescence of Fluo-3 was about 50% of the initial value. Continuous incubation for 20 h in EGTA completely prevented the maturation of the parasites, but many RBCs appeared damaged under these conditions. A less drastic protocol was thus adopted; the invaded RBCs were incubated for 2 h in an EGTA-containing medium and then were returned to the normal [Ca2+]-containing medium. Fig. 6 (B and C) shows that after 48 h, the number of infected RBCs (parasitemia) is identical in controls and in cells that have been treated for 2 h in EGTA (Fig. 6 C), indicating that the Ca2+ removal protocol did not damage the RBC. However, the percentage of immature forms was significantly increased in the Ca2+-depleted condition, and the mature form, the schizonts, was reduced by about 30%. This experiment demonstrates that maintaining a high [Ca2+] in the PV is necessary for a normal maturation of Plasmodia within the RBC, and that even a short depletion of [Ca2+] in the PV results in a substantial alteration in the maturation process.

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