![CQ transport activity of the C101F and L272F variants of PfCRT in Xenopus oocytes. ( a , b ) The upt a ke of [ 3 H]CQ into oocytes expressing PfCRT was measured in the absence (closed bars) or presence of 250 μ M VP (light grey bars; a ), 100 μ M BSD (dark grey bars; b ), or 500 μ M BSD (open bars; b ). Within each experiment, measurements were made from 10 oocytes per treatment and uptake was expressed relative to that measured in the PfCRT Dd2 -expressing oocytes under control conditions. The normalized data obtained from 4–5 separate experiments (each using oocytes from different frogs) were then averaged and are shown + SEM. Both panels show PfCRT-mediated CQ uptake, calculated by subtracting CQ uptake measured in PfCRT 3D7 -expressing oocytes ( i.e. the component of CQ accumulation attributable to diffusion; see Supplementary Fig. S3) from that measured in oocytes expressing a variant of PfCRT. In the control treatments, the rates of CQ uptake (pmol/oocyte/h; n = 9 ± SEM) in oocytes expressing PfCRT Dd2 and PfCRT 3D7 were 23.6 ± 2.3 and 1.3 ± 0.2, respectively. ‘ns’ denotes no significant difference ( p > 0.05) in CQ accumulation between oocytes expressing a PfCRT variant (in the presence or absence of VP or BSD) and that measured in the PfCRT 3D7 -expressing oocytes under control conditions.](https://www.researchgate.net/profile/Catherine-Moore-8/publication/282517330/figure/fig5/AS:281555923685385@1444139571947/CQ-transport-activity-of-the-C101F-and-L272F-variants-of-PfCRT-in-Xenopus-oocytes-a_Q320.jpg)
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1
SCIENTIFIC RepoRts | 5:14552 | DOI: 10.1038/srep14552
www.nature.com/scientificreports
Mutations in the Plasmodium
falciparum chloroquine resistance
transporter, PfCRT, enlarge the
parasite’s food vacuole and alter
drug sensitivities
Serena Pulcini1,*, Henry M. Staines1,*, Andrew H. Lee2, Sarah H. Shak3,
Guillaume Bouyer1,4,5, Catherine M. Moore1, Daniel A. Daley6, Matthew J. Hoke6,
Lindsey M. Altenhofen7, Heather J. Painter7, Jianbing Mu8, David J. P. Ferguson9,
Manuel Llinás7, Rowena E. Martin3, David A. Fidock2,10, Roland A. Cooper6,11 & Sanjeev Krishna1
Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major
determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe
P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations
(C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have
increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or
minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic
parasite line expressing the L272F variant of PfCRT conrmed this increased chloroquine sensitivity
and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation
into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport
chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These
data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant
parasite lines to chloroquine. Taken together, these ndings provide new insights into PfCRT function
and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of
many antimalarial drugs.
1Institute for Infection and Immunity, St. George’s, University of London, London SW17 0RE, UK. 2Department of
Microbiology and Immunology, Columbia University Medical Center, New York, NY 10032, USA. 3Research School
of Biology, Australian National University, Canberra, ACT 2601, Australia. 4Sorbonne Universités, UPMC Univ. Paris
06, UMR 8227, Integrative Biology of Marine Models, Comparative Physiology of Erythrocytes, Station Biologique
de Rosco, Rosco, France. 5CNRS, UMR 8227, Integrative Biology of Marine Models, Comparative Physiology of
Erythrocytes, Station Biologique de Rosco, Rosco, France. 6Department of Biological Sciences, Old Dominion
University, Norfolk, VA 23529, USA. 7Department of Biochemistry and Molecular Biology and Center for Malaria
Research, Pennsylvania State University, State College, Pennsylvania 16802, USA. 8Laboratory of Malaria and
Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville MD
20852, USA. 9Nueld Department of Clinical Laboratory Sciences, University of Oxford, John Radclie Hospital,
Oxford OX3 9DU, UK. 10Division of Infectious Diseases, Department of Medicine, Columbia University Medical
Center, New York, NY 10032, USA. 11Department of Natural Sciences and Mathematics, Dominican University
of California, San Rafael, CA 94901, USA. *These authors contributed equally to this work. Correspondence and
requests for materials should be addressed to S.K. (email: s.krishna@sgul.ac.uk)
Received: 29 May 2015
Accepted: 14 August 2015
Published: 30 September 2015
OPEN
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SCIENTIFIC RepoRts | 5:14552 | DOI: 10.1038/srep14552
Chloroquine (CQ) rapidly became one of the most useful antimalarial drugs for rst-line therapy soon
aer the Second World War. Resistance to CQ was rst reported in the late 1950s in Plasmodium fal-
ciparum. It then spread globally and forced the development of alternative regimes, culminating in the
more expensive artemisinin-based combination therapies (ACTs) used today. e locus containing the
P. falciparum chloroquine resistance transporter gene (pfcrt) was initially mapped by classical genetic
studies as being crucial to the development of CQ resistance, with this gene subsequently being identied
and its role conrmed using reverse genetic approaches1–3. CQ resistance is now emerging in P. vivax,
for which it remains the rst-line treatment4.
CQ is a diprotic weak base that accumulates in the parasite’s acidic food vacuole (FV) by diusion
and subsequent trapping by protonation. CQ interferes with the detoxication of heme in the FV, which
leads to parasite death5. Predicted to have 10 transmembrane domains (TMDs), PfCRT is located in the
FV membrane1,6 and, when mutated, increases export of CQ from the FV and its target of heme polym-
erisation7. Single nucleotide polymorphisms (SNPs) in PfCRT in eld isolates correlate with a resistance
phenotype in in vitro assays and are sensitive markers for treatment failure in patients8,9. However, these
molecular markers are not always specic because other variables such as previous exposure to malaria
can inuence treatment response in patients10.
One polymorphism at position 76 (K76T) in the rst TMD of PfCRT seems to be key to CQ resist-
ance. is substitution removes a positive charge from a predicted substrate-binding site in PfCRT, allow-
ing protonated CQ to escape from the FV down its electrochemical gradient11. Other mutations (K76I
and K76N) in this position also arise when P. falciparum is exposed in vitro to lethal concentrations of
CQ, allowing parasites to survive and supporting the critical role of this residue1,6.
e native function of PfCRT is not clear, although it has been postulated to be involved in hemoglo-
bin catabolism, possibly by mediating the transport of hemoglobin-derived peptides/amino acids from
the FV12, a hypothesis consistent with recent heterologous expression and metabolomics studies7,13,14.
PfCRT has also been proposed to function as a chloride channel, a proton pump or a regulator of
proton pumps, a general activator or modulator of transport systems (reviewed in11) or, most recently,
a proton-coupled transporter of a broad range of cationic substrates15. ere are many reasons to eluci-
date the function of PfCRT in parasites, including the suggestion that PfCRT could itself become a new
drug target16,17, or that chemosensitizing agents could be directed against PfCRT to restore the ecacy
of CQ16–18. Furthermore, CQ continues to be used in the treatment of non-falciparum malarias. It may
also regain ecacy against falciparum malaria in areas where usage has been tightly regulated, since the
withdrawal of CQ can result in dramatic decreases in the prevalence of CQ-resistant parasites19.
Here, mutations in pfcrt that alter parasite phenotype give new insights into its native function as a
transporter. e novel and pleiotropic phenotypic characteristics associated with mutated PfCRT include
altered FV morphology and changes in quinoline sensitivities. We also investigated the eect of these
changes on the parasite’s sensitivity to other antimalarial classes, such as the artemisinins, that some have
considered to act (at least in part) in the FV of the parasite20,21.
Results
New and previously described mutations in pfcrt. SNPs were identied in pfcrt in two dierent P.
falciparum lines (Fig.1a,b). e rst was discovered aer isolating amantadine (AMT)-resistant mutants
of the CQ-resistant parasite strain FCB, following selection with 80 μ M of this antiviral agent. Viable par-
asites were observed in one of four drug-pressured asks at 42 days, whereas none had emerged within
the remaining asks by 60 days. PCR amplication and sequencing of pfcrt in four clonal lines derived
from the AMT-resistant culture detected a single non-synonymous SNP, g302t. is encoded the amino
acid mutation C101F. ese lines were therefore designated FCBC101F. Position 101 is predicted to lie
within the second TMD of PfCRT (Fig.1a). is mutation was earlier observed in a CQ-resistant Dd2
parasite line derived by continuous piperaquine (PPQ) pressure22, although that study did not describe
any changes in parasite morphology.
e second parasite line, derived from the CQ-sensitive strain 3D7, was selected by blasticidin (BSD)
pressure as an inadvertent outcome of transfection experiments on an unrelated gene (that had aimed
to achieve single cross-over homologous recombination with a tagging plasmid under BSD selection)23.
Aer several weeks of selection, pfcrt cDNA transcripts of the daughter parasite line and parental 3D7
were sequenced. A mutation at position c814t in the pfcrt coding sequence, resulting in the amino acid
mutation L272F, was detected in the selected line, designated 3D7L272F, and was absent in its parent. is
substitution is positioned immediately aer the seventh predicted TMD, placing it in the FV compart-
ment (Fig.1a). To our knowledge, this mutation has not been reported previously. No other mutations
in pfcrt were detected in either of the new parasite lines.
Given that 3D7L272F arose in unusual circumstances (BSD is a general inhibitor of protein translation
and is not thought to target the FV), whole-genome sequencing was undertaken to identify further
mutations. is conrmed the presence of the c814t mutation in pfcrt and identied only 2 additional
SNPs. e rst was c5549g in PF3D7_1229100 (the P. falciparum multidrug resistance-associated pro-
tein 2, PfMRP2), resulting in a stop-gain mutation (S1850*) and the loss of 259 amino acids from the
C-terminus. e second was t1032a in PF3D7_1462400 (a conserved protein of unknown function),
resulting in a stop-gain mutation (Y344*) and the loss of 2979 amino acids from the C-terminus.
Truncation of the latter sequence has been observed in other laboratory clones of 3D724. Furthermore,
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SCIENTIFIC RepoRts | 5:14552 | DOI: 10.1038/srep14552
there was no evidence of integrated copies of the plasmid vector containing the BSD selection marker23,
which had been used during the generation of the 3D7L272F line.
Enlarged FVs of parasites with mutations in pfcrt. A monstrously swollen FV was observed at all
stages that ordinarily display a vacuole in the asexual cycle of both parasite lines FCBC101F and 3D7L272F
(Fig.2). is phenotype was stably maintained in the parasites following repeated rounds of parasite cul-
ture and cryopreservation. e enlarged FVs were already apparent in the early to mid trophozoite stages
of the FCBC101F line, when compared with FVs from FCB parental controls (Fig.2a le and right panels).
In more mature FCBC101F parasites, the FVs were strikingly clear in appearance, with hemozoin crystals
apparently marginalized to the FV periphery and opposite the developing nuclei, although live imaging
suggests that the hemozoin is distributed normally (Fig. 2b). e immature ring stages of development
were indistinguishable from those of the parental strain. Similar ndings were evident in the parasite
line 3D7L272F when compared with 3D7 (Fig.2c le and right panels). Measurement of the area of the
FV was also undertaken and expressed as a ratio of the parasite’s area to correct for parasite age (Fig.3a).
is conrmed that FCBC101F and 3D7L272F parasites have a relative FV/parasite area that is approximately
twice that of FCB and 3D7, respectively (p < 0.0001). Neither FCBC101F nor 3D7L272F parasites appeared
to be enlarged within their host red blood cells (RBCs).
e 3D7L272F line was selected for a more detailed characterization. e appearances of parasites
examined with transmission electron microscopy (TEM) were consistent with observations made with
light microscopy (Fig.3b,c), with few dierences evident between parental strains and daughter parasite
lines except for the size of the FV. Specic to this line, TEM also revealed that “knobs”, electron dense
protrusions of the RBC membrane caused by parasite infection, which are important determinants of
cytoadherence25 and which are oen lost from infected RBCs during long term parasite culture26, were
Figure 1. PfCRT mutations. (a) Schematic representation of PfCRT and positions of previously identied
polymorphisms8,71 from eld isolates (green circles) and from drug-pressured laboratory lines (purple
circles). e critical CQ resistance mutation site (K76) is shaded red, and the two residues at which
mutations are described in this study are shaded in orange (C101) and blue (L272). (b) PfCRT haplotypes
included this study.
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SCIENTIFIC RepoRts | 5:14552 | DOI: 10.1038/srep14552
displayed approximately 7.5-fold more on the host surface of 3D7L272F-infected RBCs than 3D7-infected
RBCs. is is unlikely to be directly related to the mutation in pfcrt and may be due to sub-population
selection.
Since BSD pressure has been shown to alter infected RBC permeability27–29, electrophysiological
transport studies were also undertaken to compare 3D7 and 3D7L272F-infected RBCs, although no dier-
ences were observed (Supplementary Fig. S1).
Figure 2. Representative morphology of parasite lines FCBC101F and 3D7L272F. (a) Appearance of
enlarged FVs in xed FCBC101F parasites (le panel), when compared with parental FCB parasites of similar
developmental stages (right panel). (b) Images of live FCBC101F and FCB trophozoite-stage parasites, using
bright-eld and dark-eld microscopy (le and right panels, respectively). (c) Appearance of enlarged FVs
in xed 3D7L272F parasites (le panel), when compared with parental 3D7 parasites of similar developmental
stages (right panel). e diameter of a RBC is ~7 μ m.
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SCIENTIFIC RepoRts | 5:14552 | DOI: 10.1038/srep14552
In vitro sensitivity to antimalarials. Both cell lines with mutations in pfcrt displayed altered suscep-
tibility to antimalarials when compared with the parental strains (Table 1). Using a 72 h in vitro growth
inhibition assay that yields IC50 values, FCBC101F parasites were found to be 83 fold less susceptible to
AMT (used in its selection). FCBC101F showed a 5–6 fold increase in sensitivity to CQ, yet interestingly
still retained the characteristic verapamil (VP)-reversibility of CQ-resistant parasites30. Furthermore,
compared with FCB, FCBC101F was signicantly (p < 0.01) more sensitive to quinolines (quinine (QN),
quinidine (QD) and monodesethyl amodiaquine (MDAQ)) but not the arylmethanol, meoquine (MQ).
ere was a small (29%) increase in sensitivity to artemisinin (ART; p <0.01). e FCBC101F line became
approximately 2-fold more resistant to PPQ relative to controls (p < 0.05).
In similar experiments, 3D7L272F parasites, assayed over 48 h in vitro, were ~2.5 fold more sensitive to
CQ than 3D7, with respective IC50 values of 6.1 and 15 nM (p < 0.01). e 3D7L272F parasites were also
slightly more sensitive to QN than 3D7 parasites. VP sensitivity was not examined because unlike FCB,
the 3D7 line is already CQ-sensitive. e increased sensitivity to CQ therefore indicates that 3D7L272F is
Figure 3. Morphological comparisons of parasites with mutations in pfcrt compared with their parental
controls. (a) Parasites were synchronized by sorbitol lysis and areas of FVs and parasites (approximately
38 h post-invasion) were measured and expressed as ratios (AFV/AParasite). 3D7, FCB and Dd2Dd2 (open
bars; n = 72, 31 and 42, respectively) and 3D7L272F, FCBC101F and Dd2Dd2 L272F (closed bars; n = 84, 58 and
88, respectively) parasites were analyzed and signicant enlargement of the FV was conrmed in 3D7L272F,
FCBC101F and Dd2Dd2 L272F parasites relative to 3D7, FCB and Dd2Dd2, respectively (*p < 0.0001: two-
tailed, unpaired, Student’s t-test). (b,c) Transmission electron micrographs of 3D7 and 3D7L272F parasites,
respectively, showing the food vacuole (FV) and nucleus (N). Note the enlarged electron lucent FV in
3D7L272F (suggesting changes in the process of hemoglobin degradation and formation of hemozoin crystals).
RBCs infected with 3D7L272F displayed approximately 7.5-fold more knobs (arrowheads) on the host surface
than 3D7-infected RBCs, although this is likely due to sub-population selection rather than a direct link to
the mutation in pfcrt. (insert) Detail of a knob. Bars represent 1 μ m (b,c) and 100 nm (insert).
Drug†
Mean ± SEM IC50 values for individual parasite strains/lines‡
3D7 3D7L272F FCB FCBC101F FCB + VP FCBC101F + VP
CQ 15 ± 1.8 6.1 ± 1.5* 187 ± 7.1 34 ± 1.8* 47 ± 1.2 14 ± 1.0
QN 176 ± 17 108 ± 13* 333 ± 5.8 220 ± 23* 161 ± 33 223 ± 31
QD — — 167 ± 13 62 ± 3.1* 46 ± 1.9 46 ± 2.9
MQ 87 ± 29 64 ± 17 13 ± 1.1 14 ± 0.9 8.8 ± 1.0 18 ± 2.0
AQ 25 ± 2.3 27 ± 2.1 — — — —
MDAQ 14 ± 1.5 15 ± 1.5 52 ± 4.1 18 ± 1.0* 16 ± 1.0 9.1 ± 0.7
PPQ 18 ± 4.1 22 ± 5.3 12 ± 0.6 27 ± 1.8* 13 ± 0.4 25 ± 3.1
ART 3.5 ± 1.2 4.1 ± 1.6 13 ± 0.9 9.1 ± 0.7** 9.9 ± 0.9 9.1 ± 0.5
BSD (μ M) 1.5 ± 0.6 47 ± 6.0* — — — —
AMT (μ M) — — 5.6 ± 0.6 465 ± 54* 11 ± 0.5 687 ± 32
Table 1. In vitro sensitivity of 3D7, 3D7L272F, FCB and FCBC101F (in presence or absence of verapamil,
VP) to antimalarial drugs. †CQ, chloroquine; QN, quinine; QD, quinidine; MQ, meoquine; AQ,
amodiaquine; MDAQ, monodesethyl amodiaquine; PPQ, piperaquine; ART, artemisinin; BSD, blasticidin;
AMT, amantadine; VP, verapamil (used at 0.8 μ M). ‡IC50 values are listed in nM, except where indicated,
and are show as the mean ± SEM. n = 3 independent assays (each performed as a single replicate for FCB
parasites and in quintuplicate for 3D7 parasites). Signicantly dierent mean IC50 values relative to controls
(F-test; *p < 0.05, **p < 0.01).
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SCIENTIFIC RepoRts | 5:14552 | DOI: 10.1038/srep14552
a ‘CQ-hypersensitive’ parasite line. e mean IC50 values for MQ, MDAQ, PPQ and ART were similar
between the 3D7L272F and 3D7 parasites (Table1).
Transfection studies. To conrm the phenotype observed in 3D7L272F, we engineered the L272F
mutation in pfcrt using zinc-nger nuclease mediated allelic replacement31 in the Dd2 line and compared
results with congenic controls. Figure 4a,b illustrate this strategy and provide conrmation of integra-
tion. As observed in 3D7L272F, signicant FV distension (~2 fold as measured by vacuolar area relative
to parasite area; Fig. 3a) was generated by introduction of this single amino acid change (Fig. 4c,d).
However, a signicant increase in BSD resistance was not observed between the Dd2Dd2 L272F line and its
congenic control, Dd2Dd2 (Table2), which suggests that the PfCRT L272F mutation was not primarily
responsible for the BSD resistance found in 3D7L272F parasites. e parental strain Dd2 and the congenic
control Dd2Dd2 were both CQ-resistant. However, Dd2Dd2 L272F was considerably more susceptible to CQ
and monodesethyl chloroquine (MDCQ) than the Dd2Dd2 line, although the IC50 values of the L272F
Figure 4. Introduction of PfCRT L272F into Dd2. (a) Schematic of zinc-nger nuclease (ZFN)-mediated
generation of vacuole-enlarged parasites. Dd2 parasites were rst enriched for the episomal pcrtDd2 L272F-
hdhfr or pcrtDd2-hdhfr donor plasmids (latter not shown). e donor plasmids encoded a cDNA copy of
the Dd2 pfcrt allele (dark blue, plasmid), either L272F-mutated (dark blue bump) or wild-type (not shown),
followed by a dhfr selection cassette (light grey). Each donor-enriched parasite was then transfected with
the pZFNpfcrt-bsd plasmid, expressing the genomic (light blue) pfcrt intron 1-targeting ZFN pair (ZFN L
and ZFN R, orange) and the bsd selection cassette (dark grey). ZFN-induced recombination in pfcrt yielded
either control Dd2Dd2 (not shown) or Dd2Dd2 L272F parasites (dark blue, locus). (b) PCR verication of
parental, recombinant control, and Dd2Dd2 L272F parasite clones. Primer (p) positions are shown in panel a.
(c) Light microscope analysis of representative examples of parental, recombinant control, and experimental
parasite clones. Ring morphology for each parasite Dd2, Dd2Dd2, Dd2Dd2 L272F, and GC03 was normal.
Progression through the trophozoite and schizont stages showed normal morphological development
except for the Dd2Dd2 L272F clone, which exhibited the characteristic enlarged vacuole and diuse hemozoin
phenotypes seen in 3D7L272F and FCBC101F Giemsa stained parasites. (d) Transmission electron micrographs
of Dd2Dd2 (i) and Dd2Dd2 L272F parasites (ii) showing similar cytoplasmic appearances except for the enlarged
food vacuole (FV) in Dd2Dd2 L272F. Note neither parasite exhibits knobs. is conrms similar morphological
appearances to those of 3D7 and 3D7L272F, respectively but without knob formation. N—Nucleus. Bars
represent 1 μ m.
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SCIENTIFIC RepoRts | 5:14552 | DOI: 10.1038/srep14552
mutant remained higher than the fully CQ-sensitive reference line GC03 (Table2). ART sensitivity, as
measured in these IC50 assays, was unaltered across parasites. ere were no dierences in whole-cell
electrophysiological properties between the Dd2Dd2 and Dd2Dd2 L272F parasite lines (Supplementary Fig.
S1) and the RBCs infected with Dd2Dd2 L272F parasites remained knobless (Fig.4d), suggesting that the
increased expression of knobs in 3D7L272F-infected RBCs was not related to the L272F mutation in pfcrt.
Measurements of CQ transport via the C101F and L272F variants of PfCRT. e Xenopus
oocyte system for the heterologous expression of PfCRT7 was employed to investigate the eect of the
C101F and L272F mutations on the ability of PfCRT to mediate CQ transport. e L272F and C101F
mutations were introduced into the Dd2 haplotype of PfCRT (PfCRTDd2, from the CQ-resistant strain
Dd2; Fig.1b) and L272F was also introduced into PfCRT3D7 (from the CQ-sensitive strain 3D7; Fig.1b).
e resulting variants (L272F PfCRTDd2, L272F PfCRT3D7, and C101F PfCRTDd2), as well as PfCRTDd2 and
PfCRT3D7, were expressed in oocytes. Localization of each of the PfCRT variants to the oocyte plasma
membrane was conrmed by immunouorescence assay (Supplementary Fig. S2a) and a semiquantita-
tive western blot analysis32 indicated that the dierent PfCRT proteins were present at similar levels in
the oocyte membrane (Supplementary Fig. S2b). e ability of the PfCRT variants to mediate [3H]CQ
transport was measured in an acidic medium (pH 5.5), in which the majority of CQ is protonated. e
extent to which oocytes expressing PfCRTDd2 accumulate [3H]CQ varies considerably between batches
of oocytes from dierent frogs, with the PfCRTDd2-expressing oocytes accumulating between 8 and 45
times more [3H]CQ than the control (non-injected and PfCRT3D7-expressing) oocytes. Hence, within
each experiment uptake was expressed relative to that obtained for oocytes expressing PfCRTDd2 (in
the absence of inhibitors). Non-injected oocytes and oocytes expressing PfCRT3D7 have previously been
shown to take up CQ to similar (low) levels via simple diusion of the neutral species of the drug7,32;
this represents the ‘background’ level of CQ accumulation in oocytes, which in this study was estimated
by measuring CQ uptake into PfCRT3D7-expressing oocytes (see Supplementary Fig. S3).
In the data presented in Fig.5a,b, oocytes expressing PfCRTDd2 showed an 11 to 40-fold (mean and
SEM of 21 ± 3; n = 9 separate experiments) increase in CQ uptake relative to the PfCRT3D7-expressing
control. e component of CQ accumulation attributable to diusion (i.e. the uptake of CQ measured
in PfCRT3D7-expressing oocytes) was subtracted to obtain the PfCRT-mediated component of CQ trans-
port. Supplementary Figure S3 shows the total level of CQ accumulation in each oocyte and treatment
type. e introduction of L272F or C101F into PfCRTDd2 substantially reduced the protein’s ability to
transport CQ (by ~82% and ~93%, respectively; p < 0.001, ANOVA) whereas the introduction of L272F
into PfCRT3D7 was without eect (p > 0.05). e addition of the CQ resistance-reverser VP (250 μ M)
reduced PfCRTDd2-mediated CQ transport by ~93% (p < 0.001) and also dramatically decreased CQ
uptake via L272F PfCRTDd2 and C101F PfCRTDd2 (by ~84% and ~92%, respectively; p < 0.01), such that
the accumulation of CQ in the latter two treatments was not signicantly dierent from that measured
in the PfCRT3D7-expressing controls (p > 0.05).
To investigate how BSD pressure might have produced the 3D7L272F mutant, interactions between
the PfCRT variants and BSD were assessed by measuring the uptake of [3H]CQ in the presence of
unlabeled BSD (100 or 500 μ M; Fig. 5b). e addition of BSD reduced CQ transport via PfCRTDd2 by
~39% (100 μ M; p < 0.001) and ~56% (500 μ M; p < 0.001) and, to a lesser degree, decreased CQ uptake
via L272F PfCRTDd2 (by ~22% (p > 0.05) and ~49% (p < 0.01), respectively). Neither concentration of
BSD reduced the C101F PfCRTDd2-mediated transport of CQ (p > 0.05), nor was the accumulation of
CQ in the PfCRT3D7-expressing controls aected (p > 0.05). Note that the micromolar concentrations of
the compounds used here to inhibit PfCRT are physiologically relevant given that when present in the
Drug†
Mean ± SEM IC50 values for individual parasite strains/lines‡
Dd2 Dd2Dd2 Dd2Dd2 L272F GC03
CQ 97 ± 6.8 88 ± 6.8 20 ± 1.8* 13 ± 2.3
MDCQ 497 ± 43 440 ± 33 121 ± 9.0* 26 ± 3.0
MDAQ 45 ± 5.8 35 ± 3.6 22 ± 4.0** 18 ± 1.1
PPQ 32 ± 3.1 32 ± 5.8 39 ± 1.5 23 ± 5.3
ART 18 ± 1.1 18 ± 4.6 13 ± 1.9 15 ± 4.7
BSD 456 ± 46 631 ± 35*** 708 ± 54 456 ± 59
Table 2. In vitro sensitivity of Dd2, Dd2Dd2, Dd2Dd2 L272F and GC03 to antimalarial drugs. †CQ,
chloroquine; MDCQ, monodesethyl chloroquine; MDAQ, monodesethyl amodiaquine; PPQ, piperaquine;
ART, artemisinin; BSD, blasticidin. ‡IC50 values are listed in nM and are shown as the mean ± SEM. n = 3
independent assays (each performed in duplicate). Signicantly dierent mean IC50 values between Dd2Dd2
and Dd2Dd2 L272F (F-test; *p < 0.0001, **p = 0.07) and between Dd2 and Dd2Dd2 (F-test; ***p = 0.038).
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SCIENTIFIC RepoRts | 5:14552 | DOI: 10.1038/srep14552
extracellular solution at nanomolar levels, these protonatable drugs are expected to accumulate within
the parasite’s FV via weak-base trapping to micromolar or millimolar concentrations.
Discussion
Mannaberg stained parasites with Romanowsky’s dyes and published detailed studies on the eects of
QN against P. falciparum, which described the emergence of a ‘dropsical distension’ (enlarged FV) in
mature parasites33. Here, we describe a similar peculiar phenotype of P. falciparum parasites that is visible
without the application of antimalarial drugs. is phenotype is comparable between two parasite lines
that have mutations in pfcrt in dierent positions (amino acids 101 and 272) and that have been selected
by two chemically unrelated compounds (AMT and BSD). ese mutations conrm that pfcrt encodes a
function that is critical to maintaining FV volume. In support of this function, mutations in PfCRT that
cause CQ resistance have been reported to increase FV volume34. However, the parasite lines described
in this present study have clearly enlarged FVs but with PfCRT mutations that render the parasites more
CQ sensitive than their control strains (be that either CQ-sensitive 3D7 or CQ-resistant FCB), suggesting
an alternative mechanism of FV volume regulation is induced.
An enlarged FV is also oen observed in the presence of protease inhibitors, such as E64 or leupep-
tin35. Interference with the digestion of hemoglobin leads to a buildup of darkly staining FVs in electron
micrographs and, eventually, to parasite death. e parasites described here have enlarged FVs but these
are electron lucent (Figs2 and 4), suggesting that the digestion of hemoglobin is relatively unaected
(further supported by the presence of visible hemozin within the FVs). e simplest explanation for these
observations is that the C101F and L272F mutations interfere with the transport of the natural substrates
of PfCRT out of the FV. e resulting increase in FV osmotic pressure would lead to water ingress and
produce the unusual swelling observed in the FV of the FCBC101F, 3D7L272F, and Dd2Dd2 L272F parasites.
Figure6 presents a schematic model of this process. ese morphological changes are associated with
other phenotypic changes (which are discussed below). e natural substrate(s) of PfCRT are yet to be
identied. Studies performed with other PfCRT expression systems have reported that the protein might
function as a chloride channel, a proton pump, an activator of Na+/H+ exchangers and non-specic
cation channels or, most recently, a transporter of cationic amino acids as well as a very broad range of
other cations15. However, in many of these studies the insertion of PfCRT into the foreign membrane
required its fusion to other proteins/polypeptides, and in the most recent study the additions to PfCRT
were at both the N- and C- termini, almost doubled its size, and included a protein of undetermined
function15. Moreover, in this and the previous studies, little or no interaction could be detected between
PfCRTDd2 and known inhibitors of this protein (e.g. VP). Of signicant note, the transport kinetics for
the proposed natural substrates did not dier signicantly between PfCRTDd2 and PfCRT3D7—despite
Figure 5. CQ transport activity of the C101F and L272F variants of PfCRT in Xenopus oocytes. (a,b)
e uptake of [3H]CQ into oocytes expressing PfCRT was measured in the absence (closed bars) or presence
of 250 μ M VP (light grey bars; a), 100 μ M BSD (dark grey bars; b), or 500 μ M BSD (open bars; b). Within
each experiment, measurements were made from 10 oocytes per treatment and uptake was expressed
relative to that measured in the PfCRTDd2-expressing oocytes under control conditions. e normalized
data obtained from 4–5 separate experiments (each using oocytes from dierent frogs) were then averaged
and are shown + SEM. Both panels show PfCRT-mediated CQ uptake, calculated by subtracting CQ uptake
measured in PfCRT3D7-expressing oocytes (i.e. the component of CQ accumulation attributable to diusion;
see Supplementary Fig. S3) from that measured in oocytes expressing a variant of PfCRT. In the control
treatments, the rates of CQ uptake (pmol/oocyte/h; n = 9 ± SEM) in oocytes expressing PfCRTDd2 and
PfCRT3D7 were 23.6 ± 2.3 and 1.3 ± 0.2, respectively. ‘ns’ denotes no signicant dierence (p > 0.05) in CQ
accumulation between oocytes expressing a PfCRT variant (in the presence or absence of VP or BSD) and
that measured in the PfCRT3D7-expressing oocytes under control conditions.
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multiple lines of evidence indicating that PfCRTDd2 imparts a substantial tness cost13,36–38. Furthermore,
the recent nding that much higher levels of acidic amino acids and/or short acidic peptides accumulate
within CQ-resistant parasites than in CQ-sensitive strains7,13,14 is not readily reconciled with PfCRT
functioning as a chloride channel, a proton pump, or a non-specic cation channel/transporter. ese,
plus other inconsistencies in the data, suggest that PfCRT does not function correctly when fused to
other proteins and that the natural function of PfCRT remains to be resolved.
AMT is an antiviral agent with moderate antimalarial activity that is more potent against CQ-resistant
parasites than against CQ-sensitive strains39. AMT is likely to accumulate in the FV via weak-base
trapping40 and is a low-anity inhibitor of the PfCRTDd2-mediated transport of CQ in the oocyte sys-
tem7. While the antiplasmodial target of AMT remains unclear, AMT resistance has been linked previ-
ously to novel PfCRT mutations (S163R, I356V and V369F; Fig.1) selected in parasites harboring CQ
resistance-associated alleles of pfcrt; these mutations were linked with the loss of CQ resistance in the
AMT-resistant mutants41,42. Here, a dierent single mutation (C101F) in the CQ-resistant FCB strain was
likewise associated with a gain of AMT resistance and a reduction in CQ resistance. is mutation was
identied previously in a PPQ-pressured parasite line that appeared to have acquired an unstable PPQ
resistance phenotype via multiple genetic changes22. One of two PPQ-revertant lines derived during that
study was ~2-fold more resistant to PPQ than the parental Dd2 strain, which along with a reduction in
CQ resistance, is consistent with the data reported here for FCBC101F.
It has been suggested that the S163R mutation reintroduces a positive charge into the PfCRT binding
pocket/translocation pore, thereby compensating for the loss of the positively-charged lysine residue
from position 7611 and resulting in a dramatic reduction in the ability of the protein to transport pro-
tonated CQ7. e S163R mutation also abolishes the CQ resistance-reversing eect of VP42. e C101F
and V369F mutations both entail the introduction of a bulky hydrophobic residue, rather than one car-
rying a positive charge, and it is interesting to note that VP still exerted a resistance-reversing eect in
the FCBC101F parasites (Table1)—even though they were considerably less resistant than the FCB strain
Figure 6. Hypothetical schematic model of the eects of PfCRT mutations. e FV is acidied by
a vacuolar proton pump to create a suitable environment for hemoglobin digestion. e acidic nature
of the FV also leads to near complete diprotonation of CQ, which diuses across the FV membrane
in an uncharged form (CQ) and accumulates as a charged form (either CQH+ or CQH22+, although
predominantly CQH22+). CQH22+ interferes with the polymerization of toxic heme to non-toxic hemozoin,
which leads to parasite death. In normal CQ-sensitive (CQS) parasites, PfCRT, which contains a positive
charge in its pore (K76), exports its natural substrates but little, if any, CQH22+. us, CQH22+ accumulates
in the FV and causes parasite death. In CQ-resistant (CQR) parasites, the positive change in the pore of
PfCRT is lost (K76T) and both its natural substrates and CQH22+ are transported out of the FV. As CQH22+
cannot accumulate in the FV, the parasites become resistant to the drug. In 3D7L272F parasites (where the
parent strain is already CQS), the mutation may reduce residual transport of CQH22+ out of the FV even
further or completely, leading to a greater FV accumulation of CQH22+ and CQ-hypersensitivity or some
other mechanism may be responsible for this phenomenon. e mutation also leads to a reduction in the
export of natural substrates, resulting in a build-up of these substrates. is causes water to enter the FV by
the process of osmosis, leading to swelling. In FCBC101F and Dd2Dd2 L272F parasites (where the parent strains
are CQR), the mutations reduce the export of CQH22+ back towards levels measured in CQS lines and
also reduce natural substrate export, leading to normal CQ sensitivity and FV swelling, respectively. Note
mutations in PfCRT (orange graphic) are denoted by red transmembrane or loop regions, depending on the
location of the amino acid change (see Fig.1a).
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to CQ. ese observations are consistent with our direct measurements of CQ transport via C101F
PfCRTDd2 (Fig.5), which conrmed that this protein possesses a relatively low level of CQ transport
activity that can be inhibited by VP. Likewise, our nding that the introduction of L272F into PfCRTDd2
causes a dramatic (but not complete) reduction in the protein’s capacity for CQ transport (Fig.5) corre-
lates well with the low level of CQ resistance exhibited by the Dd2Dd2 L272F line. e phenylalanine resi-
dues are likely to be proximate to the binding site and/or translocation pore of PfCRT (Fig.1) where their
bulky side chains may act to signicantly hinder the transport of certain drugs out of the FV, including
CQ, QN and QD (based on the growth assay data presented in Tables1 and 2). Another mutation that
has arisen under the AMT pressure of a CQ-resistant strain, and which also resulted in both the intro-
duction of a phenylalanine residue (V369F) and a signicant reduction in CQ resistance, did not cause
the FV to swell41. Hence, the enlarged FV phenotype described here appears to manifest only when the
bulky phenylalanine side chain is inserted at specic positions within PfCRT.
BSD is used commercially as a fungicide against a rice blast disease and acts by inhibiting protein
translation. In biological research it is used to select transformed cells. BSD resistance has previously been
linked to altered expression of clag3.1 and a decrease in the RBC membrane permeability mediated by the
new permeability pathways, NPP28,29. Neither 3D7L272F nor Dd2Dd2 L272F parasites diered in their electro-
physiological NPP characteristics when assayed by whole-cell patch-clamp methods (Supplementary Fig.
S1). is suggests that one or more clag3.1-independent BSD resistance mechanisms exist. Our results
indicate that, under the conditions of the growth assay, the L272F mutation does not cause a signicant
increase in BSD resistance when introduced in isolation into Dd2 parasites (Table 3). Nevertheless, BSD
was found to inhibit CQ uptake via PfCRTDd2 and the potency of this interaction appeared to decrease
upon the introduction of L272F (the addition of 100 μ M BSD was more eective against PfCRTDd2 than
against L272F PfCRTDd2; Fig.5). A demonstration that BSD interacts with PfCRTDd2, and to a lesser
extent with L272F PfCRTDd2, provides support for the idea that BSD also interacts with, and may be
transported by, PfCRT3D7. BSD contains two protonatable nitrogens with pKa values that are well above 7.
It is therefore likely to be accumulated within the FV via weak-base trapping to high micromolar, or even
millimolar, concentrations when present in the extracellular solution at the concentration (5.4 μ M) under
which the 3D7L272F line arose. Given that BSD inhibits protein translation, which occurs outside of the
FV, it is possible that a PfCRT3D7-mediated eux of BSD from the FV could increase the drug’s access to
its main target and that the L272F mutation diminishes this activity, such that the drug remains seques-
tered within the FV. e nding that L272F PfCRTDd2 does not confer BSD resistance when expressed
in Dd2 parasites suggests that either (1) PfCRTDd2 is already a poor transporter of BSD (noting that the
3D7 and Dd2 haplotypes of PfCRT dier by eight mutations) and a reduction in this meager transport
activity by the introduction of L272F has little eect on the accumulation of BSD within the FV, or (2)
PfCRTDd2 has a very high capacity for BSD transport, such that the presence of L272F causes only a mod-
est reduction in its ability to redistribute BSD from the FV into the cytosol. In any case, it is clear that if
L272F is directly involved in altering the parasite’s susceptibility to BSD, its eect is only evident when
one or more other changes are present. In this regard, it is worth noting that one of the two mutations
identied by whole genome analysis of the 3D7L272F line would result in a truncated PfMRP2 protein.
An understanding of the contribution of this transporter to BSD susceptibility, and its possible interplay
with the BSD transport activity of PfCRT, requires further transfection-based analysis.
A diverse range of PfCRT variants implicated in conferring CQ resistance have been shown to
exhibit CQ transport activity (to varying extents) in the oocyte system7,32. However, CQ transport via
the wild-type form of the protein (found in CQ-sensitive parasites such as 3D7) has not been detected
in this assay. Although it is possible that a very low level of CQ eux is mediated by PfCRT3D7 in situ,
and that the introduction of the L272F mutation abolishes this activity, it is perhaps unlikely that this
would result in the 2-fold dierence in the CQ IC50 observed between the 3D7L272F and 3D7 parasites.
An alternative explanation for the hypersensitivity of the 3D7L272F line to CQ and QN entails view-
ing the eect of the L272F mutation as being equivalent to the eect of an ‘anti-PfCRT’ drug. e
presence of the L272F mutation causes the FV to swell, probably because it signicantly obstructs the
PfCRT-mediated eux of solutes from this compartment. A drug that binds to the substrate-binding
site of PfCRT3D7, thereby blocking or dramatically reducing its normal activity, would achieve a similar
eect. If such an anti-PfCRT drug were applied in combination with CQ or QN, which also exert their
antimalarial eects in the FV, it is possible that an additive, or even synergistic, interaction would be
observed in 3D7L272F parasites. at is, the 3D7L272F parasites have a dis-functional FV and this could
render certain drugs more eective against them; perhaps the altered composition of the FV lumen
alters the solubilities of CQ and QN and/or their anities for heme. It is not immediately apparent
why AQ, MDAQ, and PPQ are not likewise more active against 3D7L272F parasites. However, it is worth
noting that AQ, MDAQ and PPQ are much more lipophilic than CQ and this may explain dierences
in potency43,44. Hence, the antimalarial activities of the latter drugs might be less sensitive to changes in
FV volume and composition. Alternatively, or in addition, it is also possible that extending the growth
assays to 72 or 96 h (from 48 h) would reveal dierences in AQ, MDAQ, and PPQ sensitivity between
the 3D7 and 3D7L272F parasites.
If artemisinins act mainly in the FV, a disputed suggestion45 (along with reports of predominantly
non-FV-localized artemisinin targets, e.g.46,47), then their activities may also dier between the parental
and mutant lines. ere was no signicant change in the ART sensitivity of the 3D7, Dd2 and GC03 lines
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and only a < 30% change in the IC50 value obtained for FCB (which displays an IC50 value < 15 nM).
Recent observations made in a P. berghei model using protease knockouts that alter vacuolar morphology
also leave artemisinin sensitivity unaltered48. is is also consistent with the lack of PfCRT expression
in early ring stages49, when artemisinins exert their major antimalarial action in vivo. Similarly, the MF
IC50 value was unaected in the experiments presented here. Our observations therefore relate relatively
large changes in the activities of several aminoquinolines to an enlarged FV phenotype that is caused by
specic mutations in PfCRT.
Our results show for the rst time that mutations at position 272 and 101 in PfCRT can hypersensitize
parasites to CQ and enlarge the FV, thereby extending the function of this key transporter to include
maintenance of FV morphology. We suggest that the introduction of a phenylalanine residue at either of
these positions decreases the protein’s ability to transport its physiological substrate(s) (as well as certain
drugs) and that the resulting build-up of the physiological substrate(s) causes the FV to swell. e fact
that these mutations do not reintroduce a positive charge into the predicted binding cavity/translocation
pore of PfCRT, as has been observed in other examples of laboratory parasites that revert to CQ-sensitive
status (e.g.42), indicates that there is more than one type of single mutation—and therefore more than one
mechanism—by which the CQ transport activity of PfCRT can be abrogated. is insight extends our
understanding of the structure-function of PfCRT (e.g.32). Moreover, the nding that single mutations
to the protein can result in gross changes to parasite morphology emphasizes the central role of this
transporter in the physiological processes that occur within the FV and provides a novel insight into
one of the factors constraining the evolution of PfCRT. e observation that BSD binds to, and appears
to exert a selection pressure on, PfCRT further broadens the diversity of chemotypes that are known (or
suspected) to interact with the protein. Our data encourage further studies to dene agents that could
reverse antimalarial drug resistance mediated by PfCRT by inhibiting its function.
Methods
Antimalarials and reagents. CQ, QN, QD, MQ, AQ, PPQ, ART, BSD, AMT and VP were purchased
from Sigma Aldrich Chemical Co. MDAQ was purchased from Santa Cruz Biotechnology, Inc. MDCQ
was a gi from William Ellis (Walter Reed Army Institute of Research, Silver Spring, MD). SYBR Green
I was purchased from Invitrogen Corp. Drug stocks were prepared to 10 mM in DMSO or 70% ethanol
and stored below - 20 °C.
In vitro culture and selection of parasites. P. falciparum 3D7 and 3D7L272F parasites were cul-
tured in human RBC suspensions using RPMI 1640 medium (Sigma-Aldrich; Cat. No. R0883-500ML)
supplemented with 2 mM L-glutamine, 34 mM HEPES, 0.5% (w/v) Albumax I, 0.19 mM hypoxanthine,
and 50 μ g/ml gentamycin and maintained at 37 °C under 5% CO2. For parasite clone 3D7L272F, complete
medium was supplemented with 2.5 μ g/ml blasticidin-S HCl (Invitrogen). Parasite growth was followed
by microscopic examination of Field’s stained thin blood smears. Synchronization of early trophozoite
stages was achieved by incubating infected RBCs in 5% (w/v) sorbitol for 10 to 20 min at room temper-
ature50. Following transfection studies23, parasites with abnormally enlarged FVs, as described in results,
reappeared in culture under BSD pressure aer four weeks. In order to select these parasites, the lim-
iting dilution technique was used, and cloned parasites were identied by microscopy using thin blood
smears51.
P. falciparum FCB and FCBC101F parasites were cultured in AB+ or O+ human RBC suspensions using
RPMI 1640 medium (Mediatech, Inc.) supplemented with 0.5% Albumax I, 29.8 mM sodium bicarbo-
nate, 25 mM HEPES, 0.37 mM hypoxanthine, and 10 μ g/ml gentamicin and maintained at 37 °C under
an atmosphere of 90% N2, 5% CO2, and 5% O2. AMT-resistant P. falciparum was selected by single-step
selection based on an earlier method described for CQ6. Before drug pressure, parasites of the FCB strain
were grown to 5% mixed stage parasitemia at 5% hematocrit in 50 ml of media. is starter culture was
then split equally into 4 asks, with fresh media and RBCs to bring the volume in each ask to 50 ml
and 5% hematocrit. When parasitemia of the 4 asks had returned to 5%, the media was replaced with
fresh media containing 80 μ M AMT. At ~14 fold the IC50 value determined for FCB (Table 2), this
concentration of AMT rapidly kills CQ-resistant parasites. For the rst week aer drug application, cul-
tures were monitored daily by Giemsa-stained thin blood lms. Fresh AMT-containing media changes
were performed daily. At one week, 50% of the RBCs were replaced, and fresh AMT media was added.
Cultures were then maintained every third day with fresh AMT media for the duration of the experiment
and monitored by thin smear for emergent parasites. With every second media change, 50% of the RBCs
were replaced with fresh cells. If no surviving parasites were observed aer 60 days, the experiment was
terminated. Aer 42 days, parasites were recovered from one of the 4 asks, which were then cloned by
limiting dilution52 in drug-free media. e mixed culture and four randomly chosen cloned lines were
cryopreserved prior to DNA sequencing and drug susceptibility testing.
Morphological measurements. For comparison, thin blood lms of cultured parasite samples were
made at various time points following sorbitol synchronization and stained with Field’s stain. Pictures
were taken under the same conditions for the 3D7 (parent) strain and the 3D7L272F line and analyzed
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with a Nikon Eclipse TE2000 inverted microscope. Areas were measured using ImageJ 1.44o soware
and the ratio was expressed as AFV/AParasite. No image manipulations were carried out aer recording.
For micrographs of FCB and FCBC101F, thin lms from parasite cultures were stained with 2% (v/v)
modied Giemsa (Karyomax® ; Gibco) for 30 min. Slides were washed for 60 s in owing distilled water,
air-dried and mounted with coverslips. Images were photographed in bright eld, using a Lexica DMI4000
inverted microscope under a 100X objective lens. Images were compiled in Adobe Photoshop CS5.1 and
processed equally with a warming photo lter. Live parasite cultures were placed under coverslips and
photographed under a 100X objective, using a Leica DM750 light microscope equipped with ICC50 HD
digital camera. Images were adjusted for white balance with the Leica Application Suite soware and
cropped in Adobe Photoshop CS5.1.
For experiments with Dd2 parasites, thin blood smears were xed with methanol, stained for 20 min
in 10% (v/v) Giemsa (Invitrogen), washed, and air-dried. Images were taken with an Olympus DP12
digital camera attached to an Olympus CX 41 light microscope with a 100X objective (N.A 1.4x). Images
were cropped and corrected for white balance using Adobe Lightroom 3.
Electron microscopy. Samples of 3D7, 3D7L272, Dd2Dd2 and Dd2Dd2 L272F, synchronized at the mature
trophozoite stage were xed in 4% (v/v) glutaraldehyde in 0.1 M phosphate buer and processed for
routine electron microscopy, as described previously53. Samples were post xed in osmium tetroxide,
treated en bloc with uranyl acetate, dehydrated and embedded in Spurr’s epoxy resin. in sections were
stained with uranyl acetate and lead citrate prior to examination in a JEOL1200EX electron microscope.
In vitro inhibition assays. Sensitivity to CQ and other drugs for 3D7 and 3D7L272F parasites was
determined by measurement of [3H]-hypoxanthine incorporation over 48 h, as described previously54.
Nine serial dilutions plus a control (no drug) were tested in quadruplicates and the experiment was
repeated at least three times for each drug. e assay was performed always in parallel on 3D7 and
3D7L272F parasites.
e in vitro susceptibility of FCB and the FCBC101F line of P. falciparum to antimalarial drugs was
measured in a 72 h, 96 well microplate uorescence assay using SYBR Green I detection as described55,56.
Drugs were serially diluted 2-fold in the microplates, except for AMT, which was diluted 3-fold. VP was
used at a concentration of 0.8 μ M where indicated. Synchronous (immature) ring-stage parasites were
assayed at 0.2% parasitemia and 2% hematocrit. Assays were conducted every 48 h until three independ-
ent replicates were performed. For Dd2 parasites, the same methodology was used except parasites were
also stained with 1.6 μ M Mito Tracker Deep Red.
Genotypic characterization of pfcrt gene. For 3D7 parasites, RNA was extracted from parasites
collected in RNAlater, using QIAGEN RNeasy Mini Kit, and immediately used to retro-transcribe cDNA
(QIAGEN, QuantiTect Rev. Transcription Kit). Mutation in pfcrt was investigated by PCR, as described
previously1. e same primers (Supplementary Table S1), which amplied overlapping products, were
used to sequence the products to cover the entire open reading frame (ORF) of the gene. Amplication
of the gene and its sequencing was performed twice (by Beckman Coulter Genomics). Alignment of
the reported 3D7 gene from PlasmoDB and 3D7 and 3D7L272F sequenced genes was performed using
MacVector soware (version 11.1).
For FCB parasites, 4 clonal lines of FCBC101F were used for pfcrt sequencing. All ORF sequences of
pfcrt were amplied from P. falciparum genomic DNA57. PCR products were sequenced directly using an
ABI 3730xl DNA analyzer (Applied Biosystems).
Whole genome sequencing and variant detection. Genomic DNA was isolated and prepared
from the parental P. falciparum parasite line 3D7 and 3D7L272F. A total of 10 μ g of gDNA from each line
was sheared to obtain a fragment size of ~200–400 bp using an E220 focused-ultrasonicator (Covaris)
with the following settings: 10% duty cycle, intensity 5, 200 cycles per burst, 180 s treatment length.
e resulting sheared gDNA was size selected on a 2% (w/v) low-melting agarose gel and then puried
and concentrated using MinElute purication columns followed by the QIAquick PCR purication kit
(QIAGEN). Barcoded libraries for Illumina TruSeq single-end sequencing were then constructed from
the size-selected, sheared material using NEBNext DNA Library Preparation reagents (New England
Biolabs) by following the standard Illumina (Illumina) library preparation protocol. Finally, barcoded
libraries were size selected using Agencourt AMPure XP magnetic beads (Agencourt Biosciences,
Beckman Coulter) thereby removing any adapter dimers and resulting in a highly enriched barcoded
library of 200–400 bp adapter-ligated fragments. e quality of the nal sequencing libraries was assessed
using an Agilent 2100 Bioanalyzer (Agilent Technologies) run alongside the original size-selected frag-
mented gDNA from the same preparation, and the concentration of each library was quantied using
a Quant-iT dsDNA Broad-Range Assay Kit (Invitrogen). e nal libraries were multiplexed with three
barcoded samples and 20% (v/v) PhiX control DNA (Illumina, Catalog # FC-110-3001) per lane and were
sequenced using an Illumina HiSeq 2500 Rapid Run (150 bp) system (Illumina).
Sequencing outputs were uploaded into Galaxy58, which is hosted locally at the Millennium Science
Complex at Pennsylvania State University. Sequence reads were mapped to the P. falciparum 3D7
reference genome v. 10.0 (http://plasmodb.org/common/downloads/release-10.0/Pfalciparum/) and
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pCam-BSD-PfATP6-doubleHA plasmid sequence23, using the Burrows-Wheeler alignment tool59, and
les were converted to allow for further analysis (GATK/BAM-to-SAM)60. Sequence variations were
detected by Freebayes (version 0.9.0.a) using stringent ltering parameters based on quality and read
depth61,62. en SNPe (version 3.3) was applied to annotate and determine statistical signicance of
each variant63. Genome copy number variations were detected based upon local chromosomal read depth
using CNVnator (version 0.3) and annotated with Intansv (version 0.99.3)64. Alignments and variants
were visualized using the Integrative Genomics Viewer65. Unique reads were selected and ltered for
Map Quality > 30.
Plasmid construction and generation of Dd2 recombinant parasites. e donor plasmid pcrtDd2-
hdhfr has been previously reported31. e mutation-encoding plasmid, pcrtDd2 L272F-hdhfr, was generated
by site-directed mutagenesis of pcrtDd2-hdhfr, using primers p3527 + p3528 (Supplementary Table S2).
ZFN-editing transfection methods have been previously described31. Briey, Dd2 parasites were elec-
troporated with either pcrtDd2-hdhfr or pcrtDd2 L272F-hdhfr donor plasmid66. On Day 1 post-electroporation,
they were cultured in the presence of 2.5 nM WR99210 (obtained from Jacobus Pharmaceuticals Inc.).
Once recovered, both pcrtDd2-hdhfr and pcrtDd2 L272F-hdhfr transfected parasites were electroporated a
second time with pZFNcrt-bsd separately. On Day 1 post-electroporation each transfection was cultured
with 2 μ g/ml blasticidin S (Invitrogen) and 2.5 nM WR99210 for six days and followed by addition of
only 2.5 nM WR99210, generating Dd2Dd2and Dd2Dd2 L272F parasites, respectively. Clones were established
from the bulk cultures by limiting dilution52. PCR primers for verication of parental, recombinant con-
trol, and Dd2Dd2 L272F parasite clones are shown in Supplementary Table S2.
Expression of the C101F and L272F variants of PfCRT in X. laevis oocytes and measurements
of CQ transport. Ethical approval of the work performed with the X. laevis frogs was obtained from
the Australian National University Animal Experimentation Ethics Committee (Animal Ethics Protocol
Number A2013/13) in accordance with the Australian Code of Practice for the Care and Use of Animals
for Scientic Purposes. e C101F PfCRTDd2, L272F PfCRTDd2, and L272F PfCRT3D7 coding sequences
were generated via site-directed mutagenesis using an approach described previously32. e mutations
were introduced into codon-harmonized versions of the PfCRTDd2 and PfCRT3D7 coding sequences,
which encode retention motif-free forms of these proteins that are expressed at the plasma membrane
of X. laevis oocytes7. All of the resulting coding sequences were veried by sequencing. e in vitro tran-
scription of cRNA and the harvest and preparation of oocytes were performed as outlined elsewhere67.
e oocytes were microinjected with 20 ng of cRNA and the uptake of [3H]CQ (0.25 μ M; 20 Ci/mmol;
American Radiolabeled Chemicals) was measured 3–4 days post-injection as detailed previously67. e
measurements were made over 1.5 h at 27.5 °C and in medium that, unless otherwise specied, con-
tained 96 mM NaCl, 2 mM KCl, 2 mM MgCl2, 1.8 mM CaCl2, 10 mM MES, 10 mM Tris·base (pH 5.5),
and 15 μ M unlabeled CQ. In all cases, at least three separate experiments were performed (on oocytes
from dierent frogs), and in each experiment measurements were made from 10 oocytes per treatment.
Immunouorescence and western blot analyses of oocytes expressing PfCRT.
Immunouorescence analyses were performed on oocytes 3 days post-injection using an approach
described elsewhere68. Briey, the oocytes were xed and labeled with rabbit anti-PfCRT antibody (con-
centration of 1:100; Genscript32; ) and Alexa Fluor 488 goat anti-rabbit antibody (concentration of 1:500;
Molecular Probes). e oocytes were embedded in an acrylic resin using the Technovit 7100 plastic
embedding system (Kulzer) as outlined previously69 and images of 4 μ m slices were obtained with a Leica
Microsystems inverted confocal laser microscope. At least two separate experiments were performed (on
oocytes from dierent frogs) for each treatment and slices were examined from a minimum of three
oocytes within a treatment. All of the slices taken from oocytes expressing a PfCRT variant displayed
a uorescent band above the pigment layer (consistent with the localization of PfCRT to the plasma
membrane) that was not present in non-injected oocytes.
e preparation of oocyte membranes and the semi-quantication of PfCRT protein was carried out
using a protocol described in detail elsewhere32. Protein samples prepared from oocyte membranes were
separated on a 4–14% bis-Tris SDS-polyacrylamide gel (Life Technologies) and transferred to nitrocel-
lulose membranes. e membranes were probed with rabbit anti-PfCRT antibody (1:4,000) followed by
horseradish peroxidase-conjugated goat anti-rabbit antibody (1:8,000; Life Technologies). e PfCRT
band for each variant was detected by chemiluminescence (Pierce), quantied using the Image J so-
ware70, and expressed as a percentage of the intensity measured for the PfCRTDd2 band. In all cases, at
least three separate experiments were performed (on oocytes from dierent frogs), and in each experi-
ment measurements were averaged from two independent replicates.
Curve tting and statistical analyses. Mean half-maximal inhibitory concentrations (IC50 val-
ues) were derived by plotting percent growth inhibition against log drug concentration, and tting the
response data to a variable slope, sigmoidal curve-t function for normalized data using Prism 5.0d for
Macintosh (GraphPad Soware). IC50 values represent means ± standard error from 3 independent tests.
IC50 values between mutant and parent lines were tested for statistically signicant dierences using an
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SCIENTIFIC RepoRts | 5:14552 | DOI: 10.1038/srep14552
F-test that determines whether the two dose response data sets are best described by single or independ-
ent curve ts (p < 0.05). In the case of the oocyte data, statistical comparisons were made using ANOVA
in conjunction with Tukey’s multiple comparisons test. Other data were compared using the Student’s
t-test and Fisher’s exact test as noted.
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Acknowledgments
is work was supported by the European Community’s Seventh Framework Programme, FP7/2007-2013
(Marie Curie-funded Initial Training Network InterMal, 215281-2 to SK and NanoMal, 304948 to SK
and HMS), the NIH (R01 AI50234 to DAF and R01 AI071121 to RAC), the Australian National Health
and Medical Research Council (Grant 1007035 and Fellowship 1053082 to REM) and the Burroughs
Wellcome Fund - Investigators in Pathogenesis of Infectious Disease award (1007041.02 to ML).
Author Contributions
Experimentation was undertaken by S.P., A.H.L., S.H.S., G.B., C.M.M., D.A.D., M.J.H., L.M.A., H.J.P., J.M.
and D.J.P.F. and designed by H.M.S., D.J.P.F., M.L., R.E.M., D.A.F., R.A.C. and S.K. e manuscript was
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16
SCIENTIFIC RepoRts | 5:14552 | DOI: 10.1038/srep14552
prepared by S.P., H.M.S., A.H.L., M.L., R.E.M., D.A.F., R.A.C. and S.K. All authors had the opportunity
to read and approve the manuscript.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Pulcini, S. et al. Mutations in the Plasmodium falciparum chloroquine
resistance transporter, PfCRT, enlarge the parasite's food vacuole and alter drug sensitivities. Sci. Rep.
5, 14552; doi: 10.1038/srep14552 (2015).
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