Abstract
Plasmodium falciparum, the most important etiological agent of human malaria, is endowed with a highly complex cell cycle that is essential for its successful replication within the host. A number of evidence suggest that changes in parasite Ca2+ levels occur during the intracellular cycle of the parasites and play a role in modulating its functions within the RBC. However, the molecular identification of Plasmodium receptors linked with calcium signalling and the causal relationship between Ca2+ increases and parasite functions are still largely mysterious. We here describe that increases in P. falciparum Ca2+ levels, induced by extracellular ATP, modulate parasite invasion. In particular, we show that addition of ATP leads to an increase of cytosolic Ca2+ in trophozoites and segmented schizonts. Addition of the compounds KN62 and Ip5I on parasites blocked the ATP-induced rise in [Ca2+]c. Besides, the compounds or hydrolysis of ATP with apyrase added in culture drastically reduce RBC infection by parasites, suggesting strongly a role of extracellular ATP during RBC invasion. The use of purinoceptor antagonists Ip5I and KN62 in this study suggests the presence of putative purinoceptor in P. falciparum. In conclusion, we have demonstrated that increases in [Ca2+]c in the malarial parasite P. falciparum by ATP leads to the modulation of its invasion of red blood cells.
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Abbreviations
- THG:
-
Thapsigargin
- [Ca2+]c :
-
Calcium cytosolic concentration
- RBC:
-
Red blood cell
- SmATPDase:
-
S. mansoni ATP-diphosphohydrolase
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Acknowledgements
We are grateful to Dr. Sergio Verjovski de Almeida (Chemistry Institute, São Paulo University) for kindly providing the recombinant apyrase from S. mansoni. This work was supported by grants from Brazilian Agencies FAPESP, CNPq (to C.R.S.G)-INCT-InBqmed, and Pronex Malaria-MS-DECIT.
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ESM 1
Addition of nucleosides CTP, GTP and ADP has no effect on calcium rise in trophozoites of P. falciparum. However, UTP (10 μM) is able to increase [Ca2+]c. Each experiment was carried out in triplicates at least three times. Student’s t test was applied, *p = 0.0322 (DOC 231 kb)
ESM 2
a ATP does not lead to a Ca2+ rise in absence of external calcium (adding 3 mM EGTA) in schizont parasites. Where indicated, ATP (100 μM) was added. b Addition of UTP (10 μM) does not induce an increase [Ca2+]c in trophozoite parasites in the absence extracellular of calcium. Thapsigargin addition as control is able to raise [Ca2+]c (DOC 95 kb)
ESM 3
Cultures of Plasmodium 1 h post-invasion were incubated in the presence of ATP (50 μM), KN-62 (1 μM) and apyrase (10 nM) for 17 h at 37°C. Percentage of parasitemia presented as mean ± SEM are based on three independent experiments in triplicate (DOC 262 kb)
ESM 4
Development of parasites after in vitro treatment with KN62, Ip5I or apyrase during 24 or 48 h. P. falciparum-infected RBCs do not change morphology after treatment for 24 or 48 h, showing that these inhibitors act on parasite invasion but do not have any action on parasite development inside the RBC. Slides were fixed with methanol and stained with Giemsa (DOC 1833 kb)
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Levano-Garcia, J., Dluzewski, A.R., Markus, R.P. et al. Purinergic signalling is involved in the malaria parasite Plasmodium falciparum invasion to red blood cells. Purinergic Signalling 6, 365–372 (2010). https://doi.org/10.1007/s11302-010-9202-y
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DOI: https://doi.org/10.1007/s11302-010-9202-y