Quinine localizes to a non-acidic compartment within the food vacuole of the malaria parasite Plasmodium falciparum.
ABSTRACT BACKGROUND: The naturally fluorescent compound quinine has long been used to treat malaria infections. Although some evidence suggests that quinine acts in the parasite food vacuole, the mechanism of action of quinine has not yet been resolved. The Plasmodium falciparum multidrug resistance (pfmdr1) gene encodes a food vacuolar membrane transporter and has been linked with parasite resistance to quinine. The effect of multiple pfmdr1 copies on the subcellular localization of quinine was explored. METHODS: Fluorescence microscopy was used to evaluate the subcellular localization of quinine in parasites containing different pfmdr1 copy numbers to determine if copy number of the gene affects drug localization. The acidotropic dye LysoTracker Red was used to label the parasite food vacuole. Time-lapse images were taken to determine quinine localization over time following quinine exposure. RESULTS: Regardless of pfmdr1 copy number, quinine overlapped with haemozoin but did not colocalize with LysoTracker Red, which labeled the acidic parasite food vacuole. CONCLUSIONS: Quinine localizes to a non-acidic compartment within the food vacuole possibly overlapping haemozoin. Pfmdr1 copy number does not affect quinine subcellular localization.
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ABSTRACT: Amino acid utilization is important for the growth of the erythrocytic stages of the human malaria parasite Plasmodium falciparum, however the molecular mechanism that permits survival of the parasite during conditions of limiting amino acid supply is poorly understood. We provide data here suggesting that an autophagy pathway functions in P. falciparum despite the absence of a typical lysosome for digestion of the autophagosomes. It involves PfATG8, which has a C-terminal glycine which is absolutely required for association of the protein with autophagosomes. Amino acid starvation provoked increased colocalization between PfATG8- and PfRAB7-labeled vesicles and acidification of the colabeled structures consistent with PfRAB7-mediated maturation of PfATG8-positive autophagosomes; this is a rapid process facilitating parasite survival. Immuno-electron microscopic analyses detected PfRAB7 and PfATG8 on double-membrane-bound vesicles and also near or within the parasite's food vacuole, consistent with autophagosomes fusing with the endosomal system before being routed to the food vacuole for digestion. In nonstarved parasites, PfATG8, but not PfRAB7, was found on the intact apicoplast membrane and on apicoplast-targeted vesicles and apicoplast remnants when the formation of the organelle was disrupted; a localization also requiring the C-terminal glycine. These findings suggest that in addition to a classical role in autophagy, which involves the PfRAB7-endosomal system and food vacuole, PfATG8 is associated with apicoplast-targeted vesicles and the mature apicoplast, and as such contributes to apicoplast formation and maintenance. Thus, PfATG8 may be unique in having such a second role in addition to the formation of autophagosomes required for classical autophagy.Autophagy 08/2013; 9(10). · 11.42 Impact Factor
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ABSTRACT: Plasmodium falciparum is the etiological agent of malignant malaria and has been shown to exhibit features resembling programmed cell death. This is triggered upon treatment with low micromolar doses of chloroquine or other lysosomotrophic compounds and is associated with the leakage of the digestive vacuole contents. In order to exploit this cell death pathway, we developed a high-content screen to select compounds which can disrupt the parasite vacuole, as measured by the leakage of intravacuolar Ca(2+). This assay utilizes the ImageStream 100, an imaging-capable flow cytometer, to assess the distribution of the fluorescent calcium probe Fluo-4. We obtained two hits from a small library of 25 test compounds, quinacrine and 3' ,4' -dichlorobenzamil. These hits were validated by confocal microscopy to permeabilize the digestive vacuole in laboratory strains and clinical isolates. The hits could induce programmed cell death features in both chloroquine-sensitive and -resistant laboratory strains. Quinacrine was effective at inhibiting field isolates in a 48 h reinvasion assay regardless of artemisinin clearance status. We therefore present as proof-of-concept a phenotypic screen with the potential to provide mechanistic insights to the activity of antimalarial drugs.Antimicrobial Agents and Chemotherapy 11/2013; · 4.57 Impact Factor
Quinine localizes to a non-acidic compartment
within the food vacuole of the malaria parasite
Elaine B Bohórquez1, Michael Chua2and Steven R Meshnick3*
Background: The naturally fluorescent compound quinine has long been used to treat malaria infections. Although
some evidence suggests that quinine acts in the parasite food vacuole, the mechanism of action of quinine has
not yet been resolved. The Plasmodium falciparum multidrug resistance (pfmdr1) gene encodes a food vacuolar
membrane transporter and has been linked with parasite resistance to quinine. The effect of multiple pfmdr1 copies
on the subcellular localization of quinine was explored.
Methods: Fluorescence microscopy was used to evaluate the subcellular localization of quinine in parasites
containing different pfmdr1 copy numbers to determine if copy number of the gene affects drug localization.
The acidotropic dye LysoTracker Red was used to label the parasite food vacuole. Time-lapse images were taken
to determine quinine localization over time following quinine exposure.
Results: Regardless of pfmdr1 copy number, quinine overlapped with haemozoin but did not colocalize with
LysoTracker Red, which labeled the acidic parasite food vacuole.
Conclusions: Quinine localizes to a non-acidic compartment within the food vacuole possibly haemozoin. Pfmdr1
copy number does not affect quinine subcellular localization.
Keywords: Quinine, Plasmodium falciparum, Pfmdr1, Copy number
Quinine (QN) was isolated from the bark of the Peru-
vian Cinchona tree in the mid-1800s  and quickly be-
came the treatment of choice for intermittent fever
worldwide . Although efficacious, QN use as an anti-
malarial requires a relatively long treatment regimen and
has substantial side effects. As a result, artemisinin-
based combination therapy (ACT) has now been imple-
mented as first-line treatment regimens due to drug
efficacy and better patient tolerance. However, QN
remains an important treatment option for severe mal-
aria infections [3-5]. Despite the longevity of its use as
an anti-malarial, the mechanism of action of QN has not
yet been fully resolved.
Early investigations into the anti-malarial activity of
QN demonstrated the ability to inhibit chloroquine-
induced clumping of haemozoin in the parasite food
vacuole . Subsequent evidence found that, like chloro-
quine, QN interferes with haem crystallization [7,8],
indicating that QN acts in the food vacuole. In recent
years, parasite resistance to QN has been reported in
Africa and Southeast Asia, prompting investigations into
resistance mechanisms with a focus on the food vacuole
[4,9-13]. Decreased QN sensitivity has been strongly
linked to a protein on the food vacuolar membrane,
encoded by the Plasmodium falciparum multidrug
resistance (pfmdr1) gene [10-12,14-16]. Whereas some
studies have found that pfmdr1 mutations exert a signifi-
cant effect on in vitro QN sensitivity [10,13,14], other
studies have found no association between pfmdr1 muta-
tions and QN sensitivity [17-19]. Elevated pfmdr1 gene
copy number, however, has been linked to reduced para-
site susceptibility to QN in both in vitro and clinical
* Correspondence: firstname.lastname@example.org
33301 Michael Hooker Research Center, Department of Epidemiology,
University of North Carolina at Chapel Hill, Campus Box 7435, Chapel Hill NC
Full list of author information is available at the end of the article
© 2012 Bohorquez et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Bohórquez et al. Malaria Journal 2012, 11:350
studies [12,16,18-20]. Thus, pfmdr1 has an effect on
Quinine is an aromatic alkaloid that naturally fluor-
esces under ultraviolet (UV) light. Because of its rela-
tively constant fluorescence quantum yield, QN is
commonly used in photochemistry as a fluorescence
standard . Surprisingly, the inherent fluorescence of
QN has not previously been exploited to investigate its
mechanism of action. In this study, the fluorescent prop-
erties of QN were exploited to evaluate subcellular
localization of the drug by fluorescence microscopy in
parasites containing different pfmdr1 copy numbers in
order to determine if copy number of the gene affects
Plasmodium falciparum cultures of two clonal parasite
strains (3D7 and Dd2) with different pfmdr1 copy num-
bers and quinine (QN) sensitivity were used in this
study. The 3D7 strain has one pfmdr1 copy and is sensi-
tive to QN. The Dd2 strain has four pfmdr1 copies 
and is resistant to QN. Cultures were maintained in
candle jars at 37°C under the Trager and Jensen method
for malaria parasite culturing . Media was changed
every 24 hours. Type O+ serum at 10% and red blood
cells (Research Blood Components, LLC: Boston, MA,
USA) at 2% haematocrit in malaria culture media (MCM)
 were used in culturing and experimental conditions.
Imaging of parasites using fluorescence microscopy
Live asynchronous cultures at 5-8% parasitaemia with
1μM of QN were imaged over an 11-hour period using
fluorescence microscopy. Parasitized RBC preparations
were plated onto 35mm glass bottom dishes (MatTek,
Corp.: Ashland, MA, USA) coated with Poly-L-lysine.
A coverslip was carefully applied, and the excess fluid
gently squeezed out. The coverslip was sealed with
warmed Vaseline to prevent the sample from drying out.
Time-lapse imaging of multiple parasites was carried out
with a Prior Scientific (Prior Scientific: Rockland, MA,
USA) motorized x-y stage. An incubator box main-
tained the microscope work area including the objective
at 37°C. Because P. falciparum grows well at 3% oxygen
but will tolerate levels as low as 0.5% , air was in
the incubator box, and the samples were sealed during
imaging as stated above. LysoTracker Red DND-99
(Invitrogen: Carlsbad, CA, USA) (50nM) was used to
stain the food vacuole and DRAQ5 (Biostatus Limited:
Leicestershire, UK) (1μM) was used to stain DNA .
Fluorescence images were acquired using an Olympus
FV1000 inverted IX81 microscope with a 63x 1.42 NA
oil immersion objective at the UNC-Chapel Hill Michael
Hooker Microscopy Core Facility. Serial sections with
step sizes of 0.20 or 0.25μm were gathered using sequen-
tial scanning at 405nm (QN), 559nm (LysoTracker Red),
595±25nm and 705±50nm emission filters for QN,
LysoTracker Red, and DRAQ5 respectively. Confocal pin-
hole was set to maximum (800μm, ~8 Airy units). Laser
transmitted light differential interference contrast (DIC)
images were collected at an optimum z-level separately.
Image stacks were deconvolved using the iterative blind
deconvolution method with Volocity Image Analysis
software, version 5.52 (Perkin Elmer: Waltham, MA,
USA). Occasionally, Tetraspec 0.1μm beads (Molecular
Probes, OR) were added to the sample and used for
deconvolution with measured point spread functions.
Three-dimensional (3-D) opacity volume rendering and
3-D co-localization analysis was carried out with Volocity.
Quinine localizes to the parasite food vacuole
Two-dimensional (2-D) fluorescence and DIC images of
both 3D7 (one pfmdr1 copy) and Dd2 (four pfmdr1 cop-
ies) parasites showed that quinine (QN) co-localizes with
haemozoin, which suggests localization in the parasite
food vacuole (Figure 1). Similar results were found using
a Leica SP2 microscope with a 351nm excitation laser
(data not shown). The co-localization was highly repeat-
able and seen in four fields in at least 10 experiments.
Parasites were also imaged at earlier time points follow-
ing the addition of QN (beginning at one hour of expos-
ure through 11 hours). Quinine fluorescence was found
in the food vacuole as early as one hour of exposure
and remained there for the duration of the experiment
(Figure 2). This effect appears to be independent of
pfmdr1 copy number as experiments in both strains ren-
dered similar results. The same pattern was seen regard-
less of whether the infected red cells were maintained
on the heated microscope stage or in a candle jar,
indicating that photobleaching was not a factor. The
parasites did not significantly progress through the
intraerythrocytic cell cycle during this 11-hour period
consistent with a previously observed maturation delay
in the intraerythrocytic cell cycle following QN exposure
at IC50 and IC99 concentrations for 12 hours . Thus,
the apparent halt in cell cycle progression observed in
the current study is likely due to the previously reported
QN-induced maturation delay phenomenon.
To further characterize subcellular localization of QN,
co-localization of QN with either LysoTracker Red,
which localizes to acidic compartments, or DRAQ5,
which localizes to the nucleus, was investigated using 3-D
expected, no co-localization with DRAQ5 was seen. How-
ever, QN was found to localize in a separate but adjacent
compartment from that in which LysoTracker Red was
fluorescence images. As
Bohórquez et al. Malaria Journal 2012, 11:350
Page 2 of 6
localized (Figure 3). This 3-D imaging method does not
allow for haemozoin to be visualized. The pattern of co-
localization was the same for 3D7 and Dd2 parasites, des-
pite their differences in pfmdr1 copy number. These
observations suggest that QN localizes to a distinct, non-
acidic compartment within the parasite food vacuole, pos-
sibly overlapping with haemozoin in parasites with both
low and high pfmdr1 copy number.
Although QN was the first therapeutic compound
used to treat malaria infection , its mechanism of
action has never been fully resolved [6-8]. Some evi-
dence suggests that parasite resistance to QN is asso-
ciated with mutations and/or elevated copy number of
the pfmdr1 gene, which encodes for a transporter pro-
tein found in the membrane of the parasite food vacuole
Figure 1 Quinine localizes with haemozoin in the parasite food vacuole. Asynchronous parasites of PQuinine localizes with haemozoin in
the parasite food vacuole. Asynchronous parasites of P. falciparum strains 3D7 (one pfmdr1 copy) and Dd2 (four copies) were incubated with QN
to determine its localization. The scale bar represents 5μm. White arrows point to haemozoin crystals in the trophozoite food vacuole. QN (blue)
overlapped with haemozoin in both strains and thus localized to the food vacuole.
Bohórquez et al. Malaria Journal 2012, 11:350
Page 3 of 6
[9,10,12-14,16,18,20]. Here, the natural fluorescent prop-
erties of QN were exploited to obtain insight into the
mechanism of action of the drug. Although knowledge
of QN’s fluorescent properties has been around since the
late-1800s , this is the first study to employ the QN’s
fluorescence for imaging in the malaria parasite.
Fluorescence microscopy was employed to image QN
subcellular localization in two P. falciparum strains that
contained different pfmdr1 copy numbers. Quinine con-
sistently overlapped with the haemozoin crystals in both
strains when evaluated by 2-D microscopy (Figure 1).
However, upon 3-D reconstruction of serial z-stack
Figure 2 Quinine localization over time in P. falciparum. Asynchronous parasites were incubated with quinine (blue) to determine
subcellular localization of the drug over an 11-hour period. White arrows point to haemozoin crystals in the food vacuole of trophozoites.
Quinine overlapped with haemozoin crystals at all time points assessed. The scale bar represents 5μm.
Figure 3 3-D reconstruction of P. falciparum parasites. Z-stack images of each parasite shown in Figure 1 were used for 3-D volume
rendering and colocalization analysis. No QN colocalization with LysoTracker Red was observed in either the 3D7 (left) or the Dd2 (right) strain.
Because LysoTracker Red is highly specific for acidic organelles, QN appears to be in a separate and non-acidic compartment of the food
vacuole. QN is shown in blue, LysoTracker Red in red, and the DNA stain DRAQ5 in green.
Bohórquez et al. Malaria Journal 2012, 11:350
Page 4 of 6
images, QN was found to reside in a distinct compart-
ment, which is contiguous to, but separate from, the
compartment stained by LysoTracker Red. The lack of
co-localization with the acidotropic dye suggests that
QN resides in a non-acidic compartment within the food
vacuole, possibly the same one occupied by haemozoin.
This would be consistent with previous reports that
quinolone compounds including QN interact with hae-
mozoin crystals directly or with enzymes involved in the
haemozoin crystallization process, as previously reported
[7,8,29-33]. In summary, these findings suggest that QN
is localized in a non-acidic compartment in the food
vacuole, possibly that which contains haemozoin.
This study underscores the importance of utilizing the
3-D reconstruction software in imaging studies, since
the localization of QN into this novel compartment
would not have been detected otherwise. A recent study
revealed that QN-haem adducts exhibit fluorescence at
least seven-fold greater than QN alone . Thus, it is
possible that QN exists in other areas of the food vacuole
but cannot be visualized due to the fluorescence intensity
of the QN-haem adducts present in these parasites.
Although there was no apparent difference in localization
in strains containing different pfmdr1 copy numbers, the
possibility that the pfmdr1 gene has a role cannot be
ruled out. Single nucleotide polymorphisms in pfmdr1
have also been associated with decreased sensitivity to
QN [10,13,14]. Because the protein encoded by pfmdr1
is a membrane transporter that pumps solutes into the
food vacuole, it is possible that mutations within the
pfmdr1 gene could affect the transporter function of
the protein by altering the conformation or function of
the transporter protein.
In summary, this study is novel because it is the first
to exploit quinine fluorescence to study the intracellular
distribution of the drug. Here, QN was shown to enter a
distinct, non-acidic compartment inside the parasite food
vacuole. These results are important because they provide
visual support for the hypothesis that QN interferes with
haemozoin production, which could guide future studies
to investigate a possible interaction between QN and
enzymes involved in the haemozoin formation process.
Quinine localizes to a non-acidic compartment within
the food vacuole and pfmdr1 copy number does not
affect QN subcellular localization. These results support
previous findings that QN acts in the parasite food vacu-
ole, perhaps interfering with haemozoin formation. This
is the first study to provide evidence for QN localization
in a sub-compartment of the food vacuole.
The authors declare that they have no competing interests.
EBB participated in the design of the study, carried out all of the
fluorescence microscopy experiments and data analysis, and drafted the
manuscript. MC participated in the acquisition and analysis of microscopy
data. SRM conceived of the study and participated in its design. All authors
read and approved the final manuscript.
Funding for this project was provided by NIH grant 1-R21-AI076785-01A1.
13113 Michael Hooker Research Center, Department of Microbiology and
Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC
27599, USA.26129 Thurston Bowles, Michael Hooker Microscopy Facility,
University of North Carolina at Chapel Hill, Campus Box 7248, Chapel Hill, NC
27599-7248, USA.33301 Michael Hooker Research Center, Department of
Epidemiology, University of North Carolina at Chapel Hill, Campus Box 7435,
Chapel Hill NC 27599-7435, USA.
Received: 14 May 2012 Accepted: 18 October 2012
Published: 22 October 2012
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