Background
Plasmodium falciparum is the main causative agent of malaria, killing hundreds of thousands of people annually [
1]. Resistance to anti-malarial drugs is widespread, and promising anti-malarial drugs that are effective and affordable for people with low income are slow in development. For decades, chloroquine (CQ) was the safest, most affordable and effective drug against malaria, saving the lives of millions of people until resistance emerged [
2]. To date, its mode of action is still not fully understood. Some researchers suggest that CQ could have more than one intracellular target, making it more difficult for parasites to develop resistance [
3‐
5].
To effectively use CQ as an anti-malarial treatment in areas where CQ resistance has been reported, it is imperative to gain more insight into its mechanism of action. The small size and uncharged form of CQ at neutral pH makes it difficult to use for molecular biological experiments to determine its accumulation in intracellular compartments or its affinity to other molecules.
Chloroquine transport studies have mainly been performed using radiolabelled CQ [
6‐
8]. The great advantage of this method is that the intrinsic properties of CQ remain unaltered. However, it is not possible to study intracellular distribution and trafficking of radiolabelled CQ in intact parasites. Therefore, fluorescently labelled CQ analogues provide a new means to evaluate intracellular activity of this drug. To date, there are two fluorescently labelled chloroquine analogues commercially available: LynxTag-CQ™
BLUE and LynxTag-CQ™
GREEN (BioLynx Technologies, Singapore, Singapore). While the literature cites that CQ
BLUE was mainly found to be fluorescent in the parasite cytosol [
9‐
11], only one study has been published with CQ
GREEN. This study showed CQ
GREEN accumulation in the DV of CQ-sensitive (CQS)
P. falciparum parasites and yeast-derived microsomes expressing the
P. falciparum chloroquine resistance transporter (PfCRT) [
12].
For this study, CQGREEN was compared to unmodified CQ and investigated whether the fluorescently tagged CQ analogue can be used to obtain insight into intracellular CQ trafficking and localization. A better knowledge of the differences in CQ accumulation, distribution, uptake and efflux between CQS and CQ-resistant (CQR) strains is imperative to understand drug resistance. The findings show that, although CQ and CQGREEN show comparable IC50 values in CQS and CQR parasites, discrepancies were seen between CQGREEN and unmodified CQ in their expected intracellular localization. A strong CQGREEN fluorescence was mainly seen in the cytosol of both CQS and CQR strains.
Methods
Parasite strains and culture conditions
Two CQS (3D7, HB3) and two CQR (FCB, Dd2) strains were used for all experiments. Parasites were cultured continuously, as described by Trager and Jensen [
13], with modifications. Briefly, parasites at 5% haematocrit were propagated in culture medium containing RPMI 1640 (Life Technologies, Burlington, ON, Canada) supplemented with 25 mM HEPES, 2 mM
l-glutamine, gentamicin (20 µg/ml) (Life Technologies, Burlington, ON, Canada), 100 µM hypoxanthine (Sigma-Aldrich, Oakville, ON, Canada), and 0.5% AlbuMAX I (Life Technologies, Burlington, ON, Canada). Parasites were maintained at 37 °C with an atmosphere of 5% CO
2, 3% O
2 and 92% N
2. A
+ red blood cells were obtained from the Interstate Blood Bank (Memphis, TN, USA). Giemsa-stained blood smears were prepared daily to monitor parasite growth. For synchronization, parasites were treated with 5%
d-sorbitol (BioShop Canada, Burlington, ON, Canada) for 10 min at 37 °C; sorbitol was removed and parasites were washed once before returning them back into culture.
Cytotoxicity assays
Cytotoxicity assays were performed as described previously [
14‐
17], with modifications. Cultures of 0.5% parasitaemia and 2% haematocrit were incubated in 100 µl culture medium per well in a 96-well plate assay. A drug dilution series of 1:3 was prepared, starting with 1 µM as highest substrate concentration. Plates were incubated at 37 °C, 5% CO
2 and 3% O
2 for 72 h, then frozen and stored at − 80 °C.
Readouts of the assay were performed using the SYBR Green I detection method. For this, plates were thawed at room temperature and 100 µl 2× lysis buffer (20 mM Tris pH 7.5, 5 mM EDTA, 0.008% saponin, 0.08% Triton X-100, and 0.2 µl SYBR Green I/ml) was added to each well. Plates were incubated in the dark for at least 1 h. Fluorescence intensity was determined using a Synergy H4 plate reader (Fisher Scientific, Nepean, ON, Canada) with 485 nm excitation and 520 nm emission wavelengths. IC
50 values were determined by fitting concentration response curves with a custom-made procedure for IGOR Pro 6.2 based on a R script kindly provided by Le Nagard [
18,
19].
Fluorescence of CQGREEN at varying pH
To determine if fluorescence intensity of CQGREEN is altered at varying pH, buffer solutions were prepared ranging from pH 5.0–8.0 based on a modified Ringer’s solution (122.5 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl2, 0.8 mM MgCl2, 11 mM d-glucose). Buffer solutions contained 10 mM MES for pH 5.0–6.5 or 10 mM HEPES for pH 7.0–8.0. In a 96-well plate, 100 µl buffer solutions of varying pH containing 1 µM CQGREEN were prepared in triplicate. Fluorescence intensity was measured at 37 °C using the Synergy H4 fluorimeter (Bio-Tek, Winooski, VT, USA). Excitation spectra ranging from 400 to 520 nm were measured with fixed emission at 540 nm, and emission spectra ranging from 500 to 630 nm were measured with fixed excitation at 488 nm. Quantification was done using Microsoft Excel 2013.
Live cell imaging
Intracellular CQGREEN accumulation at different concentration and pH was analysed in intact parasitized red blood cells. For this, 3D7 trophozoite stage parasites were incubated for 30 min at 37 °C with 25, 50, 500 nM or 2.5 µM CQGREEN in Ringer’s solution with pH 7.4 or buffer solutions with pH 7.2 or 5.2. Images were taken using a 488 nm argon laser (12.5 mW, 0.8%) on a Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with a water-corrected objective (C-apochromat 63×/1.20 W Korr M27). Emission range was set to 500–600 nm. Localization of CQGREEN within the parasite under various pH conditions was determined using the ZEN 2010 software (Carl Zeiss MicroImaging, Oberkochen, Germany).
For long-term incubation with CQGREEN, early trophozoite stage 3D7 parasites were incubated with 100 nM, 300 nM and 500 nM CQGREEN for 7 h in culture medium at 37 °C, 3% O2, 5% CO2. Parasites were then transferred onto a microscope chamber and imaged using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany), a 63× water corrected objective (C-apochromat 63×/1.20 W Korr M27) and a 488 nm laser (12.5 mW, 0.8%). Emission range was set to 500–600 nm. A constant temperature of 37 °C was maintained during the measurements using a stage-top incubator (Tokai Hit, Shizuoka-ken, Japan). Images were analysed with the ZEN 2010 software (Carl Zeiss MicroImaging, Oberkochen, Germany).
To analyse CQ
GREEN uptake of CQS and CQR strains, synchronized trophozoite stage parasites were washed in Ringer’s solution and transferred onto a microscope chamber. Parasites were allowed to settle for 5 min, then the solution was aspirated and replaced with new Ringer’s solution containing 500 nM CQ
GREEN. If verapamil was added, parasites were preincubated with 1 µM VP for 15 min at 37 °C, then transferred onto a microscope chamber. For the time lapse measurement, 500 nM CQ
GREEN were added to the Ringer’s solution with or without 1 µM VP. Images were taken every 3 s for a time span of 500 s using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany) and a 63× water corrected objective (C-apochromat 63×/1.20 W Korr M27). Excitation was done using a 488 nm laser (12.5 mW, 0.8%), and an emission range from 500 to 600 nm. Regions of interest (ROI) were set for the parasite cytosol, DV, infected RBC cytosol and uninfected RBC. Fluorescence of ROIs was determined for each time point using ImageJ 1.47q (National Institutes of Health, USA). Uptake rates were calculated for the cytosol and DV during the saturation phase and analysed using IGOR Pro 6.2 by fitting influx to ƒ = y
0 + a(1 − e
−bx) and initial rates (v
0) to y = mx + b, as described previously [
20]. Graphs were created using IGOR Pro 6.2.
Discussion
The exact mechanisms responsible for chloroquine resistance have eluded researchers for decades. It has long been described that CQR strains accumulate two to sevenfold less CQ than CQS strains [
35]. Nevertheless, CQR strains are still able to tolerate higher intracellular CQ concentrations than CQS strains before irreversible cell damage occurs [
3,
7,
33,
36]. Researchers have advocated that inhibition of haemozoin formation by CQ is not sufficient to explain its effect on parasite killing [
37]. Furthermore, a possible role of CQ in the parasite’s cytosol has also been proposed [
5]. A fluorescently labelled CQ analogue could provide insight into the intracellular distribution of CQ that is not attributed to diffusion alone. This study set out to examine CQ uptake in live parasites using CQ
GREEN.
IC
50 values were compared in CQS and CQR strains after exposure to CQ or CQ
GREEN. A decrease in the efficacy for CQ
GREEN may suggest that the CQ analogue does not reach its site of action as efficiently as its native form or binds to its target less efficiently. Calculated IC
50 values in CQS strains showed that CQ was twice as effective as CQ
GREEN in parasite growth inhibition. In comparison, similar IC
50 values for CQ and CQ
GREEN were determined in CQR strains. In a previous study by Loh and colleagues [
12], IC
50 values in 3D7 were approx. fivefold higher for CQ
GREEN compared to CQ, and nearly doubled in the CQR strain K1. Thus, the parasites used for this study seemed to be more sensitive to CQ
GREEN exposure than reported in earlier publications. In both studies, drug resistance could be reversed by chemosensitizers such as verapamil, suggesting that CQ
GREEN’s mode of action is similar to CQ.
Although CQGREEN seemed slightly less effective than CQ in the IC50 assays for CQS strains, its Bodipy fluorescent tag makes it suitable for live cell imaging. This was confirmed by fluorometric readings, where a strong fluorescence emission signal for CQGREEN was measured at a 488 nm excitation wavelength. Spectral scans showed a strong fluorescence signal at the measured pH spectrum ranging from pH 5–8 with a moderate increase in fluorescence at acidic pH.
Accumulation of CQ
GREEN fluorescence was found in only 5% of the parasite’s DV compared to the cytosol during live cell imaging at any of the tested CQ
GREEN concentrations, ranging from 25 nM to 2.5 µM. Considering that the CQ
GREEN IC
50 was determined at 24 nM, any of the tested concentrations for live cell imaging would have been sufficient to kill the parasites. If CQ and CQ
GREEN had its primary target in the parasite’s DV, as suggested by several studies [
38‐
42], then we would have expected a higher CQ
GREEN fluorescence in the DV.
Protonation of CQ, or its analogues, influence their membrane permeability and thus their intracellular distribution [
43,
44]. Treatment of intact
P. falciparum-infected erythrocytes with pH buffered solutions ranging from pH 5–8 showed that protonation did not play a role in the intraparasitic CQ
GREEN distribution. One study used microsomes to resemble events occurring in the DV and reported accumulation of CQ
GREEN fluorescence in these microsomes [
12]. They did not use intact parasites in their experiments and the data does not support their assumption that CQ
GREEN accumulates equally well in the DV of live parasites as it does in microsomes [
12].
Fluorometric measurements were performed to verify CQGREEN fluorescence at low pH. Slightly higher fluorescence was observed for CQGREEN at acidic pH (5.0) compared to neutral pH (7.0). Therefore, if CQGREEN accumulated in the DV, a fluorescent signal would be expected. The absence of fluorescence in the DV suggests that CQGREEN does not reach the DV. Two reasons for this are possible. First, cleavage of the fluorescent Bodipy moiety from CQ may already occur in the parasite cytosol. This would allow CQ to accumulate in the DV, while the Bodipy moiety remains in the cytosol. In this case, the fluorescence signal does not specify the subcellular localization of CQ but rather only the fluorochrome. Second, the Bodipy moiety alters the intrinsic properties of the substrate and, therefore, prevents its accumulation in the DV. A simple way to elucidate whether the Bodipy moiety gets cleaved is determining fluorescence properties of conjugated versus free acid Bodipy-FL. Since there is a fluorescence shift between free acid Bodipy-FL alone (peak at 512 nm) and the Bodipy-tagged CQGREEN (peak at 520 nm), cleavage of the Bodipy moiety from CQGREEN during the cellular uptake would result in a fluorescence shift to 512 nm when parasites are treated with CQGREEN. For this study, intact P. falciparum-infected RBCs were treated with CQGREEN to analyse the potential cleavage of the Bodipy moiety. Although a proportional increase in fluorescence with higher parasitaemia was measured, no shift in the fluorescence peak was observed. Thus, CQGREEN remained intact and functional in live parasites prior to parasite lysis for fluorescence measurements.
Despite the unexpected localization of CQ
GREEN fluorescence mainly in the parasite cytosol and to a lesser extent in the DV, uptake rates were analysed in two CQS and two CQR strains. Although the fluorescent signal obtained from the DV was weak compared to the cytosol, it was sufficient to calculate the uptake rate for this compartment. CQS strains had approx. twofold higher CQ
GREEN uptake rates for the cytosol and 2.5-fold higher rates for the DV compared to CQR strains. Addition of verapamil to the CQR strains did not alter CQ
GREEN uptake rates, suggesting that PfCRT does not play a role in its uptake. This is consistent with the hypothesis that PfCRT is involved in CQ efflux from the DV and is not involved in its uptake [
20]. Decreased CQ uptake rates in CQR strains compared to CQS strains may be explained through reduced availability or affinity to an intracellular target, such as free haem [
4]. Thus, accumulation of CQ in the DV may contribute to parasite killing, but additional drug targets in the parasite cytosol could also play a role.
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