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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

Extracellular ATP triggers proteolysis and cytosolic Ca2+ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Laura Nogueira Cruz, Maria Aparecida Juliano, Alexandre Budu, Luiz Juliano, Anthony A Holder, Michael J Blackman, Célia RS Garcia
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-69) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

LNC designed the study, collected the data and prepared the first draft. MAJ and LJ synthesized the FRET peptides. AB performed confocal experiment (1B) and wrote the draft related to it. AAH and MJB provided the monoclonal antibody MAb25.1 and analysed the data. CRSG designed the study and prepared the first draft. All authors put forward different ideas, contributed to the interpretation of the data, helped with early drafts and contributed to and agreed the final draft. All authors read and approved the final manuscript

Abstract

Background

Plasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host.

Methods

Using the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca2+ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated.

Results

The protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 μM) and PPADS (50 μM) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 μM), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 μM) to isolated parasites previously loaded with Fluo4/AM in a Ca2+-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 μM), TNP-ATP (50 μM) or the purinergic blockers KN-62 (10 μM) and Ip5I (10 μM). Incubating P. berghei infected cells with KN-62 (200 μM) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 μM) led to an increase in rings forms (82% ± 4, n = 11) and a decrease in trophozoite forms (18% ± 4, n = 11).

Conclusions

The data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway.
Zusatzmaterial
Additional file 1: Extracellular adenosine but not GTP triggers intracellular protease activity in P. berghei and P. yoelii (A and B). Bar graph analyses of peptide hydrolysis after GTP (50 μM) (0.99 ± 0.09, n = 5, P = 0.63 and 1.06 ± 0.05, n = 9, P = 0.365) or adenosine (10 μM) (1.51 ± 0.11, n = 7, P = 0.006 and 2.15 ± 0.11, n = 3, P < 0.0001) in P. berghei and P. yoelii, respectively. P values were calculated by comparison with control data (1.05 ± 0.08, n = 6 and 1.12 ± 0.03, n = 6, respectively). Isolated parasites (108 cells ml-1) were incubated in MOPS buffer with 1 mM calcium in a 1 ml cuvette. The fluorescence was measured continuously 1 min after addition of the peptide Abz-AIKFFARQ-EDDnp (10 μM). (PDF 65 KB)
12936_2011_2077_MOESM1_ESM.PDF
Additional file 2: Dose response effects of adenosine on [Ca2+] c rise in P. yoelii and P. berghei . (A and B). Analyses of Ca2+ concentration in P. berghei and P.yoelii Fluo4/AM labelled isolated parasites (108 cells ml-1) after addition of adenosine (10 and 15 μM) (1.59 a.u. ± 0.07, n = 18, P = 0.007 and 1.5 a.u. ± 0.062, n = 8, P = 0.002, respectively) in P. berghei or adenosine (10 μM) (1.78 a.u. ± 0.16, n = 8, P = 0.004) in P. yoelii. P values were calculated by comparison with control data (1.05 a.u. ± 0.01, n = 3 and 0.99 a.u. ± 0.064, n = 5; respectively). Bar graphs represent means with SEM. (PDF 63 KB)
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Authors’ original file for figure 1
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