Cell Death of P. vivax Blood Stages Occurs in Absence of Classical Apoptotic Events and Induces Eryptosis of Parasitized Host Cells

Abstract

Elucidation of pathways regulating parasite cell death is believed to contribute to identification of novel therapeutic targets for protozoan diseases, and in this context, apoptosis-like cell death has been reported in different groups of protozoa, in which metacaspases seem to play a role. In the genus Plasmodium, apoptotic markers have been detected in P. falciparum and P. berghei, and no study focusing on P. vivax cell death has been reported so far. In the present study, we investigated the susceptibility of P. vivax to undergo apoptotic cell death after incubating mature trophozoites with the classical apoptosis inducer staurosporine. As assessed by flow cytometry assays, staurosporine inhibited parasite intraerythrocytic development, which was accompanied by a decrease in cell viability, evidenced by reduced plasmodial mitochondrial activity. However, typical signs of apoptosis, such as DNA fragmentation, chromatin condensation, and nuclear segregation, were not detected in the parasites induced to cell death, and no significant alteration in metacaspase gene expression (PvMCA1) was observed under cell death stimulus. Interestingly, dying parasites positively modulated cell death (eryptosis) of host erythrocytes, which was marked by externalization of phosphatidylserine and cell shrinkage. Our study shows for the time that P. vivax blood stages may not be susceptible to apoptosis-like processes, while they could trigger eryptosis of parasitized cells by undergoing cell death. Further studies are required to elucidate the cellular machinery involved in cell death of P. vivax parasites as well as in the modulation of host cell death.

Keywords: P. vivax; cell death; metacaspase.

Figure 1

Inhibition of P. vivax-blood stage development by staurosporine. Percoll-enriched trophozoites were incubated for 24 h in the absence (control) or presence of staurosporine (4 µM and 8 µM), and then parasite development (A) and viability (B) were measured by flow cytometry assays using Syto-16 and rhodamine 123 (RD123), respectively. Data are mean fluorescence intensity (MFI) of gated positive cells in which DNA content (Syto-16; A) and mitochondrial activity (RD123; B) were determined. Results are presented as means ± standard error (SE) of five independent experiments, normalized to percentage of non-treated parasites (control: 100%). *: p < 0.05; **: p < 0.01; ***: p < 0.001 compared with control.

Figure 2

Staurosporine induces non-apoptotic cell death in P. vivax parasites. Percoll-enriched trophozoites were incubated for 6–24 h in the absence (control) or presence of staurosporine (Stau; 4 µM or 8 µM) and then apoptotic events were examined. (A) Cytometric analysis of TUNEL assay after 24 h incubation using DNase-treated parasites as positive control. (B) Representative electron micrographs of staurosporine-induced cell death analysis. Control parasites showed normal morphology, with regular nuclei (N), preserved cytoplasm, and close juxtaposition of plasma and parasitophorous vacuole membranes. Staurosporine-treated parasites presented marked retraction and loss of cytoplasmic content as well as irregular nucleus shape, but absence of detectable chromatin condensation and nuclear segregation. (C) Gene expression levels of PvMCA1 under cell death stimulus (4 µM staurosporine), as evaluated by qPCR with P. vivax β-tubulin gene as endogenous control. Results are presented as means ± standard error of three independent experiments, and no significant difference was observed between control and staurosporine-treated parasites.

Figure 3

Staurosporine-induced parasite cell death positively modulates eryptosis of parasitized red blood cells (pRBCs). Percoll-enriched trophozoites were induced to cell death for 24 h with staurosporine (4 µM and 8 µM), and eryptosis of pRBCs (Syto-16-positive cells) and nRBCs (Syto-16-negative cells) was evaluated by annexin V staining (A,C) and cell size measurement (B, pRBCs) in flow cytometry analysis. (A) Levels of phosphatidylserine-exposed pRBCs (annexin V+), normalized to percentage of non-treated parasites (control: 100%). (B) Cell size of eryptotic pRBCs (annexin V+) compared to non-eryptotic pRBCs (annexin V-; 100%). (C) Levels of phosphatidylserine-exposed nRBCs (annexin V+), normalized to percentage of non-treated parasites (control: 100%). Data are presented as means ± standard error of four independent experiments. *: p < 0.05; **: p < 0.01, compared with annexin V- (B) and control (A), respectively.