Antimalarial Exposure Delays Plasmodium falciparum

Intra-Erythrocytic Cycle and Drives Drug Transporter

Genes Expression
Maria Isabel Veiga1,2*, Pedro Eduardo Ferreira1,2, Berit Aydin Schmidt1, Ulf Ribacke3, Anders Björkman1,
Ales Tichopad4,5, José Pedro Gil1,2,6
1 Malaria Research Lab, Department of Medicine, Karolinska Institutet, Stockholm, Sweden, 2 Drug Resistance and Pharmacogenetics Group, Institute of Biotechnology
and Bioengineering, Centre of Molecular and Structural Biomedicine, University of Algarve, Faro, Portugal, 3 Department of Microbiology, Tumor and Cell Biology (MTC),
Karolinska Institutet, Stockholm, Sweden, 4 Institute of Biotechnology AS CR, Prague, Czech Republic, 5 Physiology Weihenstephan, Technical University Munich, Freising-
Weihenstephan, Germany, 6 Laboratory of Molecular Anthropology and Health, Department of Anthropology, Binghamton University, Binghamton, New York, United
States of America

Abstract
Background: Multi-drug resistant Plasmodium falciparum is a major obstacle to malaria control and is emerging as a
complex phenomenon. Mechanisms of drug evasion based on the intracellular extrusion of the drug and/or modification of
target proteins have been described. However, cellular mechanisms related with metabolic activity have also been seen in
eukaryotic systems, e.g. cancer cells. Recent observations suggest that such mechanism may occur in P. falciparum.

Methodology/Principal Findings: We therefore investigated the effect of mefloquine exposure on the cell cycle of three P.
falciparum clones (3D7, FCB, W2) with different drug susceptibilities, while investigating in parallel the expression of four
genes coding for confirmed and putative drug transporters (pfcrt, pfmdr1, pfmrp1 and pfmrp2). Mefloquine induced a
previously not described dose and clone dependent delay in the intra-erythrocytic cycle of the parasite. Drug impact on cell
cycle progression and gene expression was then merged using a non-linear regression model to determine specific drug
driven expression. This revealed a mild, but significant, mefloquine driven gene induction up to 1.5 fold.

Conclusions/Significance: Both cell cycle delay and induced gene expression represent potentially important mechanisms
for parasites to escape the effect of the antimalarial drug.

Citation: Veiga MI, Ferreira PE, Schmidt BA, Ribacke U, Björkman A, et al. (2010) Antimalarial Exposure Delays Plasmodium falciparum Intra-Erythrocytic Cycle and
Drives Drug Transporter Genes Expression. PLoS ONE 5(8): e12408. doi:10.1371/journal.pone.0012408
Editor: Georges Snounou, Université Pierre et Marie Curie, France
Received May 20, 2010; Accepted August 4, 2010; Published August 25, 2010
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This work was supported by project grants from the Swedish Development Cooperation Agency - Department for Research Cooperation (SWE 2005 -
0017, SWE 2005 - 4596, SWE-2007-174 and SWE-2005-4027). MIV and PEF are recipients of PhD grants from Fundação para a Ciência e Tecnologia (FCT)/Ministério
da Ciência e Ensino Superior, Portugal - MCES (ref. SFRH/BD/28393/2006 and ref. SFRH/BD/28368/2006, respectively). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]

Introduction phenomenon is related with other observations by us [6] and

others [7,8] showing that antimalarials can interfere with the cell
Plasmodium falciparum malaria remains a major disease burden in cycle development of the parasite, potentially constituting a
the developing world [1], chemotherapy being the foremost general first step towards high grade resistance to antimalarial
available tool for its control. Efficacious malaria treatment is drugs.
presently dependent on the efficacy of artemisinin combination Drug resistance has been widely associated with the action of
therapy (ACT), the global replacement of the previous mainstays, efflux pumps, able to transport drugs out of the relevant cellular
chloroquine and sulfadoxine-pyrimethamine [1,2]. Unfortunately, compartment, and thereby displacing them from their molecular
recent data suggests that P. falciparum resistance to ACT might be targets [9]. This action is expected to be potentiated through an
presently developing, both to its long half-life components but also increase in the availability of transporter proteins through
– and worryingly – against the artemisinin based components enhanced gene transcriptional activity. In P. falciparum two tran-
[3,4]. The latter phenomenon has been mainly defined as an in vivo sporters have been robustly associated with antimalarial resistance:
significant decrease in parasite reduction rate, manifested clinically PfCRT (chloroquine resistance transporter), a drug metabolite
by markedly longer parasite clearance times [5]. The molecular superfamily transporter coded by the pfcrt gene (MAL7P1.27)
basis of this phenomenon is unclear, starting from the fact that [10,11], and the Pgh (P-glycoprotein homologue, coded by pfmdr1
most of these observations are not associated with altered (PFE1150w)) an ATP-binding cassette (ABC) transporter super-
artemisinin IC50s in vitro [3]. It is conceivable that this family member [12]. Two other ABC transporters potentially

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 P. falciparum Cycle/Expression

associated with P. falciparum drug resistance can be added - the Results

homologues of the multidrug resistance-associated protein (MRP)
type [13,14,15] coded by the pfmrp1 (PFA0590w) and pfmrp2 Parasites genetic characterization
(PFL1410c) genes. Polymorphisms in these drug transporters have We have studied in parallel three P. falciparum clones, one MQ
been associated with altered in vitro and/or in vivo responses to sensitive (W2) and two (3D7 and FCB) with decreased sensitivity.
ACT components [16,17,18,19,20,21], indicating that their The pfmdr1, pfcrt, pfmrp1 and pfmrp2 ORF were fully sequenced to
common action might constitute a basis for wide range multidrug determine the genetic variability between the clones. The three
resistance phenotypes able to manage the new combinational clones, show two different main pfmdr1 polymorphisms: a single
therapy challenges faced by the parasite. Besides single nucleotide nucleotide polymorphism at amino acid position 86 (N86 in 3D7,
polymorphisms (SNPs), enhanced locus expression can drive 86Y in W2 and FCB) and gene copy number variation (W2 and
decreased drug sensitivity, a phenomenon well documented for 3D7 harbouring one copy [25], with FCB carrying two copies
pfmdr1, where gene duplication events are strongly associated to [23]). Numerous single nucleotide polymorphism differences were
mefloquine (MQ) resistance [22,23] and lumefantrine decreased seen in the three remaining genes (GenBank accession numbers:
sensitivity [24]. GU797309, GU797310, GU797311, GU797312, GU797315,
The synthetic arylaminoalcohol MQ constitutes one of the GU797316).
central partner drugs in artemisinin combination therapy, widely
used in South East Asia and South America. In SE Asia, the in vivo Plasmodium falciparum cell cycle progression alterations
delayed clearance has been observed not only in artesunate driven by mefloquine
monotherapy explorative regimens, but also associated to the In vitro MQ 50% inhibitory concentration (IC50) of W2, 3D7 and
regular mefloquine-artesunate ACT [3]. FCB clones were in average from 6 independent assays 1063.3nM,
We hypothesize that the observed clearance delay is associated 50616.9nM and 50614.1nM respectively. As for IC99 the average
with this phenomena, contributing for a survival advantage in a results was 44613.9nM, 146636.1nM, 146650.6nM for W2, 3D7
fraction of the parasite infection, by allowing an extended time for and FCB respectively. These concentrations were used for
the induction and expression of key drug transporters, leading to a challenging the clones in the assay performed.
pivotal decreased intracellular drug exposure in the first phase of Exposure to MQ induced an unexpected and unambiguous
the treatment course. delay in the cell cycle progression of the three tested parasites, as
Using MQ as a relevant and convenient reference ACT evaluated by Giemsa staining under light microscopy for the four
antimalarial, we have focused on monitoring changes in the cell counted stages (Figure 1). The degree of delay effect was clone
cycle progression rate of three P. falciparum clones with different dependent.
MQ susceptibilities, while in parallel detecting variations in At MQ IC99 a pronounced morphology arrest was seen in the
transcript abundance of the pfmdr1, pfcrt, pfmrp1 and pfmrp2 genes. three clones remaining essentially at the experimental initial ring

Figure 1. Stage morphology development throughout time upon MQ exposure. Parasite stage percentage (Y axis) of W2, 3D7 and FCB
clones with continuous exposure at MQ IC50 and MQ IC99 throughout 48 hours time course (X axis). Four morphological stages were differentiated
using light microscope: early rings (brown), late rings/early trophozoites (orange), trophozoites (light green) and schizonts (dark green).
doi:10.1371/journal.pone.0012408.g001

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 P. falciparum Cycle/Expression

stage with picnotic features. The quantification of end point RNA available data at PlasmoDB [28] (Figure 2, black curves), using as
indicated the presence of viable cells, an observation supported for endogenous control seryl-tRNA synthetase gene. In brief, pfmdr1, pfcrt
all tested parasites by their growth recovery 7–10 days after drug and pfmrp2 displayed the lowest levels of expression at approxi-
withdrawal (Figure S1). mately 24 hours (middle/late trophozoite stage) and highest
Each gene has its own stage morphology expression profile, amplitudes around 42–48 hours post invasion. pfmrp1 revealed
which once induced by drugs, it was previously shown that the the previously described clearly different expression profile
expression is strictly correlated with the cell morphology [26,27]. [29,30], with a peak of expression during the development of
The MQ induced cell morphology delay was also detectable trophozoites (12–24 hours post-invasion).
through the analysis of the transcript accumulation patterns Comparing relative transcript abundance (R6SD) between the
(Figure 2, green and red curves). Applying the described non- three clones at onset (Figure 2, time-point 0h), pfmdr1 mRNAs
linear regression model to the cycle (figure 3), significant showed higher levels in the FCB clone (18.1562.84), as compared
differences in the degree of cell morphology progression were to W2 (14.1860.99) and 3D7 (4.9060.21) with a fold difference of
seen with MQ IC99 exposure in the three clones (p,0.05; figure 4: 1.3 and 3.7 respectively. Pfcrt showed to be less expressed in the
red lines). The calculated rate at IC50, compared to control was W2 (3.8660.15) compared with 3D7 (7.4061.90) and FCB
clone dependent. The effect was less pronounced in W2 parasites (6.1060.88) with a fold difference of 0.5 and 0.63 respectively. As
with approximately 15% delay (p = 0.23) vs. ,40% for the MQ for the mrp genes, pfmrp2 gene showed higher expression for 3D7
tolerant clones FCB and 3D7 (p,0.01) (Figure 4 and Table 1). (8.4061.95) compared with W2 (5.0660.13) and FCB
Similar cell cycle morphology delay events were also observed (3.8161.03) with a fold difference of 1.7 and 2.2 respectively
upon quinine exposure (IC50 and IC90) in exploratory 12 hour while pfmrp1 at time-point 0 the results was 3.5 fold difference
follow up assay (Figure S2). between FCB (0.00769E-4) and W2 (0.00261E-4) and 1.75 fold
comparing with 3D7 (0.00466E-4).
Basal gene expression of transporter genes in W2, 3D7
and FCB Specific drug transporter genes induction by mefloquine
The pfmdr1, pfcrt, pfmrp1 and pfmrp2 cell cycle patterns of relative The fact that the drug exposure led to a proliferative delay in
transcript abundance from control (i.e. non drug exposed) the parasites stage created a new challenge on how to differentiate
experiments for the reference 3D7 clone were similar to the gene transcription levels affected by cell morphology from the

Figure 2. Drug transporter gene expression throughout time upon MQ exposure. Parasites cell cycle time course of drug transporters gene
expression at basal level (control, black line) and after exposure to MQ IC50 (green lines) and MQ IC99 (red lines). X axis represent time-points in hours
and Y axis relative gene expression quantification using pf07_0073 as endogenous control gene. Error bars represent standard deviation from 3 in
vitro replicates.
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 P. falciparum Cycle/Expression

Figure 3. Non linear regression equation I (for cell morphology) and II (for gene transcripts). y0 = offset, corresponding to the initial
(experimental to) status of the parameter under analysis (e.g. number of parasites at ring stage); a = wave amplitude; b = wave period (the reciprocal
wave frequency); b21 = wave frequency; c = wave phase; x = time; d = damping constant factor (to account to dampening variations in amplitude of
gene expression). cm: cell morphology; gt: gene transcripts.
doi:10.1371/journal.pone.0012408.g003

direct action of the drug on the transcript accumulation of the Discussion

studied genes. We solved this challenge through two approaches:
(1) From the few possible comparative time-points (control vs. MQ Upon continuous MQ exposure we have documented a dose
exposure time-points that had statistically non distinguishable dependent delay of the parasite cell cycle, particularly among the
parasite morphology proportion differences (p.0.05)), transcript two MQ less sensitive P. falciparum clones (3D7 and FCB). This
abundance values were compared. The levels of significant gene observation prompts the discussion of its biological importance in
expression modulation, within each clone upon MQ exposure, terms of drug resistance response.
ranged from 0.6 to 5.8 fold: pfmdr1 (0.6–1.5 fold), pfcrt (0.8–1.3 Mechanistically, the phenomenon of drug resistance is essen-
fold), pfmrp2 (0.8–2.7 fold) and pfmrp1 with the highest MQ driven tially associated with a decrease in the number of effective
induction (0.7–5.8) (Table 2). (2) To complement this limited opportunities for the therapeutic drug to effectively engage to its
comparative point by point verification of drug direct action, target(s). Below a certain threshold of interaction rate, the
which gave rise to few analyzable points, another analytical pharmacodynamic effect of the drug will be reduced to a degree
approach based on a non-linear regression analysis of gene that makes it clinically ineffective (hence associated to a treatment
expression over time was conducted for the MQ IC50 experiments. failure). This can be achieved in the cell by decreasing drug
The calculated fold difference confirmed that the drug exposure concentration in the target cellular compartment through the
was generally associated with mild changes in the expression of the action of transmembrane transporters. To this a reduced
genes (figure 5). Considering each gene at IC50 exposure: (a) The availability of the target can be added, particularly through a
pfmdr1 gene, was significantly more induced in the less MQ decrease in metabolic activity, potentially related with a slow down
sensitivity clones 3D7 (1.35 fold, p = 0.029) and FCB (1.49 fold, of the cell cycle.
p,0.001), than in the more sensitive W2 (1.23 fold). Comparisons In several clinically relevant biological systems – notoriously in
between the two less sensitive parasites showed that FCB can cancer cells - drug exposure leads to a noticeable slowing down in
induce this gene significantly more than 3D7 (p = 0.003). (b) As for cell cycle progression, even to the point of stalling [31,32]. In
pfmrp1, our data show no statistical difference between the clones. parallel, drug driven cell cycle arrest in P. falciparum has been
(c) Concerning pfcrt gene, the W2 and 3D7 parasites appear to lack consistently described by us and others for several antimalarial
the capacity to significantly induce pfcrt upon MQ exposure (1.06 drugs [6,7] including MQ [8] and very recently chloroquine [33].
fold and 0.92 fold) compared to FCB (1.25 fold). (d) Pfmrp2, P. falciparum dormant states are essentially refractory to antima-
appears to significantly induce the less MQ sensitivity clones 3D7 larial action [6] presumably due to its low metabolic activity status.
(1.32 fold, p = 0.003) and FCB (1.46 fold, p = 0.023) compared Upon these observations, we speculate that the observed cell cycle
with W2 (0.96 fold). delay behavior might functionally constitute a first stage towards

Figure 4. Effect of MQ in P.falciparum stage development. Wave form smoothing function best fitted over the early ring stage cell count
(Figure 1, brown color). The wave frequency value (time needed to complete a cell cycle) for each fitted function (control, MQ IC50 and IC99) was used
for the ratio calculation delay between treated and non treated parasite cultures (Table 1). Statistical comparisons (t-tests) of treatment effects
(control vs. MQ IC50 and IC99) were performed with the associated error of wave frequency value of different experiments per clone compared with
control.
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 P. falciparum Cycle/Expression

Table 1. Cell cycle development and gene expression of significantly affects the parasite response to MQ and other drugs
parasites challenged with MQ IC50. [22,23,36,37]. Interestingly, the clones herein observed with the
largest induction of transporter genes transcripts in response to
MQ were also the less sensitive to the drug (3D7, FCB), suggesting
Cell cycle a role of increased expression in the overall parasite drug response.
progression Pfcrt pfmdr1 pfmrp2 pfmrp1 Our observations of significant parasite cycle delay upon drug
Clones %* (SE) %{ (SE) %{ (SE) %{ (SE) %{ (SE) exposure points this as a significant confounding factor in the
W2 85.3 (12.9) 91.5 (5.6) 107.9 (9.5) 81.2 (9.8) 104.9 (11.8) analysis of parasite drug driven gene expression. Hence, we
propose a simple non-linear regression model as a novel analytical
3D7 64.3 (8.1) 56.6 (16.6) 98.8 (6.4) 96.4 (14.9) 107.3 (22.9)
tool to distinguish drug specific gene induction from the effect of
FCB 61.2 (10.4) 86.1 (6.3) 109.8 (11.2) 107.5 (26.8) 77.5 (24.5)
the time changed gene expression variation associated to the cell
*
- Cell cycle development percentage was calculated as the ratio of the time cycle. This approach could be applied in future studies, as well as
needed to complete a cycle obtained from the fitted wave function (Figure 3, revisiting previous data that did not consider the phenomenon of
equation I) of control and MQ IC50 experiments (Figure 4). drug driven cell cycle delay in P. falciparum.
{
- Gene expression percentage was calculated as the ratio of the time needed In conclusion, MQ is able to induce expression of pfmdr1, pfcrt,
to complete a cycle obtained from the fitted damped wave function (Figure 3,
equation II) of control and MQ IC50 experiments (data not shown).
pfmrp1 and pfmrp2 which, although mild, are potentially significant
doi:10.1371/journal.pone.0012408.t001 in aiding the parasite to evade antimalarial drug action. This
occurs in the context of a prompt MQ driven cell cycle delay that
this dormant state. The observed capacity of the different parasites potentially constitutes a further parallel mechanism for the parasite
clones to full recover growth, after 48 hours of intense MQ IC99 populations to avoid, or at least delay, antimalarial action.
exposure, in different recrudescence time indirectly supports this
suggestion. Materials and Methods
The capacity to delay the intra-erythrocytic developmental
cycle could therefore be an operational contributor for drug P. falciparum parasite clones
resistance, particularly when considering drugs with short half- P. falciparum 3D7 clone was obtained from Prof. D. Walliker
lives, like artemisinins and quinine. Such a response could help the (Department of Animal and population genetics, University of
parasite to withstand the effects of the drug at its peak serum Edinburgh, UK), while parasite clones W2 (MRA-157) and FCB
concentration, increasing the chances of survival as the drug (MRA-309) were obtained from Malaria Research and Reference
concentration rapidly decreases in the circulatory system, due to Reagent Resource Center (MR4, ATCC Massanas Virginia). The
elimination. The delay in the cycle might also be of importance for parasite lines were selected on the basis of their sensitivity to
allowing time for a parallel action: the specific induction of drug mefloquine hydrochloride (Sigma-AldrichH, St.Louis, MO, USA)
transporters able to reduce the intra-cellular (or intra-compart- and their genetic background (sequences submitted to GenBank
ment) concentrations of the drug. This action can in turn gain time accession numbers: GU797309, GU797310, GU797311,
for the process of metabolic shut off leading to a drug action GU797312, GU797313, GU797314, GU797315, GU797316).
refractory state based on the scarcity of drug targets.
Our results show a gene expression induction of generally less Drug susceptibility determinations
than 2 fold after MQ challenge, suggesting that these genes are The inhibitory concentration of 50 and 99% for the three clones
actually inducible by this drug, though to a limited extent. Similar was assessed using an Histidine-Rich Protein 2 based Double-Site
induction levels have been observed for pfmdr1 (1.7 fold, when Sandwich Enzyme-Linked Immunosorbent Assay [38] followed by
normalized for hsp86) in a northern blot based approach, after a 6h nonlinear regression analysis (http://malaria.farch.net).
MQ treatment [34]. This data is also coherent with a trend of low
pfdhfr gene transcriptional response previously reported upon P. falciparum in vitro culture
exposure to the experimental antifolate WR99210 [26], as well as The parasites were kept in culture using conventional methods
to chloroquine [27]. It is to note that the observed relatively low [39] in human O+ RBCs and Malaria Culture Medium,
induction of transcription seen in response to MQ does not containing RPMI 1640 culture medium supplemented with 10%
exclude more significant responses upon exposure to other L-glutamine, 0.05% gentamicine (GibcoH/InvitrogenTM, Carls-
xenobiotics. Accordingly, treatment of the P. falciparum clone bad, CA, USA) and 10% human AB+ serum. Large scale culture
3D7 with phenobarbital (a classical drug elimination inducer) for of each clone was produced with 3% parasitaemia in 2.5%
48h was associated to an extensive increase in pfmdr1 transcripts, hematocrite and synchronized in early ring stage at least twice by
and to a concordant reduction in drug susceptibility [35]. applying the MACSH system (Miltenyi Biotec, Bergisch Gladbach,
These observations raise the question on how important mild Germany). The batch was then divided for three experiments: one
changes in gene expression can be in terms of the parasite response control culture (no drug added) and two cultures with MQ IC50
to the drug. When drug resistance is mainly based on direct and IC99.concentration followed by spreading it in culture plates
modifications of the target, as in the case with antifolates, an (2mL/well). At the time of reinvasion, early rings (time-point 0),
increase in drug quantity of the latter will probably be irrelevant, two Giemsa-stained smears were made for examination of
since it is unlikely that the number of drug molecules will parasites stages, while three replicate wells of parasite culture
overcome the number of target proteins (considering a regular were harvested for RNA extraction. This procedure was followed
stoichiometric relationship of action). In the case of transporter for the three experiments, in six hour time-point intervals up to
proteins - assuming that they are not themselves targets for the 48 hours (total of 9 time-points analyses including time 0). The
drug - the situation is markedly different. One protein can smears were fixed with methanol and stained with 5% Giemsa and
transport a large number of molecules per time unit, and a simple visualization under light microscopy (10006 magnification). Their
2 fold increase in this capacity may well have an impact on the examination included counting 100 parasites, twice per slide (2
effect of the drug. This is supported by in vivo and in vitro studies slides per time point), distinguishing four main morphological
showing that a two fold gene copy number amplification of pfmdr1 stages: early rings, late rings/early trophozoites, trophozoites and

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 Table 2. Gene transcripts time points comparison in MQ exposed parasites with identical stage proportions.

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Comparable
Comparable time time points
Clones points{ Chi-square pfcrt pfmdr1 pfmrp2 pfmrp1

W2 Control IC50 X2 Fold diff SD t-test P-value Fold diff SD t-test P-value Fold diff SD t-test P-value Fold diff SD t-test P-value
diff* mean diff mean diff mean diff mean
12 12 0.06 1.2 20.8 0.2 26.1 0.00* 1.2 22.5 0.6 25.2 0.01* 1.5 21.1 0.1 217.2 0.00* 0.7 0 0 6 0.00*
24 30 0.08 1.3 20.3 0.1 24.7 0.01* 1.5 22 0.3 28.1 0.00* 1.3 20.2 0.1 22.4 0.08 1.2 0 0 22.1 0.1
36 42 0.11 1.1 20.3 1.5 20.3 0.81 0.7 11.4 5.1 2.8 0.05* 1.1 20.4 0.4 21.5 0.22 1 0 0 0 1
36 48 0.09 1 20.1 1.5 0 0.97 0.6 14.4 5.4 3.3 0.03* 1.2 21 0.5 22.4 0.07 0.6 0 0 1 0.38
Control IC99

6
0 6 0.95 1.2 20.8 0.2 25.4 0.01* 1.2 23 0.9 24.1 0.02* 0.9 0.7 0.2 5 0.01* 3.2 0 0 217 0.00*
6 18 0.5 0.9 0.5 0.4 1.5 0.2 1 0.4 1.6 0.3 0.79 1.2 20.8 0.5 21.9 0.13 2.4 0 0 24.9 0.01*
42 6 0.16 0.8 0.9 0.2 6.4 0.00* 0.6 9.8 1.1 11.4 0.00* 0.8 0.8 0.2 5.9 0.00* 1.3 0 0 25.4 0.01*
48 6 0.58 1.2 20.8 0.3 23.8 0.02* 1.1 21.4 0.8 22.1 0.1 1 0.1 0.2 0.7 0.52 1.6 0 0 28.5 0.00*
3D7 Control IC99
42 6 0.43 0.5 0.7 2.1 0.4 0.72 0.6 20.2 0.6 20.4 0.71 0.9 3.2 3.1 1.2 0.28 0.4 0 0 21.9 0.13
48 12 0.64 1.6 0.2 0.7 0.3 0.79 1 20.5 0.6 21 0.37 2.7 21.2 0.4 23.8 0.02* 5.2 0 0 21.8 0.15
48 18 0.65 1.1 3.2 1.1 3.7 0.02* 0.8 4 2.4 2 0.11 1.7 0.3 1.1 0.3 0.76 5.8 0 0 4.4 0.01*
FCB Control IC50
6 12 0.09 0.9 1.7 1.5 1.4 0.24 0.9 2.1 1.4 1.9 0.14 1.3 20.9 1 21 0.37 1.2 0 0 20.5 0.63

*: Fold difference is represented in bold for the significant (t-test, p,0.05) gene expression MQ induction.
{
: For each clone, the four morphological stages count in all time points from control experiments and MQ exposed were compared (Chi-square). Only the time points with same proportion of stages (no statistical difference,
p.0.05) are included in the table for MQ gene expression induction analysis with different ICs.
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 P. falciparum Cycle/Expression

Figure 5. Drug transporter genes induction by MQ IC50. Results appear as fold difference in gene expression normalized with cell cycle stage
development achieved after smoothing the data with wave functions. Error bars represents SE of the difference.
doi:10.1371/journal.pone.0012408.g005

schizonts. A total count of 400 parasites per time-point, per calculated as the ratio (R) between the transformed Ct values of
experiment was performed. The experimental design was equally target gene and internal control reference gene (PF07_0073),
applied for the three clones analyzed (W2, 3D7 and FCB). taking in account the amplification efficiency for each gene [41].
Calculations were conducted using the SAS 9.1 system for
Molecular analysis WindowsTM.
Parasite cultures collected for RNA extraction were centrifuged
at 0.8 g-force for 2 min. 1.85 mL of supernatant were removed Determination of the drug exposure specific induction
and 150 mL of PBS plus 300 mL of Lysis buffer (Applied effect: time-point gene expression fold differences and
BiosystemsTM, Fresno, CA, USA) were added. The mix was kept the non-linear regression model approach
at 220uC until RNA extraction. RNA extraction was carried out In order to distinguish the changes in cDNA abundance
using an ABIPRISMH6100 Nucleic Acid PrepStationH (Applied associated with the parasite cell cycle progression from the ones
BiosystemsTM, Fresno, CA,USA) according to the recommenda- associated with drug exposure, two different approaches were
tions of the manufacturer. Total RNA quality and quantity was performed:
measured using the AgilentRNA 6000 Pico total RNA assay in an I. Direct comparisons. Fold difference in transcript abun-
Agilent2100 BioanalyserTM (Santa Clara, CA, USA). Sample dance was calculated in control vs. MQ exposed cultures only in
concentrations with RNA Integrity Number .5 were normalized time-points showing a statistically equal fractional composition of
for each clone before cDNA synthesis (High-Capacity cDNA the four microscopically determined stages (Fisher test, df = 3,
Reverse Transcription Kit (Applied Biosystems, Fresno, CA, p.0.05).
USA)). Real-time analyses were performed in an ABIPRISMH II. Non linear regression analysis. A wave function applied
7900HT Sequence Detection System (Applied BiosystemsTM, to the number of total parasites ring stage count throughout all time-
Fresno, CA, USA) with gene specific MGB TaqManH probes and points for each clone (equation I – figure 3) was achieved
primers. TaqManH probes and primers for target gene pfmdr1 were (goodness of fit was R2.0.9) (SigmaPlotTM 9.0). Specific parameters
as previously published [22]. New designs were developed for pfcrt, of this function were used to quantify the parasite delays in cell
pfmrp1, pfmrp2 and for endogenous control gene [30,40] seryl-tRNA morphology (cm) imposed by MQ exposure. The wave frequency
synthetase (PF07_0073) (Table S1). Amplification reactions were (bcm21) from each fitted function (control, MQ IC50 and MQ IC99
done in quadruplicate in 384 well plates with 10ml containing exposed), corresponds to the time needed to complete a cycle and it
TaqManH Gene Expression Mastermix (Applied BiosystemsTM, was used to calculate the cell cycle delay based in the observation of
Fresno, CA, USA), 300nM of each forward and reverse primer, the morphology development of the parasite between MQ exposed
100nM of TaqManHprobe and 2 mL of amount-normalized (ICn) and control (Rmorphology = bcm21ICn/bcm21control). Statistical
cDNA. The thermal cycle profile was 50uC for 2 min, 95uC for comparisons (t-tests) were performed with the associated error of the
10 min and forty cycles of 95uC for 15 s and 60uC for 1 min. The wave frequency parameter ‘‘bcm21’’.
amplification efficiency (E) was estimated for each gene and used A similar approach, of applying a wave function, was performed
as a correction factor for relative gene expression quantification. in the gene transcripts data (gt). During the 48 hours exposure to
All experimental threshold cycle values (Ct) were first transformed MQ IC50, the associated delay in the cycle gene expression was
to adjust the RNA concentration adding to the Ct value the log2 calculated by comparing the wave frequency parameter ‘‘bgt21’’
RNA concentration of each clone. Relative gene expression was (Rgene transcripts = bgt21IC/bgt21control) obtained through a best fit of

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 P. falciparum Cycle/Expression

the gene expression data, this time in a damped wave function Figure S2 Cell cycle morphology progressions upon quinine
(equation II – figure 3). Statistical comparisons (t-tests) were exposure (IC50 and IC90) after 12 hour follow up. Parasite stage
performed with the associated error of the wave frequency percentage (Y axis) of W2, FCB and 3D7 strains after 12 hours (X
parameter ‘‘bgt21’’. axis) with continuous exposure of quinine IC50 and IC90. The
The use of these equations allowed us to determine whether the counting of the parasites included 100 parasites, twice per slide (2
expression profiles were solely stage-driven, or included a slides per different ICs exposure), distinguishing four main
significant fraction associated with drug exposure: morphological stages using light microscope: early rings, late
rings/early trophozoites, trophozoites and schizonts.
MQ driven inductionðfold differenceÞ Found at: doi:10.1371/journal.pone.0012408.s002 (0.01 MB

PDF)
~ Rgene transcripts {Rcell morpho log y z1:
Table S1 TaqManH probes and primers sequence.
By its nature, this approach was only possible to apply to Found at: doi:10.1371/journal.pone.0012408.s003 (0.03 MB
experimental outputs where sufficient data points were available DOC)
for enabling fitting a sine function, being hence only applicable to
gene transcripts data concerning IC50 and not to the IC99 drug Acknowledgments
level exposure.
We greatly appreciate Prof. Ralph M Garruto for the valuable discussion
and suggestions. We also thank Prof. Yngve Bergqvist and Doctor Daniel
Supporting Information Blessborn from Högskolan, Dalarna for chromatographic measurement of
mefloquine concentrations. MR4 for providing us with malaria parasites
Figure S1 Cell viability after MQ IC99 exposure. Parasite strains
contributed by DE Kyle and TE Wellems. P. falciparum 3D7 malaria
viability after challenged with continuous MQ IC99 (W2, 44nM; parasites were kindly supplied by the late Prof. D. Walliker.
3D7 and FCB, 146nM) for 48 hours analyzed by Histidine-Rich
Protein 2 Double-Site Sandwich Enzyme-Linked Immunosorbent.
All strains had a growth recovery of 7–10 days after drug
Author Contributions
withdrawal. Error bars are SE of rate between day0 HRP2 and Conceived and designed the experiments: MIV PEF UER AB JPG.
collected day. Performed the experiments: MIV PEF BAS JPG. Analyzed the data: MIV
Found at: doi:10.1371/journal.pone.0012408.s001 (0.06 MB PEF UER AT JPG. Contributed reagents/materials/analysis tools: AB
JPG. Wrote the paper: MIV PEF UER AB AT JPG.
PDF)

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