Background
With 3.3 billion of people at risk to be infected and an estimated death toll of 429,000 in 2015 [
1] malaria remains a great threat in tropical countries. Artemisinin combination therapies, although highly effective, have met with the development of parasite resistance in recent years, exacerbating the need for the identification of new anti-malarial drugs [
2].
In
Plasmodium falciparum, Ca
2+ signalling regulates different physiological processes involved in the life cycle of parasite, such as erythrocyte invasion [
3,
4], merozoite egress from the infected erythrocyte [
5] and parasite development [
6]. As well as in higher eukaryotic cells, different Ca
2+ stores have been found in
Plasmodium: endoplasmic reticulum, acidocalcisomes, mitochondria and the digestive vacuole. Acidocalcisomes are acidic organelles rich in Ca
2+ and other cations bound to phosphate polymers. A Ca
2+-ATPase, a bafilomycin-sensitive vacuolar V-H
+-ATPase, a V-H
+-PPase and a Ca
2+–H
+ antiporter localize on the membrane of these organelles, regulating Ca
2+ balance [
7]. It has been proposed that another acidic organelle of
P. falciparum, the food vacuole, could be a Ca
2+ store sensitive to thapsigargin, bafilomycin and NH
4Cl [
8]. Ca
2+ homeostasis in
Plasmodium is mainly regulated by two Ca
2+-ATPase, the thapsigargin-insensitive PfATP4 [
9] and the thapsigargin-sensitive sarco/endoplasmic reticulum Ca
2+-ATPase (SERCA) orthologue PfATP6 [
10]. Scheibel and co-workers showed that both Ca
2+ and calmodulin antagonists inhibit the growth of
P. falciparum [
11]. Moreover, the identification of SERCA as a target of artemisinins [
10] highlights the crucial role of Ca
2+ signalling in the life cycle of the parasite.
In higher eukaryotic cells, different intracellular second messengers finely regulate the spatio-temporal fluctuation of cytosolic Ca
2+ concentration, mobilizing calcium from different intracellular stores. Inositol 1,4,5-bisphosphate (IP3) [
12], cyclic ADP-ribose (cADPR) [
13] and nicotinic acid adenine dinucleotide phosphate (NAADP) [
14] have been so far identified as Ca
2+-mobilizing second messengers in higher eukaryotic cells. The second messenger NAADP was described for the first time as a potent Ca
2+ mobilizing agent in sea urchin eggs [
14] and subsequently in ascidian and starfish oocytes [
15,
16], in plants [
17] and in higher eukaryotic cells [
18‐
21], suggesting a highly conserved feature in evolution for this molecule.
In
Apicomplexa, second messengers involved in Ca
2+ mobilization from intracellular organelles have been poorly investigated. At present only a few works have investigated the role of IP3 and cADPR in Ca
2+ release in
P. falciparum [
22‐
24] and none focused on NAADP, possibly for the absence in the parasite genome of sequences homologous to the putative two pore channels (TPCs) NAADP-receptor. Recently, Ned-19 has been identified by virtual screening as a specific inhibitor of NAADP-induced calcium signalling in sea urchin eggs and pancreatic beta cells, and was shown to bind NAADP receptors as a fluorescent probe [
25]. Subsequently, Ned-19 mediated Ca
2+ signalling inhibition and its biological effects have been reported in different mammalian cells. Ned-19 has been shown to inhibit histamine-induced secretion of von Willebrand factor (vWF) in endothelial cells [
20], endothelin-1 (ET-1)-induced contraction of smooth muscle cells [
26], NAADP-induced acrosome reaction in mammalian spermatozoa [
27] and exocytosis of cytolytic granules in cytotoxic T Lymphocytes and VEGF-induced neoangiogenesis [
28,
29].
The aim of this study was to investigate the effect of Ned-19 on the blood stage development of P. falciparum. Results show for the first time that Ned-19 specifically impairs both the growth and the ability to transform into multinucleate schizonts of the asexual trophozoite stages, suggesting a crucial role of NAADP in life cycle progression. This work also shows that Ned-19 inhibits spontaneous Ca2+ oscillations in early ring and trophozoite stages, which also suggests an important role of NAADP in Ca2+ homeostasis of P. falciparum.
Methods
Plasmodium falciparum parasites and cultures
Parasites from clone 3D7 [
30] were cultured in 0
+ human red blood cells at 5% haematocrit in RPMI 1640 plus hypoxanthine 50 mg/mL, HEPES 25 mM, 0.225% sodium bicarbonate and 10 mg/mL gentamicin, supplemented with 10% heat inactivated human serum. Parasites were kept at 37 °C, in a 2% O
2, 5% CO
2 and 93% N
2 atmosphere. Percoll cushion and sorbitol treatment for parasite synchronization were performed as described [
31,
32] previously. In parasite synchronization, sorbitol treatment of newly invaded parasites from Percoll purified schizonts was performed 3 h after Percoll treatment to obtain a parasite synchronization window of maximum 3 h. For gametocyte production, asynchronous parasites were grown to high parasitaemia (>8%) and culture medium was doubled at this point. The day after, 50 mM N-acetylglucosamine was added to medium and maintained for 3 days, until no asexual parasites were detected in the culture. Stage II gametocytes were detected 48 h after the addition of N-acetylglucosamine, while mature stage V appeared from 9 days after the treatment.
Parasitaemia was measured through Giemsa staining of culture blood smears (counting of at least 2000 RBCs) or FACS using CYBRGreen staining as previously described (counting of at least 50,000 cells) [
33]. FACSAria I (BD Biosciences, Erembodegem, Belgium) equipped with three lasers (488, 635 and 407 nm violet solid state laser) was used to determine parasitaemia to a precision of 0.1%. The results were analyzed by BD FACSDiva Software version 6.1.3 (BD Biosciences).
Ned-19 and Ned-20 treatments
Ned-19 (Tocris bioscience) was resuspended in a stock solution of sterile dimethyl sulfoxide (DMSO) at 100 mM and kept at −20 °C until added to the Plasmodium cultures at the specified concentrations. In the case of Ned-20 the stock solution was kindly provided by Grant Churchill (Oxford University) at 10 mM and kept at −20 °C until use. Control cultures were incubated with a DMSO concentration equivalent to that of the treated cultures. Differences in parasitaemia between treated and untreated cultures were evaluated through Student’s t test.
Microscopy
Parasite cultures were incubated with 200 μM Ned-19 and 1 μM Lysotracker Green DND-26 (ThermoFisher Scientific) for 30 min at 37 °C in agitation and observed with a fluorescence microscope. A Zeiss Observer.Z1 inverted microscope was used to visualize live samples. Images were acquired using a Zeiss AxioCam MRm Rev. 3 FireWire camera through a Zeiss C-Apochromat 63x/1, 20 objective. Filters used to detect fluorescence were EX: 365–395, EM: 445–450 (Ned-19) and EX: 440–470, EM: 525–550 (Lysotracker Green DND-26). Giemsa-stained smears were examined to confirm stages of the synchronized parasites: proportions >90% of trophozoites, schizonts and ring forms were, respectively, observed at 32, 46 and 48 h after synchronization.
Electron microscopy
Parasite culture samples enriched in infected red blood cells by MACS (Magnetic-activated cell sorting) were fixed overnight in cold 2.5% glutaraldehyde in 0.1 M cacodylate buffer, postfixed in 2% osmium tetroxide for 2 h and treated for 30 min with 1% tannic acid in 0.05 M cacodylate buffer. Pellets were then dehydrated in ethanol and processed for Epon embedding. Ultrathin sections were contrasted in lead hydroxide and analyzed in a Hitachi 7000 transmission electron microscope.
Calcium imaging
In Ca
2+ imaging experiments, protocol was adapted from [
6], using the ratiometric Fura-2-AM as calcium reporter. Samples from cultures at high parasitaemia (5–10%) of the desired parasite stage were generated through parasite synchronization as follows: for early rings, parasites were harvested 50 h after the initial Percoll treatment, while for early trophozoites parasites were harvested 26 h after Percoll treatment. Cultures where then washed in BSA− medium for Ca
2+ imaging (RPMI 1640 medium without phenol red supplemented with 25 mM HEPES, 24 mM sodium bicarbonate, 0.5 g/L
l-glutamine and 50 mg/L hypoxanthine) and resuspended at 5% haematocrit in loading medium [BSA− medium supplemented with 1:100 PowerLoad (ThermoFisher Scientific)] with 3 μM Fura-2-AM (ThermoFisher Scientific) at 37 °C for 150 min in agitation. Loading medium was then washed away and cells were resuspended at 2.5% haematocrit in BSA+ medium [BSA− medium supplemented with 0.5% Albumax I and 25 mg/mL gentamicin (Sigma)]. Ned-19 100 μM or 0.1% DMSO was added to 1 mL of this cell suspension. This was plated in glass-bottomed 30 mm dishes previously coated with poly-
l-lysine and incubated in a 2% O
2, 5% CO
2 and 93% N
2 atmosphere for 45 min. Unbound cells were washed by gently rinsing the surface of the plate and substituting supernatant with BSA+ medium with Ned-19 or DMSO. Medium volume was then adjusted to 1 mL of BSA+ with Ned-19 or DMSO. This treatment resulted in cells treated with Ned-19 or DMSO for at least 45 min prior to the beginning of measurements, and the Ned-19 and DMSO treatments being kept during calcium imaging.
Ca2+ mobilization was measured in presence or absence of Ned-19. Plates with Fura-2-loaded cells were placed into a culture chamber at 37 °C on the stage of an inverted fluorescence microscope (Nikon, TE2000E), connected to a cooled CCD camera (512B Cascade, Princeton Instruments, AZ). Samples were illuminated alternately at 340 and 380 nm using a random access monochromator (Photon Technology International, NJ) and emission was detected using a 510 nm emission filter. Images (1 set of emission at 340 and 380 nm every 1.5 s) were acquired using Metafluor software (Universal Imaging Corporation, Downingtown, PA).
Images were analysed by generating square 4 × 4 mm ROIs (region of interest) encompassing a single parasite and recording the intensity of the emission for the ROI at 340 and 380 nm. Background was normalized for each ROI using a ROI the size of the full image, and ratio (R) was calculated as F(340)/F(380) for each ROI and time-point, obtaining time-courses for each ROI. R values were then normalized as R/Rmin−1.
OCTAVE free software with the script findpeaksfit.m by T.C. O’Haver [
34] was used to identify and measure height of peaks, given as R/R
min−1 values. Oscillation series were subdivided into fragments of 100 s each and script was run with settings as follows: x, y, SlopeThreshold = 0.0005, AmpThreshold = 0, smoothwide = 3, peakgroup = 3, smoothtype = 3, peakshape = 1, extra = 0, NumTrials = 0, autozero = 1, fixedparameters = 0, plots = 0. Differences between the height of peaks in DMSO and Ned-19 treated parasites were evaluated through unpaired Student’s t test analysis. For DMSO treated rings, N (number of time-courses) = 6, n (total number of peaks detected) = 622. For Ned-19 treated rings, N = 6, n = 265. In the case of trophozoites, for DMSO treated parasites N = 8, n = 349, for Ned-19 treated parasites N = 3, n = 150.
Discussion
This study investigated for the first time the effect of the NAADP antagonist Ned-19 on P. falciparum development, revealing that this compound inhibits the parasite asexual cycle and suggesting a potential role of NAADP in regulating P. falciparum Ca2+ signalling.
While the effects of inositol 1,4,5-bisphosphate (IP3)-induced [
22,
23], cyclic ADP-ribose (cADPR) and ATP-induced [
38] Ca
2+ release have been previously described in
P. falciparum [
24] and in
Toxoplasma gondii [
39], the role of NAADP in this process and in parasite physiology has never been investigated in apicomplexan parasites. Moreover, the receptors involved in second messenger-induced Ca
2+ release are unknown, since the orthologue of both IP3 receptors (IP3Rs) and ryanodine receptors (RyRs) have not been identified and two pore channels (TPCs) NAADP-receptor homologous sequences have not been found in the genome of
P. falciparum [
40]. Results of the present work show that a highly specific inhibitor of NAADP-induced Ca
2+ release (Ned-19) blocks the transition from early to late trophozoite stages and schizont maturation in
P. falciparum, supporting the notion that Ca
2+ signalling plays a crucial role in different stages of
P. falciparum asexual cycle. This supports previous findings [
6] describing a pivotal role of Ca
2+ signalling in the development of
P. falciparum. In that study, the stage-specific spontaneous Ca
2+ oscillations in the intra-erythrocytic stages of
P. falciparum were inhibited by a specific inhibitor of IP3 (2-APB), resulting in developmental defects and leading to parasite death [
6]. Interestingly, in that report and in the present work, the decrease in Ca
2+ oscillations caused by 2-APB and Ned-19 at the early ring stage was not sufficient to block parasite development, while ability to affect Ca
2+ oscillations later at the trophozoite stage was in both cases associated to an inhibitory effect on the growth of these parasites. The ability of Ca
2+ signalling in regulating cellular response in different stages of parasite life cycle relies on a family of Ca
2+-dependent protein kinases. Among these, PfCDPK1 (calcium-dependent protein kinase-1) is expressed during the intraerythrocytic schizogony and in the sporozoite stage, and it is crucial for the viability of
P. falciparum. Kato and co-workers identified purkalfamine as an inhibitor of PfCDPK1 able to block parasite development at the late schizont stage [
41]. It is known that CDPK1 is crucial also in the sexual life cycle stages of
Plasmodium berghei, regulating zygote development [
42]. Other studies showed that another Ca
2+-dependent protein kinase, PfCDPK5, controls parasite egress from erythrocytes [
43].
These data altogether highlight the potential impact of Ca
2+ signalling antagonists as new antimalarial drugs able to block different stages of the life cycle of
P. falciparum. Accordingly, different research groups recently focused their efforts in performing large scale drug screening to identify new compounds able to target Ca
2+-dependent protein kinases or more generally Ca
2+-signalling in
Apicomplexa [
44‐
46]. In this context, the results of this study introduce NAADP signalling as a new potential target for the development of drugs able to impair Ca
2+ homeostasis in
P. falciparum.
Spontaneous Ca
2+ oscillations have been poorly investigated in
P. falciparum. It has been shown that a specific inhibitor of IP3 (2-APB) was able to block Ca
2+ oscillations in ring and trophozoite stages [
6], while a selective melatonin receptor antagonist, luzindole, was able to inhibit spontaneous Ca
2+ oscillations mainly in the ring stages of the parasite [
37]. The result that Ned-19 affects Ca
2+ oscillations in early rings and trophozoites suggests that both NAADP and IP3 are involved in this process. This result is consistent with the observations that NAADP induces Ca
2+ oscillations in different eukaryotic cell types, from sea urchin eggs [
19] to pancreatic acinar cells [
47‐
49], through a two-pool mechanism involving both endoplasmic reticulum-independent NAADP-sensitive stores and the endoplasmic reticulum IP3- and cADPR-sensitive stores [
50,
51]. In this model, NAADP is crucial in priming Ca
2+-induced Ca
2+-Release from endoplasmic reticulum (CICR), through a mechanism of overloading and spontaneous Ca
2+ release from these stores [
50].
Besides using Ned-19 as a specific inhibitor of NAADP signalling, this compound was used to fluorescently tag the NAADP receptor in living cells [
25]. These experiments showed the co-localization of the Ned-19 and the Lysotracker fluorescent signals in different stages of the life cycle of
P. falciparum, suggesting that yet to be identified NAADP-receptors may localize on acidic compartments also in
P. falciparum and possibly in
Apicomplexa, similarly to several mammalian cell types [
18,
20]. As however different acidic organelles are labelled by Lysotracker dye in
P. falciparum (acidocalcisomes, digestive vacuole and lysosomes), further experiments are needed to identity the Ned-19 positive organelles.
Authors’ contributions
PSC and GG designed and conducted the experiments and drafted the manuscript. AF contributed to design calcium oscillation experiments. FP prepared and analyzed electron micrographs, contributed to design experiments and discussed the manuscript. PA and AF contributed to the experimental design and discussed the manuscript. All authors read and approved the final manuscript.