From the Department of Infectious Diseases, St. George's Hospital Medical School, Cranmer Terrace, London SW17 ORE, United Kingdom
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ABSTRACT |
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Asexual stages of Plasmodium
falciparum cause severe malaria and are dependent upon host
glucose for energy. We have identified a glucose transporter of
P. falciparum (PfHT1) and studied its function and
expression during parasite development in vitro. PfHT1 is a
saturable, sodium-independent, and stereospecific transporter, which is
inhibited by cytochalasin B, and has a relatively high affinity for
glucose (Km = 0.48 mM) when expressed
in Xenopus laevis oocytes. Competition experiments with
glucose analogues show that hydroxyl groups at positions C-3 and C-4
are important for ligand binding. mRNA levels for PfHT1, assessed
by the quantitative technique of tandem competitive polymerase chain
reaction, are highest during the small ring stages of infection and
lowest in gametocytes. Confocal immunofluorescence microscopy localizes PfHT1 to the region of the parasite plasma membrane and not to host
structures. These findings have implications for development of new
drug targets in malaria as well as for understanding of the
pathophysiology of severe infection. When hypoglycemia complicates malaria, modeling studies suggest that the high affinity of PfHT1 is
likely to increase the relative proportion of glucose taken up by
parasites and thereby worsen the clinical condition.
Malaria is one of the most important pathogenic protozoa and is
responsible for more than 1 million deaths each year. Antimalarial drug
resistance is developing rapidly in different geographical areas and is
severely curtailing therapeutic options (1). Asexual multiplication of
intraerythrocytic Plasmodium falciparum is a prerequisite
for the development of clinical symptoms and lethal outcome in malarial
infection (2). At this stage, parasites are dependent on glucose from
the host as a source of energy (3) and metabolize glucose
anaerobically. Increases in metabolic demands are associated with
enlargement of parasites within erythrocytes before the stage of
nuclear division (4). There is an approximately 100-fold increase in
the utilization of glucose by maturing parasites when compared with
uninfected erythrocytes. This induced increase in uptake of glucose is
accompanied by increased production and export of lactic acid by
infected cells (4).
Studies on intact infected erythrocytes have suggested that P. falciparum obtains its glucose through an equilibrative mechanism (5) which may involve a saturable carrier associated with the parasite
itself (6). However, these types of studies assess mechanisms of
glucose transport indirectly, because multiple membrane systems are
involved in analysis. More detailed assessment of the enzymatic
characteristics of a proposed glucose transporter from the malarial
parasite can only be individually assessed in heterologous expression systems.
Previously, we have hypothesized that asexual stages of parasites
encode substrate-specific transporters that are located in the region
of the developing parasite's plasma membrane (7). These transporters
are presumed to act in conjunction with important changes in the
permeability properties of the infected erythrocyte membrane to
regulate the uptake of substrates by parasites. As a preliminary test
of this hypothesis, we microinjected Xenopus laevis oocytes
with mRNA obtained from cultured P. falciparum and
demonstrated significantly increased uptake of several substrates or
analogues of metabolism including 2'-deoxy-D-glucose
(2-DOG)1 and lactate (7).
We have now identified a parasite-encoded hexose transporter that is
localized to the region of the parasite plasma membrane within the
infected red cell. Quantitation of mRNA encoding this transporter
during the asexual and gametocyte stages of the parasite's life cycle
suggests that its expression is under developmental control. Functional
studies on this transporter in Xenopus oocytes have
confirmed that it is a facilitative transporter with relatively high
affinity for glucose. These findings have identified a key parasite-encoded substrate transporter that is a potentially novel drug
target, as well as establishing the value of a heterologous expression
system for the study of malarial transport proteins.
Identification and Cloning of PfHT1 Sequence--
Sequence data
were obtained through early release from The Institute for Genomic
Research at www.tigr.org and/or NCBI at www.ncbi.nlm.nih.gov by
searching using TBLASTN and published sequences for GLUT1 (human facilitative glucose transporter) (8) and SGLT1 (rat
sodium-dependent glucose transporter) (9). PCR on genomic
DNA from parasite clone 3D7 using Pfu polymerase
(Stratagene, La Jolla, CA) was carried out with primers designed to
introduce BglII restriction sites and a strong eukaryotic
Kozak consensus (AATAATG to CACCATG, where the
initiation codon is underlined). The product was subcloned into
BglII sites in pSPGT1, which contains 5'- and
3'-untranslated Xenopus Expression of PfHT1 in Xenopus Oocytes, Kinetic Analyses, and
Studies with Competitors and Inhibitors--
X. laevis
oocytes were prepared and used in uptake assays as described previously
(7). cRNA encoding PfHT1 transcribed (MEGAscriptTM SP6, Ambion, Austin,
TX) from XbaI-linearized template (10 ng in 25 nl of water)
or RNase-free water was injected into oocytes, and uptake studies were
carried out 18-48 h later. For kinetic studies, uptakes of
2'-deoxy-[6-14C]D-glucose (2-DOG) (58 mCi·mmol
To determine the Ki (half-maximal inhibition
constant for carrier transport) for 6'-deoxy-D-glucose
(6-DOG), radiolabeled D-glucose was at a concentration of 3 µM and uptakes were for 10 min with a range of 6-DOG
concentrations. Data were analyzed using a one-site competition model
and were corrected for uptake into water-injected controls.
mRNA and Southern Blot Analyses--
Radioactive probes for
Southern and Northern blot analyses used full-length PfHTI
sequence. To construct a competitor plasmid for quantitative PCR
(tandem competitive PCR (TC-PCR) (11), we used oligonucleotide
restriction-site mutagenesis to generate a 515-base pair fragment of
PfHT1 containing a novel, asymmetrical EcoRI
site. This product, corresponding to nucleotide positions 1001-1515 of
the open reading frame of PfHT1, was ligated into a
construct containing a similarly mutated
Cultures of P. falciparum (clone 3D7) were synchronized to
an 8-h time window by sequential Percoll centrifugation and sorbitol lysis (13). Samples for RNA analysis were collected every 8 h into
RNA IsolatorTM (GenoSys Biotechnologies, Cambridge, UK). Gametocyte-enriched culture was harvested in a similar fashion. Contamination of parasite cultures with Mycoplasma spp. was
excluded by PCR (Stratagene).
To quantify the amount of cDNA for PfHT1 (or for Generation of Anti-peptide Antibodies and
Immunolocalization--
Peptide sequence KDICSENEGKKNGKSG (Hpep1,
corresponding to residues 6-21 in PfHT1, GenoSys Biotechnologies) was
selected for immunolocalization studies, because it lies in the
amino-terminal nonmembranous segment of PfHT1 and shows no homology
with any other glucose transporter or malaria protein. After
conjugation to keyhole limpet hemocyanin, rabbit polyclonal antibodies
were generated by immunizing rabbits with peptide (150 µg) in
Freund's complete adjuvant, followed by four equivalent doses
administered in Freund's incomplete adjuvant at 2-week intervals and a
final dose of peptide alone.
P. falciparum cultures (10% infected red cells) were washed
in serum-free RPMI 1640 and fixed onto slides in methanol at
For Western blotting, parasites were washed in RPMI 1640 medium and
lysed in hypotonic medium (10 mM
Na2HPO4/KH2PO4, pH 7.4) containing proteinase inhibitors (phenylmethylsulfonyl fluoride (1 mM),
N Sequence Analysis of PfHT1--
A continuous open reading frame
with significant homology to GLUT1, a human facilitative glucose
transporter, was identified on contig 7290 of chromosome 2 of P. falciparum (see "Experimental Procedures") and designated
PfHT1. Hybridization of genomic P. falciparum
Southern blots, using PfHT1 sequence as probe at low stringency, did not reveal any other closely related sequences (data
not shown). PfHT1 predicts a 504-amino acid polypeptide (56.4 kDa) with 12 membrane-spanning helices and many features, including its length, which are in common with members of the facilitative glucose transporter superfamily. There is 20-29.8% identity (51.5-58.5% similarity) between PfHT1 and other
representatives from this family, which include examples from both
prokaryotes and eukaryotes (Fig. 1).
PfHT1 contains several residues and motifs essential for exofacial and
endofacial ligand binding and substrate transport, some of which are
shown in Fig. 1. There is one potential N-glycosylation site
in the first extracellular loop of PfHT1. PfHT1 does not contain any
tandemly repeated motifs, which are characteristic of many malarial
proteins.
Functional Characterization of PfHT1--
To establish and
characterize the function and biochemical properties of PfHT1 we
introduced a strong eukaryotic Kozak consensus sequence at the start of
PfHT1 and cloned it into a vector used for expression
studies in Xenopus oocytes (see "Experimental
Procedures"). cRNA transcribed in vitro and microinjected
into oocytes induced large increases in uptake of D-glucose
and 2-DOG. For example, in one experiment the mean uptake of 2-DOG in
PfHT1-injected oocytes was 19.2 pmol/oocyte/h and in water-injected
controls it was 0.13 pmol/oocyte/h, an ~150-fold increase in uptake.
To confirm that PfHT1 belongs to the family of hexose transporters, we
examined its enzymatic characteristics. The uptake of permeants (2-DOG
and glucose) was saturable at relatively low concentrations of glucose
(<5 mM) and permitted detailed kinetic analyses. For
2-DOG, Km = 1.31 mM and
Vmax = 535 pmol/oocyte/h and for
D-glucose Km = 0.48 mM and
Vmax = 143 pmol/oocyte/h (Fig.
2a). PfHT1 has a much higher
affinity for both these permeants when compared with GLUT1, the
principle erythrocyte transporter, which has a Km
(2-DOG) = 6.9 mM (15) (also expressed in oocytes).
PfHT1 is stereospecific for D-glucose, because an excess of
L-glucose in the medium used for uptake assays did not
interfere with uptake of either 2-DOG or D-glucose
(Fig. 2b). There is no requirement for extracellular sodium,
because substitution of Na+ ions with choline did not
interfere with the function of PfHT1. These findings are consistent
with the primary sequence of PfHT1, which predicts it to be a
facilitative hexose transporter.
Structural Requirements in Substrates for Transport by
PfHT1--
We studied the structural requirements for ligand
interactions between PfHT1 and hexoses by using a variety of
competitors and inhibitors. The hydroxyl groups at C-3 and C-4
positions in glucose are important in uptake by PfHT1 as an excess of
3'-deoxy-D-glucose or D-galactose (the 4-epimer
of glucose; both at 10 mM) competed relatively poorly when
glucose or 2-DOG were used as permeants (Fig. 2b). In
particular, the oxygen atom at the C-3 hydroxyl moiety is likely to be
a hydrogen bond recipient, because 3-O-methylglucose restored the capacity to inhibit transport of both permeants. In
contrast, D-mannose (the 2-epimer of glucose; 10 mM) competed well with both 2-DOG and D-glucose
for PfHT1 uptake into oocytes. The mean (±S.E.) Ki
for 6-DOG (using D-glucose as permeant) in three
independent experiments was 2.2 ± 0.86 mM (data not
shown). The relatively small effects of changes in the C-2 and C-6
positions in these experiments suggest that these positions contribute
little to PfHT1 functionality. Fructose consistently inhibited uptake of D-glucose and 2-DOG by 25-35%, suggesting that in
contrast to GLUT1, PfHT1 may transport fructose. PfHT1 is also
inhibited by cytochalasin B (50 µM) and contains a
tryptophan residue implicated in binding to this inhibitor (Fig.
1).
Stage-specific Expression of PfHT1 mRNA--
mRNA obtained
from asynchronous P. falciparum contains a single species
(~2.6 kilobases) when hybridized to PfHT1 at low
stringency (Fig. 3). We characterized the
pattern of PfHT1 mRNA expression in greater detail using
synchronized cultures of asexual stages, as well as gametocytes. The
technique of TC-PCR was used to quantitate precisely the amount of
PfHT1 mRNA relative to that of a housekeeping gene,
The expression of PfHT1 is clearly under strict
developmental control during the life cycle of P. falciparum
with an early peak of mRNA expression at the small ring stage (8 h
time point, Fig. 4, lower panel). The 8-h time point
represents parasites developing 8-16 h after invasion of erythrocytes,
with the majority developing at ~12 h.
At 16 h after invasion, there is a fall by ~1 log10
unit in the ratio of PfHT1/ Immunolocalization of PfHT1--
There was no staining of the
infected erythrocyte membrane or red cell cytosol (Fig.
5, a-c) when polyclonal
antibodies raised to a Hpep1 were applied to infected erythrocytes.
Staining of the parasite within the erythrocyte outlined the region of
the parasite plasma membrane and was most intense when parasites were free of surrounding structures (Fig. 5, d-f). In some
preparations, staining was also associated with the pigment body.
Staining was abolished by pre-incubation of antiserum with excess
cognate peptide (Hpep1, Fig. 5, g-i) but not after
incubation with irrelevant peptide (not shown).
PfHT1 was also recognized on Western blots made from parasites lysed
with hypotonic buffer in Triton-soluble (Fig.
6), and to a lesser extent, in
Triton-insoluble fractions. Recognition was abolished by pre-incubation
of antiserum with excess Hpep1 (not shown). The estimated molecular
mass of PfHT1 (62 kDa), which contains a potential
N-glycosylation site, is consistent with its predicted mass
(56.4 kDa).
Glucose is the main energy source for asexual stages of P. falciparum (3). The isolation of a novel parasite-encoded hexose transporter is therefore of fundamental biological interest. A number
of findings suggest that PfHT1 is the major hexose transporter for
intraerythrocytic parasites. We have established that PfHT1 is a single copy gene with no close homologues of PfHT1
discernible from examination of both Southern and Northern blots under
conditions of low stringency for hybridization and washing. PfHT1 is
expressed as a single transcript (Fig. 3) that varies considerably in
abundance during the life cycle of P. falciparum. The
pattern of expression of PfHT1 mRNA, which peaks sharply
8 h after invasion of the red cell (Fig. 4), is consistent with
anticipation of a rapid rise in glucose consumption as ring forms
mature to become trophozoites (4). A sustained secondary rise in
PfHT1 mRNA levels may serve to maintain glucose supply
as parasites begin to divide. Thus the variation in abundance of
mRNA encoding PfHT1 is consistent with the changes in glucose
utilization as asexual stage parasites mature in cells. Interestingly,
gametocytes expressed the lowest relative abundance of PfHT1
mRNA suggesting that high levels of glucose uptake may not be
critical at this stage of the infection.
When expressed in Xenopus oocytes, PfHT1 mediates
sodium-independent uptake of hexoses (Fig. 2). The uptake of glucose
analogues in intact P. falciparum-infected erythrocytes is
also sodium-independent (5) and saturable (6). Furthermore, the
predicted Km for 6-DOG uptake into parasites (~5
mM) derived after modeling from studies in infected
erythrocytes (6) is consistent with our Ki estimate
for 6-DOG (2.2 mM). Taken together these observations
support assignment of PfHT1 as the principal hexose transporter in
asexual stages of parasite development. A number of controversial
mechanisms have been proposed to explain the increase in permeability
of host-derived structures to metabolites of parasites (16-19).
Whatever the nature of these important mechanisms, primary regulation
of nutrient uptake and export of products of metabolism takes place
across the parasite plasma membrane, because axenically grown parasites
are capable of completing the asexual stage of development (20).
Localization by confocal microscopy and Western blotting experiments
demonstrate that PfHT1 is associated with the parasite plasma membrane
and not the erythrocyte membrane. PfHT1 therefore provides an example
of a molecular mechanism for transport of substrates into the parasite,
in which substrate-specific transporters act in conjunction with
alterations in surrounding membrane structures to supply nutrients to
the parasite (7).
Heterologous expression systems avoid the difficulties of studying
transport across the multiple membranes of the infected erythrocyte and
will become increasingly important as the malaria-sequencing initiative
evolves. The high AT content of parasite DNA does not interfere with
expression in the Xenopus oocyte system.
The isolation of a parasite-encoded facilitative hexose transporter
permits studies focussing on differences between the well characterized
human hexose transporter family of sequences (GLUT1 to GLUT5) and the
newly isolated malarial homologue. We have already shown that PfHT1
requires an appropriately positioned hydroxyl group in the C-4 position
of hexoses, a property which distinguishes it from human GLUT1 (21).
Such differences may be used as a basis for the design of compounds
selectively toxic to the parasite.
The >10-fold higher affinity for D-glucose shown by PfHT1
compared with GLUT1 may result in important physiological consequences. Infected erythrocytes adhere to microvasculature when parasites mature
giving rise to organ-specific complications such as cerebral malaria
and the metabolic complications of hypoglycemia and lactic acidosis
(2). The enzymatic properties of PfHT1 suggest that there is potential
for infected erythrocytes to divert this substrate away from host
tissues. This may be particularly important in cerebral microvessels,
because the brain relies on glucose, which is transported by GLUT1
through the blood-brain barrier (22). The magnitude of this metabolic
diversion is further predicted to increase during episodes of
hypoglycemia (plasma glucose Our characterization of a P. falciparum-encoded high
affinity hexose transporter identifies an attractive potential drug
target because antimalarials used in the management of severe malaria take many hours to achieve maximum inhibition of glucose utilization (13). Inhibitors of PfHT1 may be rapidly parasitocidal as well as
interfering with pathophysiological processes in cerebral malaria.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-globin sequences (10). The final
product was verified by sequence analysis.
1) and
[U-14C]D-glucose (310 mCi·mmol
1) (Amersham Pharmacia Biotech, Amersham, UK)
were measured after 5 min and corrected for uptake into water-injected
controls. Uptakes are linear during this interval (data not shown).
Kinetic parameters were estimated by using a Michaelis-Menten model
(PRISMTM v.2, GraphPad Software Inc., San Diego, CA). For studies with
competitors and inhibitors (all from Sigma), uptakes were carried out
for 30 min with radiolabeled permeants (14.3 µM 2-DOG, or
2.69 µM D-glucose) and 35 µM
unlabeled D-glucose.
-tubulin sequence (a
"housekeeping" gene) (12).
-tubulin),
cDNA was mixed with a series of dilutions of competitor plasmid, and after PCR the product was digested with EcoRI and
quantified on a GDS 7600 system (Ultra-Violet Products, Upland, CA).
After correction for heteroduplex formation (12), the plasmid
competitor/cDNA ratio was calculated for individual reactions and
analyzed as exemplified in Fig. 5. To exclude the possibility of DNA
contaminating mRNA preparations, PCR was carried out across an
intron in
-tubulin sequence.
70° C. Additionally, aliquots were applied to slides coated with
poly-L-lysine and permeabilized with acetone (14).
Antiserum (20 µl) was incubated (1 h at 37° C) with an equivalent
volume of PBS, PBS containing cognate peptide (50 mg/ml), or an
irrelevant peptide (50 mg/ml) before application to slides. After
washing (once in PBS and three times in PBS with fetal calf serum, 1%
(v/v)), secondary fluorescein isothiocyanate-conjugated swine
anti-rabbit antibody (10 µl) was applied for 1 h and washing was
repeated. Fluorescence was visualized on a Zeiss Axiovert 100 confocal microscope.
-p-tosyl-L-lysine
chloromethyl ketone (0.2 mM),
N-tosyl-L-phenylalanine chloromethyl ketone (0.1 mM), antipain (0.1 mg/ml), leupeptin (0.01 mg/ml); all from
Sigma). After extraction with Triton X-100, samples were resolved by
10% SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose, incubated with primary antibody, and washed, and the
blot developed according to manufacturer's instructions (ECL,
Amersham). Blocking was with fat-free milk (5% in PBS).
RESULTS
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Fig. 1.
Sequence analysis of PfHT1. Top
panel, schematic model for the membrane arrangement of PfHT1 based
on Mueckler's original proposal (8). Numbering (M1-M12) refers to
transmembrane helices. A potential N-glycosylation site is
indicated (69NCS). Regions (a-f) highlighted in
red are shown in greater detail in the bottom panel. Bottom
panel, examples of aligned sequences conserved between PfHT1 and
sequences from the sugar transporter superfamily from phylogenetically
distant organisms. Accession numbers in GenBankTM for each
sequence with the percentage of identical residues compared with PfHT1
are as follows: AraE, Escherichia coli arabinose transporter
(P09830; 27.2%); Sman1, Schistosoma mansoni glucose
transporter protein 1 (L25065; 24.5%); GLUT1, human glucose
transporter 1 (K03195; 29.8%); THT1, Trypanosoma brucei
hexose transporter 1 (M81386; 20.0%); Hxt1, Saccharomyces
cerevisase glucose transporter 1 (P32465; 26.6%); PfHT1, P. falciparum hexose transporter. Asterisks indicate
residues that are conserved throughout these sequences. Residues
highlighted in red are of functional significance as determined by
mutagenesis experiments (23-26). Regions a and d
contain a GRR/K motif in positions characteristic of this superfamily
of transporters. Regions b and c contain highly
conserved glutamine residues that participate in exofacial ligand
binding. Region e contains a proline residue that is
required for conformational flexibility, and a tryptophan residue that
is involved in binding to cytochalasin B. Region f contains
a tryptophan residue essential for transport activity (26).
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Fig. 2.
Expression of PfHT1 in Xenopus
laevis oocytes. Panel a, kinetic analyses.
Initial mean (8 oocytes per concentration, mean ± S.E.) uptake
rates of 2-DOG (left panel) and D-glucose
(right panel) are plotted against concentration of
substrate. Panel b, studies with competitors and inhibitors.
Uptakes (8 oocytes per condition) were carried out with radiolabeled
permeants 2-DOG (left panel) or D-glucose
(right panel). Results are expressed as mean ± S.E.
percentage of uptake of substrate for each condition compared with
uptake in uncompeted oocytes. PfHT1, malarial hexose transporter
without competitor or inhibitor; DEPC, RNase-free water-injected
control oocytes; L-Gluc, L-glucose (10 mM); No Na+, sodium-free Barth's medium where
Na+ is replaced with equimolar choline chloride;
D-Mann, D-mannose (10 mM);
D-Fruct, D-fructose (10 mM); 3-DOG,
3'-deoxy-D-glucose (10 mM); 3-O-MG,
3'-O-methyl-D-glucose (10 mM);
D-Galac, D-galactose (10 mM);
6-DOG, 6'-deoxy-D-glucose (10 mM); CytoB,
cytochalasin B (50 µM); Phdz, phloridzin (500 µM); Phrt, phloretin (150 µM).
-tubulin (12) (Fig. 4). The precision
and reproducibility of this ratiometric method were confirmed by
assessing the gene copy number of PfHT1 relative to that for
-tubulin using genomic DNA as template in five independent TC-PCR
experiments (mean ± S.E. ratio = 1.07 ± 0.12, Fig. 4,
lower panel).
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Fig. 3.
Northern blot of PfHT1. mRNA
(4 µg) obtained from asynchronous parasites (clone 3D7) was
hybridized to PfHT1, washed at low stringency (2× SSC,
0.1% SDS, at 40° C for 30 min), and visualized on a PhosphorImager
after overnight exposure.
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Fig. 4.
Quantitative mRNA studies. Top
left panel, example of results of TC-PCR for PfHT1 with track 1, competitor alone; track 2, cDNA alone; and tracks 3-10, increasing
one-fourth log concentrations of competitor plasmid with constant
cDNA concentrations (5 ng) in each PCR. Products derived from
cDNA remain undigested by EcoRI, whereas those derived
from competitor are cleaved. Top right panel, same as for
top left panel, but with -tubulin. Middle
panel, quantitation of bands from respective top panels
(C, competitor-derived product; T,
cDNA-derived product). C/T ratios are analyzed by linear
regression, where the X-intercept represents equivalence in
concentrations of cDNA and competitor (here, 4.55 and 4.49 for
PfHT1 and
-tubulin, respectively, giving a ratio of
PfHT1/
-tubulin = 0.83, T = 40 h, see below).
Bottom panel, the ratio of PfHT1 to
-tubulin cDNA in
samples obtained from synchronized parasite cultures following invasion
(0 h). Samples were collected at eight hourly intervals. The ratios of
mRNAs at the 8- and 16-h time points were estimated in two
independent experiments. Gam, gametocyte-rich preparations
(70% gametocytes).
-tubulin mRNA. Levels of
mRNA for PfHT1 again increase relative to
-tubulin
when parasites develop into more mature stages. Trophozoites and
meronts express intermediate levels of mRNA for PfHT1
compared with the preceding peak and trough levels. Gametocytes express
the lowest ratio of PfHT1/
-tubulin. To confirm the early
peak in PfHT1 expression, TC-PCR was repeated for the 8- and
16-h time points and each ratio was closely reproduced, further
demonstrating the precision of this assay (Fig. 4, lower panel).
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Fig. 5.
Immunolocalization of PfHT1 with
anti-Hpep1. Panels a-c, bright-field,
immunofluorescence, and combined images of intraerythrocytic P. falciparum (mature trophozoites) stained with anti-Hpep1 antibody.
pb, pigment body; mem, parasite-host interface. Panels d-f,
images corresponding to panels a-c of free
parasites (mature trophozoite). Panels g-i,
images corresponding to panels a-c with
anti-Hpep1 antibody pre-incubated with excess of cognate peptide.
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Fig. 6.
Western blot of PfHT1. Track
1, Triton-soluble fraction (4 µg) of infected erythrocytes (ring
stages, 62%; trophozoites, 35%; meronts, 3%) lysed in hypotonic
buffer and incubated with anti-PfHT1 antibody after transfer to
membrane. Track 2, equivalent amount of material from
uninfected erythrocytes treated in the same way as for track
1.
DISCUSSION
2.2 mM). These
observations may also explain how parasites continue to develop even
when glucose delivery becomes rate-limiting for cerebral metabolism,
for example during episodes of hypoglycemia which frequently (~20%
of cases) (2) complicate the condition of cerebral malaria.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michael Barrett and Professor George Griffin for discussions, and Dr. Gwyn Gould for the pSPGT1 construct.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ131457.
Wellcome Trust Clinical Training Fellow.
§ Wellcome Trust Senior Research Fellow in Clinical Science. To whom correspondence should be addressed. Tel.: 44-181-725-5836; Fax: 44-181-725-3487; E-mail: s.krishna{at}sghms.ac.uk.
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ABBREVIATIONS |
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The abbreviations used are: 2-DOG, 2'-deoxy-D-glucose; PfHT1, Plasmodium falciparum hexose transporter 1; GLUT1, human facilitative glucose transporter 1; 6-DOG, 6'-deoxy-D-glucose; PCR, polymerase chain reaction; TC-PCR, tandem competitive PCR; Ki, half-maximal inhibition constant for carrier transport; PBS, phosphate-buffered saline.
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REFERENCES |
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