Extracellular vesicles carrying lactate dehydrogenase induce suicide in increased population density of Plasmodium falciparum in vitro

Abstract

Even with access to sufficient nutrients and atmosphere, Plasmodium falciparum can barely be cultured at maximum growth capacity in vitro conditions. Because of this behavior, it has been suggested that P. falciparum has self-regulatory mechanisms in response to density stress. Only recently has this process begun to be acknowledged and characteristics of a programmed cell death been assigned to the parasite at high parasitaemia in vitro cultures. In searching for death signals within the parasite community, we have found that extracellular vesicles (EVs) of P. falciparum from high parasitaemia cultures are able to induce programmed cell death processes in the population. A comparative proteomic analysis of EVs from low (EV_L) and high (EV_H) parasitaemia cultures was conducted, pointing to lactate dehydrogenase from P. falciparum (PfLDH) as the only parasite protein overexpressed in the later. Although the major function of P. falciparum lactate dehydrogenase (PfLDH) is the conversion of pyruvate to lactate, a key process in the production of energy in most living organisms, we investigated its possible role in the mechanism of parasite density control by intercellular signaling, given that PfLDH had already been listed as a component of extracellular vesicles of P. falciparum. In this study we present evidence of the EV-associated PfLDH regulation of parasite population by inducing apoptosis in highly parasitized cultures.

Figure 1

Highest parasitaemia growth. Cultures coming from 1%, 5.5%, 13.2% and 20.5% parasitaemias were split to 1% and monitored for 48 h.

Figure 2

Growth inhibition of P. falciparum by EVs. The growth of parasites after distinct EV treatments was measured by flow cytometry. The average and S.D. of 3 replicas for each treatment is shown. (mean ± s.e.m.; n = 3 replicas), ****P < 0.0001 versus PBS and 3D7 growth control (Bonferroni’s test). Chloroquine was used as a drug control.

Figure 3

Inhibition of PfLDH rescues the population regulation of EVs. Growth fold change was measured after distinct EV treatments and the addition of gossypol by flow cytometry. The average and S.D. of 3 replicas for each treatment is shown. (mean ± s.e.m.; n = 3 replicas), *P < 0.05 versus PBS control (Bonferroni’s test).

Figure 4

ROS detection after addition of EVs and 0.25 µM gossypol. MFI was measured by flow cytometry after diverse EV and gossypol treatment. The average and S.D. of 3 replicas for each treatment is shown. (mean ± s.e.m.; n = 3 replicas), ****P < 0.0001 versus PBS control (Bonferroni’s test).

Figure 5

Phosphatidylserine translocation after challenge with EVs.

Figure 6

Caspase activity triggered by EV_H. Caspase activity detection after EV treatment. MFI was measured after distinct EV treatments by flow cytometry. The average and S.D. of 3 replicas for each treatment is shown. (mean ± s.e.m.; n = 3 replicas), *P < 0.05, **P < 0.01, EV_L versus EV_H (Bonferroni’s test).

Figure 7

Rescued growth of P. falciparum after challenge with EV_H in the presence of a pancaspase inhibitor. Growth fold change was measured by flow cytometry after distinct EV treatments with the addition or not of the pancaspase inhibitor Z-VAD-FMK. The average and S.D. of 3 replicas for each treatment is shown. (mean ± s.e.m.; n = 3 replicas), ***P < 0.005 and *P < 0.05 versus PBS control (Bonferroni’s test).

Figure 8

APAD bioassay to measure PfLDH enzymatic activity caused by the addition of EVs. Absorbance was measured after distinct EV treatments at O. D. 650 nm. The average and S.D. of 4 replicas for each treatment is shown. (mean ± s.e.m.; n = 4 replicas), ****P < 0.0001 versus uRBC + PBS control (Bonferroni’s test).

Figure 9

Diagram of the hypothesized action of PfLDH in iRBCs after being transported by EVs. Density stressed cultures release EVs which cargo (PfLDH) is able to affect low density populations inducing death through apoptotic events triggered by this enzyme. PfLDH could inhibit the conversion of lactate to pyruvate producing an excess of the former which is detrimental to the parasite. Also, PfLDH could increment the production of ROS and the induction of apoptotic events leading to the suicide of the individual parasite.