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J. Biol. Chem., Vol. 278, Issue 35, 33593-33601, August 29, 2003
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From the
Department of Microbiology and
Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, the
¶Departments of Chemistry and of Biochemistry
& Molecular Biology, and Program in Tumor Biology, Georgetown University,
Washington, D. C. 20057, the ||Weatherall Institute
of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford,
OX3 9DS, United Kingdom, and the **Institute of
Pathology, Case Western Reserve University, Cleveland, Ohio 44106
Received for publication, March 4, 2003 , and in revised form, June 16, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Central to the action of this drug is the process of hemoglobin degradation, thought to occur primarily in the digestive vacuole (DV) of the infected red blood cell (iRBC). This serves to provide amino acids and to deplete the host cell cytosol of hemoglobin, in order to sustain parasite intracellular growth (3, 4). This process liberates soluble heme (Fe(II) protoporphyrin IX), whose inherent toxicity is neutralized via its rapid incorporation, as an oxidized (Fe(III)) iron-propionic acid-coordinated hematin dimer, into a crystalline lattice known as hemozoin (4–6). Chloroquine is thought to bind to hematin dimers, thereby interfering with heme detoxification and hemozoin formation, resulting in parasite death (6–12). Chloroquine access to heme, the kinetics of heme detoxification and hemozoin formation, and the presence of a pH gradient across the DV membrane have all been proposed to play an important role in determining CQ accumulation and activity inside the DV (reviewed in Refs. 11 and 13). Although there is much debate about the pharmacological basis of CQR, it is clear that resistant parasites accumulate less CQ than their sensitive counterparts and that this reduced accumulation can be partially reversed by verapamil (VP) (14–16).
Point mutations in pfcrt (P. falciparum chloroquine resistance transporter) have recently been implicated as the key determinant of CQR. Evidence in favor of this includes the following: (i) these mutations segregate with CQR in a P. falciparum genetic cross (17–19); (ii) mutant pfcrt haplotypes show an excellent association with CQR in laboratory-adapted field isolates from multiple geographically distinct regions (19–21); (iii) pfcrt point mutations are often associated with an increased risk of CQ treatment failure (21–24); (iv) microsatellite markers support a recent, worldwide sweep of mutant pfcrt haplotypes under CQ pressure (25); and (v) allelic exchange data have recently provided conclusive evidence that mutant pfcrt alleles prevalent in Asia, Africa, South America, and the Oceanic region can confer CQR to a CQ-sensitive (CQS) clone (26).
In a recent pfcrt allelic exchange study (26), one notable finding was that expression of the mutant haplotypes in the recombinant lines resulted in acquisition of CQ IC50 values that were 70–90% of those observed with non-transformed reference lines expressing the same pfcrt haplotypes. One possible explanation is that mutant pfcrt can account for the bulk but not all of the CQR phenotype and that other genes are required to elevate the CQ IC50 values in the CQ-resistant lines. Another explanation is that the pfcrt-modified lines had lower CQ IC50 values as a result of their reduced levels of PfCRT protein expression (as observed by RNA and protein analysis (26)). This presumably stemmed from the genetic modification of the functional locus, which involved removal of the introns and manipulation of the 3'-untranslated region (UTR).
To address the question of whether pfcrt expression levels can influence CQR independent of point mutations, we have implemented an allelic exchange strategy designed to introduce a truncated 3'-UTR into the functional pfcrt locus. This "knockdown" approach has been shown in higher eukaryotic systems to reduce levels of transcription and translation by decreasing mRNA stability and was used in Plasmodium berghei to assess the contribution of CS expression levels on sporozoite morphology (27–29). The impact of this change on pfcrt expression, acridine orange (AO) accumulation (a putative marker of vacuolar pH), and response to CQ and related hemebinding antimalarials is presented.
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Vector Construction and Parasite Transfection—The
pfcrt allelic exchange fragment was PCR-amplified from 7G8 genomic
DNA using the primer combination p4/p5. This yielded a 1.28-kb fragment
comprising exons 8–13 as well as the proximal 148 bp of pfcrt
3'-UTR. This fragment was cloned into the plasmid pHDWT, which uses as a
selectable marker the human dihydrofolate reductase (hdhfr) gene
flanked by promoter and terminator elements from the hrp3 and
hrp2 genes, respectively
(34). The resulting 7.1-kb
transfection plasmid, pHD/pfcrt
3'-UTR, was
electroporated into 7G8 P. falciparum ring stage parasites.
Plasmid-transfected parasites were selected by addition of 5 nM
WR99210 to the culture medium starting 48 h post-transfection
(34). Parasite clones were
obtained by limiting dilution and identified using the MALSTAT (Flow Inc.)
assay reagent specific for P. falciparum lactate dehydrogenase
(35,
36).
Nucleic Acid Analysis—P. falciparum genomic DNA was purified as described previously (37). Plasmid integration into the 3'-UTR of pfcrt was detected by PCR using the primer combinations p6/p7 and p8/p9 (Table I and Fig. 1B). Integration into the hrp3 and hrp2 loci was assessed using the primer combinations p10/p11 and p12/p13, respectively. Amplification of non-recombinant, endogenous pfcrt sequence was assayed using the primers p6/p9. PCR conditions included a primer extension temperature of 62 °C to account for the high A-T content in P. falciparum genomic DNA (38).
For stage-specific Northern blots, cultures were doubly synchronized by
sorbitol treatment. Total RNA was prepared using TRIzol® (Invitrogen) and
solubilized in deionized formamide
(39). RNA samples (10 µg
per lane) were size-fractionated on a 1% agarose gel supplemented with 5
mM guanidine thiocyanate and transferred to Hybond N+
nylon membranes (Amersham Biosciences). Membranes were hybridized with
32P-labeled 1.3-kb pfcrt or 1.0-kb P. falciparum
EF-1
cDNA probes, generated using the primers p14/p15 and p16/p17,
respectively. Autoradiography images were captured using a Fuji film FLA2000
phosphorimaging device and quantified for data analysis.
For reverse transcriptase-PCR assays, parasite lines were synchronized using successive rounds of gelatin flotation and sorbitol lysis. Cultures with >85% ring stage parasites were harvested by lysis with 0.15% saponin, and total RNA was extracted using TRIzol®. First strand cDNA was synthesized from 1 µg of total RNA, using the SuperscriptTM pre-amplification system (Invitrogen) with oligo(dT) priming.
For Southern blot analysis, genomic DNA was digested with EcoRI
and HaeIII, electrophoresed overnight at 2.5 V/cm in 0.8% (w/v)
agarose gels (
1.5 µg of DNA/lane) alongside a dideoxygenin
(DIG)-labeled DNA molecular mass ladder (Roche Applied Science), transferred
to Nylon membranes, and UV cross-linked. A 0.9-kb region of pfcrt
(exons 12 and 13, plus 0.6 kb of 3'-UTR sequence) and a 0.6-kb region of
hdhfr were PCR-amplified using the primers p18/p19 and p20/p21,
respectively, and labeled with DIG (Roche Applied Science). Hybridizations
were performed at 60 °C, and membranes were washed at a maximum stringency
of 0.3x SSC/0.1% SDS at 60 °C, prior to CDP-Star detection, and
autoradiography.
Western Blotting and Immunoelectron Microscopy—For whole parasite extracts, synchronized parasite cultures rich in late rings and early trophozoites were lysed using 0.15% saponin. Parasite pellets were washed in 1x PBS, resuspended in SDS-PAGE loading buffer and the proteins resolved on 10% (w/v) polyacrylamide gels prior to being transferred to polyvinylidene difluoride membranes. Western blots were performed using affinity-purified anti-PfCRT antibodies (19) and detected using the Renaissance Western blot Chemiluminescence Reagents (PerkinElmer Life Sciences). Autoradiographic data were analyzed by densitometry using Image version 1.6.2 (National Institutes of Health, available at rsb.info.nih.gov/nih-image/). Percent reductions, presented as mean ± S.E., were determined by normalization of the data from each parasite line at the different loading amounts. DV extracts were prepared and analyzed by Western blotting as described previously (40, 41).
Immunoelectron microscopy was performed as previously described (42). Anti-PfCRT antibody-labeled samples were stained with uranyl acetate and lead citrate and examined in a Zeiss CEM 902 electron microscope.
Drug Assays—In vitro drug responses were calculated from 72-h [3H]hypoxanthine incorporation assays (34). Levels of incorporation were measured using a 1450 Microbeta liquid scintillation and luminescence counter (Wallac). Chloroquine (dihydrochloride "Aralen" formulation) was from Winthrop-Breon, whereas quinine (sulfate formulation) and mefloquine (hydrochloride formulation) were from Sigma. Drug inhibition concentrations were calculated by regression analysis of dose-response curves. Mann-Whitney t-tests were used to assess for statistical significance.
Intracellular pH Measurements—Internal/external AO ratios
were measured for individual intraerythrocytic parasites under constant
perfusion, using single-cell photometric (SCP) analysis of synchronized
trophozoites following previously described methodologies and using custom
equipment (43). We stress
these SCP experiments are not analogous to other published experiments that
follow the response of trapped fluorophores via time-dependent laser confocal
microscopy (44,
45). As described in detail
elsewhere
(43),2
our SCP experiments use illumination power that is two to three orders of
magnitude lower than laser confocal microscopy, dynamic thresholding
computational methods that are unavailable for commercial laser confocal
systems, and substantial recovery from illumination after each data point
acquisition (3-s recovery in total darkness per 0.133-s data acquisition).
These data have been interpreted in previous reports
(19,
20,
42,
43) as a valid measure of DV
pH, because of the following: 1) the observed semilogarithmic Henderson
Hasselbach behavior of AO diffusion under the experimental conditions; 2) the
K+-specific abrogation of this behavior upon addition of nigericin;
3) the reversible change in AO ratio upon subtle changes in external
that do not change internal pH,
again in accordance with Henderson Hasselbach predictions; and 4) the use of
illumination powers that are two to three orders of magnitude below those
shown to affect the integrity of DV membranes in a time-dependent fashion
(45).2
Nonetheless, AO ratios have been suggested by others to be an unreliable
marker for DV pH (44,
45). Thus, so long as this
controversy remains unsettled, in the present work we avoid explicit
calculation of DV pH using these ratio data.
To directly test whether the present SCP experiments monitored AO
localizing within the DV, co-localization studies of AO and hemozoin were
performed using laser scanning confocal microscopy (cf.
Fig. 5C, under
"Results"). Laser confocal images were acquired in
"z-axis series" mode using an Olympus Fluoview confocal
microscope and an Olympus UPlanApo 100x oil immersion objective
(numerical aperture 1.35) as detailed
elsewhere.2 In brief,
the 488-nm line of an argon laser (20% output) was used for excitation, and we
acquired a set of 15 conjugate transmittance and fluorescence images upon
successive 0.2-µm displacements along the z-axis. We first focused
transmittance in the absence of laser illumination until hemozoin was clearly
visible within the center of the synchronized late stage trophozoite parasite
DV, thus putting the focal plane within the DV. We then defined this
z-axis position as 0. A series of conjugate fluorescence and Nomarski
transmittance images were then rapidly acquired in successive 0.2-µm
z-axis displacements relative to 0, such that we were sure to scan
through the parasite. After archiving the image pairs, we determined the
largest hemozoin diameter in the Nomarski transmittance set via histogram
analysis. The conjugate fluorescence image partner to this transmittance image
was then subjected to histogram analysis using the same parameters. Each image
(transmittance and fluorescence versions) defined the same
0.25-µm-thick optically defined slice (slightly larger than 0.2 µm
due to the pinhole setting) corresponding to the DV of a late stage
trophozoite. All fluorescent light recorded from this slice thus resided
within 0.12 µminthe + or – direction relative to the visualized
hemozoin and within the x and y dimensions shown in
Fig. 5 under
"Results."
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RESULTS |
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163 and 513 bp downstream of the
pfcrt stop
codon.3 Using a P.
falciparum transient transfection reporter assay
(31), we tested whether
3'-UTR truncation prior to one or both of these sites would lead to
reduced gene expression. pfcrt 3'-UTR sequences either 1033,
625, or 148 bp in length were cloned into pHLH-1
(30), downstream of the
firefly luciferase gene and in the place of the hrp2 3'-UTR
sequence, to form the plasmids pHLcrt/1033, pHLcrt/625, and pHLcrt/148,
respectively. In these constructs, luciferase transcription initiates from an
hrp3 promoter sequence. Luciferase values, assessed 72 h
post-electroporation, showed that truncation of the pfcrt
3'-UTR from 1033 to 625 bp or to 148 bp yielded 64 or 92% reduction of
luminescence units, respectively, relative to the full-length 1033-bp fragment
(Fig. 1A).
Production of pfcrt "Knockdown" Clones by Allelic
Exchange—Based on these reporter assays, we designed a transfection
plasmid, called pHD/pfcrt 3'-UTR, to replace the
full-length pfcrt 3'-UTR in the chromosomal locus with the
truncated 148-bp sequence. This plasmid contained the truncated 3'-UTR
preceded by the last six exons of pfcrt. Allelic exchange of
pHD/pfcrt 3'-UTR into the parasite genome via homologous
recombination and single site-crossover was predicted to generate a functional
pfcrt locus that retained the original pfcrt promoter and
coding sequence with 3'-UTR regulation provided by the 148-bp truncated
sequence (Fig. 1B).
This approach was also predicted to generate a downstream, disrupted remnant
pfcrt sequence that lacked a promoter and the first seven exons of
the endogenous gene. For this experiment, we transfected the 7G8 line to test
whether expression levels of the 7G8-type pfcrt allele (which appears
to have originated independently in South America and Oceania
(19,
20,
25,
46)) had a direct impact on
the P. falciparum CQR phenotype.
Electroporation of pHD/pfcrt 3'-UTR into 7G8
parasites generated episomally transformed lines that were maintained by
selection with WR99210. Passaged lines were periodically screened by PCR for
homologous recombination and plasmid integration into the pfcrt
(Fig. 1B) or
hrp2 or hrp3 loci (not shown). One line was found to contain
a mixed population of parasites that had integrated into either the
pfcrt or the hrp2 loci (data not shown). Clones were
generated from this line (at day 90 post-electroporation) by limiting dilution
(35), leading to the
identification of K2C3 and K2F10. These "transcription knockdown"
clones were confirmed by PCR analysis to be recombinant solely for
pfcrt integration and were PCR-negative for wild type pfcrt
(Fig. 1C).
Southern blot hybridization confirmed that the endogenous pfcrt locus had been replaced by the 3'-UTR-truncated locus in the K2C3 and K2F10 clones (Fig. 1D). Using a pfcrt probe, hybridizing bands of 10.2/6.8 kb and 6.5/2.7 kb, respectively, were detected upon EcoRI and HaeIII digestion of genomic DNA from the knockdown clones. This contrasted with the 10.0- and 7.6-kb bands generated by these two enzymes in non-transfected 7G8 genomic DNA. The visible lack of the wild type 7.6-kb HaeIII band in the K2C3 and K2F10 clones provided further evidence for complete pfcrt allelic replacement. These restriction patterns were consistent with integration of a single plasmid copy into the pfcrt locus, because integration of two or more tandem copies would have generated an additional 6.9-kb band and a 1.5-kb band upon EcoRI and HaeIII digestion, respectively. Results obtained using an hdhfr probe also showed hybridizing bands of the expected size in K2C3 and K2F10 (data not shown).
Expression Levels in Knockdown Mutants—To assess the impact
of 3'-UTR truncation on pfcrt expression from the chromosomal
locus, we performed Northern blots on tightly synchronized RNA preparations
from the knockdown and 7G8 parental clones. For this, we collected RNA samples
from early rings, late rings, trophozoites, and schizonts. Results showed
significantly reduced pfcrt transcript levels in the knockdown clones
K2C3 and K2F10, relative to 7G8 (Fig.
2A). Timing of maximal transcription remained equivalent
between the lines, peaking in the early ring stages and rapidly decreasing to
undetectable levels by the trophozoite stage. These results suggest that the
timing of transcription was largely determined by the pfcrt promoter
region, which remained unchanged in these different clones. Densitometric
analysis of the early ring stage results, comparing ratios of pfcrt
to EF-1 signal intensity between the different clones, predicted a
52–55% decrease in pfcrt transcription in the knockdown clones.
This assumes that EF-1 levels of transcription remained unchanged
between these clones. The reduction in pfcrt transcript size (from
4.2 kb in the wild type locus to 3.7 kb in the knockdown clones) suggests that
the wild type transcript ends about 0.65 kb downstream of the pfcrt
stop codon (as compared with 0.15 kb in the knockdown clones). Given that the
coding sequence is 1.25 kb, this would predict that the endogenous
pfcrt transcript contains about 2.3 kb of 5'-UTR sequence and
thus starts 0.65 kb downstream of the neighboring cg3 gene
(18). Reverse
transcriptase-PCR analyses of RNA prepared from synchronized late ring and
early trophozoite stages gave results consistent with the Northern data (not
shown).
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To compare PfCRT protein expression levels between parental and recombinant lines, Western blots were performed with lysates of synchronized parasite cultures and affinity-purified anti-PfCRT antibodies (19). Densitometric analysis (averaged between the different sets of loadings, Fig. 2B) predicted a reduction in PfCRT expression levels of 32 ± 10% for K2C3 and 34 ± 12% for K2F10 (mean ± S.E.), as compared with nontransfected 7G8 PfCRT. These data were confirmed with Western blot analysis of purified DV, which showed an estimated 70–80% reduced expression of PfCRT in the knockdown clones (data not shown). These data suggest a possibly even greater impact of the truncation on decreasing the amount of PfCRT being inserted into the DV membrane.
Localization of PfCRT to the DV of pfcrt Knockdown Parasites—Related studies in other organisms have found that truncation of 3'-UTR sequences can occasionally result in mislocalization of the gene product (27). To address this possibility, we performed immunogold electron microscopy using affinity-purified anti-PfCRT antibodies. This localized PfCRT solely to the membrane region surrounding the DV in the knockdown and 7G8 lines (Fig. 3). Quantitation of PfCRT antibody-conjugated gold particles in 10 cryosectioned parasite DVs gave an estimated 40–80% decrease in antibody labeling and provided additional complimentary evidence for decreased PfCRT expression in the DV of knockdown clones.
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Response of pfcrt Knockdown Parasites to Chloroquine, Quinine, and Mefloquine—72-h [3H]hypoxanthine incorporation assays revealed that the reduction in PfCRT expression levels was accompanied by a significant attenuation of the CQR phenotype, with the knockdown clones showing up to a 38% reduction in CQ IC50 values when compared with 7G8 (Fig. 4A). Essentially the same percentage decrease in IC50 values was observed when CQ was tested in the presence of the CQR reversal agent VP (tested at 0.8 µM, Fig. 4A). This indicated that the degree of VP reversibility was essentially unchanged in the knockdown clones, even though the level of CQR was reduced. This degree of VP reversibility was noticeably lower than in the CQ-resistant FCB line (Fig. 4A), which carries a pfcrt haplotype frequently found in Asian and African CQ-resistant lines (19, 20). Conclusive evidence that the extent of VP reversibility is largely determined by the pfcrt haplotype was recently obtained using allelic exchange (26). The decreased level of CQR in the knockdown clones was apparent across multiple, partially inhibitory CQ drug dilutions (Fig. 4, D and E). These clones nonetheless had IC50 values for CQ with or without VP that were closer to 7G8 than to the VP-insensitive CQS parasite line 106/1 (Fig. 4A). These drug assay data were consistent with the observation that only 7G8 and FCB, but not the pfcrt knockdown clones or 106/1, could be propagated long term in culture medium supplemented with 80 nM CQ (data not shown).
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Quantitation of the response of K2C3 and K2F10 to the other putative heme-binding antimalarials quinine and mefloquine indicated no statistically significant change in the respective IC50 values compared with 7G8 (Fig. 4, B and C). Verapamil was found to have a partial reversal effect on the quinine response but no effect on mefloquine.
AO Perfusion Experiments with pfcrt Knockdown Parasites— Single-cell photometry studies of P. falciparum-infected RBC with AO have consistently shown that CQ-resistant parasite lines expressing mutant PfCRT have a steeper intracellular [AO]/extracellular [AO] slope, relative to CQS lines (19, 20, 42, 43). These results suggest but do not prove that PfCRT mutations alter DV ion transport physiology. Here, measurements of the change in AO fluorescence in single cells, as a function of time and of [AO] in the perfusate, showed a dramatic change in the knockdown clones, which now had profiles similar to the CQS line 106/1 (Fig. 5A). This contrasted with the relationship observed for 7G8 and the other CQ-resistant line FCB. Calculation of the mean fluorescence intensity data for each line, versus external [AO], confirmed that the knockdown clones grouped with 106/1, and not with 7G8 or FCB (Fig. 5B). These data implied a direct impact of PfCRT expression levels on the internal versus external AO relationship. One interpretation of these data is that lower compartmental pH accompanies reduction in the level of mutant PfCRT expression. Using different equipment and methodologies, other researchers have questioned whether AO localizes to the DV and/or whether illumination affects its subcellular disposition (Refs. 44 and 45, respectively). To address this question, we used laser scanning confocal microscopy to compare AO and hemozoin localization. Results (Fig. 5, C and D) show that the AO fluorescence signals, as measured in successive 0.2-µm optical sections along the z-axis, coincide with the hemozoin localization to within 0.5 µm. Thus, hemozoin is contained within an irregular sphere defined by AO fluorescence that extends no more than 0.5 µm in any direction from hemozoin. This is well within the confines of the DV that can attain a diameter of up to 2.5–3.0 µm in the mature, trophozoite-stage intra-erythrocytic parasite (4).
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30–70% relative to the
non-transformed parental line. Our data suggest that differences in pfcrt expression levels may account in part for the two to 4-fold differences in CQ IC50 values that have been reported between CQ-resistant lines carrying identical pfcrt haplotypes (18–20, 46, 48–50). Undoubtedly, other genes are also involved, including pfmdr1, which was recently shown by allelic exchange to be able to modulate the CQR phenotype in a parasite line expressing mutant pfcrt (51). Interestingly, several studies have found altered levels of pfmdr1 expression in mutant lines selected under continued drug pressure (52–54). pfmdr1 also contains several point mutations that in some, though not all studies, show a correlation with CQR (reviewed in Ref. 55). Thus, point mutations and expression levels of both pfcrt and pfmdr1 have now been found to have an impact on the degree of CQ susceptibility. Both genes encode proteins localized on the membrane of the DV (19, 42, 56), the organelle wherein CQ exerts its activity, implying that altered functional properties of the DV membrane are key to the CQR mechanism.
How does mutant PfCRT contribute to CQR? One possibility could involve an effect on pH values and pH gradients within the intact intra-erythrocytic parasite that might critically alter CQ accumulation or pH-dependent binding of CQ to relevant targets. In the present study, we found a significant decrease in accumulation of the pH sensor AO in the pfcrt knockdown clones. These clones, designed to differ from the CQ-resistant 7G8 line only at the pfcrt locus, now closely aligned with CQS parasites (Fig. 5). These results support the general idea that PfCRT might be involved in ion-dependent DV transport processes that affect intracellular pH (19, 41). They are also consistent with earlier reports predicting that the DV of CQ-resistant parasites was more acidic (19, 20, 42, 43), however, we stress this suggestion awaits confirmation by a non-AO-dependent method for quantifying DV pH. Heme aggregation and crystallization assays have provided evidence that a more acidic DV pH would significantly reduce CQ accumulation by (i) increasing the rate of conversion of heme to hemozoin (10) and (ii) promoting acid aggregation of free heme (43). These two acid-activated processes could effectively titrate out the soluble hematin receptor for CQ and thus confer resistance. Independent assays are required to unequivocally prove this interpretation, which has been challenged by others based on AO measurements made with different methods (44). Along these lines, recent ratiometric DV pH measurements with the fluorophore NERF, which do not suffer from the same limitations that accompany AO-based analyses, provide additional evidence that CQS parasites have a higher DV pH than do CQ-resistant parasites.2
Regardless, importantly, our data also reveal that changes in AO partitioning do not strictly correlate with changes in the degree of susceptibility, because the knockdown parasites remained CQ-resistant, albeit at borderline levels, with CQ IC50 values of 75–80 nM. These values are slightly below the estimated CQR lower limit of 80–100 nM (58, 59). An alternative interpretation of our photometric data is that mutant PfCRT might transport AO (60, 61) and that the rate of AO transport can be affected by point mutations and expression level changes in this protein.
Another possible explanation for the contribution of pfcrt to CQR is that the point mutations might enable PfCRT to directly interact with the drug, thereby perhaps competing with hematin for CQ binding and reducing intracellular CQ accumulation. This would be expected to result in reduced CQ-hematin interaction and enable the CQ-resistant parasite to continue its normal process of heme detoxification. The ability of VP to reverse CQR could reflect VP binding to mutant PfCRT and interfering with PfCRT-CQ interactions. Efforts are underway to discriminate between the hypothesis of an indirect role for PfCRT based on its effect on intracellular physiology (including pH changes), versus the acquisition of a drug transport function. These efforts include expressing PfCRT in yeast vesicles (41) and Xenopus laevis oocytes4 to assay for endogenous PfCRT function and possible interactions with drug.
We note that, in this present study, reduction of mutant PfCRT expression rendered the parasites more susceptible to CQ yet had no major impact on their degree of susceptibility to either mefloquine or quinine (Fig. 4). These results contrast with previous reports that PfCRT point mutations can significantly alter parasite susceptibility to all three compounds (26, 42) and suggest that expression differences and sequence changes affect parasite response to heme-binding antimalarials in separate ways. These data also indicate that the means by which PfCRT affects response to mefloquine and quinine is distinct from its capacity to confer CQR.
In parallel with this experiment, we attempted to disrupt pfcrt function using "knockout" transfection constructs. Although episomally transformed parasites were obtained, pfcrt disruption was never detected above trace levels using sensitive PCR screens (K. Waller and D. Fidock, data not shown). Our findings suggested that disruption of pfcrt is deleterious to normal parasite viability and may indicate that this gene is critical for parasite propagation. We also note that, despite multiple attempts, we were unable to isolate and clone knockdown parasites in the CQ-resistant Dd2 line, even though these could be detected in the bulk cultures, probably because of a slow growth phenotype. Slower growth was also apparent in the 7G8 knockdown lines described herein (data not shown). These combined observations suggest an important role for PfCRT in helping maintain normal parasite growth inside the iRBC. Microarray and proteomic studies are underway to test whether reduced PfCRT expression has led to compensatory adjustments in the expression levels of functionally related genes, as an approach to identifying genes that work with PfCRT in helping to regulate parasite intracellular physiology.
To our knowledge, this is the first report in which genetic modification of a Plasmodium gene has been employed to specifically study the effects of gene expression levels on drug-resistant phenotypes. Application of this technology offers the attractive possibility of probing gene function in the absence of generating gene knockouts, which may significantly reduce parasite viability or in fact be lethal to the parasite, and obviates the prima facie need to identify and target individual mutations or domains. RNA interference represents a less labor-intensive alternative, and two reports provide evidence that this may work in P. falciparum (62, 63). However, concerns remain because of the lack of critical RNA interference proteins in the P. falciparum genome (64), including the RNA-dependent RNA polymerase Dicer. Alternative forms of RNA-dependent gene regulation may nonetheless exist in P. falciparum, as evidenced by the discovery of unique RNA-protein interactions that were found to affect antimalarial antifolate susceptibility (65) as well as the finding of abundant antisense transcripts in this organism (66). An improved understanding of P. falciparum gene regulation and its application to the study of drug resistance and pathogenesis can facilitate efforts to translate post-genomic research into practical solutions to combat malaria.
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FOOTNOTES |
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Present address: The Dept. of Cardiology, Albert Einstein College of
Medicine, Bronx, NY 10461.
To whom correspondence should be addressed. Tel.: 718-430-3759; Fax:
718-430-8711; E-mail:
dfidock{at}aecom.yu.edu.
1 The abbreviations used are: CQ, chloroquine; CQR, chloroquine resistance;
DV, digestive vacuole; iRBC, infected red blood cell; VP, verapamil; PfCRT,
P. falciparum chloroquine resistance transporter; CQS, chloroquine
sensitive; IC50 and IC90, 50% and 90% inhibitory
concentrations, respectively; 3'-UTR, 3'-untranslated region; AO,
acridine orange; PBS, phosphate-buffered saline; hdhfr, human
dihydrofolate reductase gene; hrp3, histidine-rich protein 3 gene;
hrp2, histidine-rich protein 2 gene; EF-1, P.
falciparum elongation factor-1 gene; DIG, dideoxygenin; SCP,
single-cell photometric.
2 T. Bennett, A. D. Kosar, L. M. B. Ursos, S. Dzekunov, A. B. S. Sidhu, D. A.
Fidock, and P. D. Roepe, unpublished data.
3 T. Nomura and T. Wellems, unpublished data.
4 D. Fidock, S. Krishna, P. Roepe, and M. Akabas, unpublished data.
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