Disruption of a Plasmodium falciparum Multidrug Resistance-associated Protein (PfMRP) Alters Its Fitness and Transport of Antimalarial Drugs and Glutathione
2009; Elsevier BV; Volume: 284; Issue: 12 Linguagem: Inglês
10.1074/jbc.m806944200
ISSN1083-351X
AutoresDipak Kumar Raj, Jianbing Mu, Hongying Jiang, Juraj Kabát, S. P. Singh, Matthew Sullivan, Michael P. Fay, Thomas F. McCutchan, Xin‐zhuan Su,
Tópico(s)Pharmacological Effects and Toxicity Studies
ResumoATP-binding cassette transporters play an important role in drug resistance and nutrient transport. In the human malaria parasite Plasmodium falciparum, a homolog of the human p-glycoprotein (PfPgh-1) was shown to be involved in resistance to several drugs. More recently, many transporters were associated with higher IC50 levels in responses to chloroquine (CQ) and quinine (QN) in field isolates. Subsequent studies, however, could not confirm the associations, although inaccuracy in drug tests in the later studies could contribute to the lack of associations. Here we disrupted a gene encoding a putative multidrug resistance-associated protein (PfMRP) that was previously shown to be associated with P. falciparum responses to CQ and QN. Parasites with disrupted PfMRP (W2/MRPΔ) could not grow to a parasitemia higher than 5% under normal culture conditions, possibly because of lower efficiency in removing toxic metabolites. The W2/MRPΔ parasite also accumulated more radioactive glutathione, CQ, and QN and became more sensitive to multiple antimalarial drugs, including CQ, QN, artemisinin, piperaquine, and primaquine. PfMRP was localized on the parasite surface membrane, within membrane-bound vesicles, and along the straight side of the D-shaped stage II gametocytes. The results suggest that PfMRP plays a role in the efflux of glutathione, CQ, and QN and contributes to parasite responses to multiple antimalarial drugs, possibly by pumping drugs outside the parasite. ATP-binding cassette transporters play an important role in drug resistance and nutrient transport. In the human malaria parasite Plasmodium falciparum, a homolog of the human p-glycoprotein (PfPgh-1) was shown to be involved in resistance to several drugs. More recently, many transporters were associated with higher IC50 levels in responses to chloroquine (CQ) and quinine (QN) in field isolates. Subsequent studies, however, could not confirm the associations, although inaccuracy in drug tests in the later studies could contribute to the lack of associations. Here we disrupted a gene encoding a putative multidrug resistance-associated protein (PfMRP) that was previously shown to be associated with P. falciparum responses to CQ and QN. Parasites with disrupted PfMRP (W2/MRPΔ) could not grow to a parasitemia higher than 5% under normal culture conditions, possibly because of lower efficiency in removing toxic metabolites. The W2/MRPΔ parasite also accumulated more radioactive glutathione, CQ, and QN and became more sensitive to multiple antimalarial drugs, including CQ, QN, artemisinin, piperaquine, and primaquine. PfMRP was localized on the parasite surface membrane, within membrane-bound vesicles, and along the straight side of the D-shaped stage II gametocytes. The results suggest that PfMRP plays a role in the efflux of glutathione, CQ, and QN and contributes to parasite responses to multiple antimalarial drugs, possibly by pumping drugs outside the parasite. Genes encoding ATP-binding cassette (ABC) 2The abbreviations used are: ABC, ATP-binding cassette; CQ, chloroquine; QN, quinine; ART, artemisinin; PQ, piperaquine; PRQ, primaquine; MQ, mefloquine; CQS, CQ-sensitive; CQR, CQ-resistant; MRP, multidrug resistance-associated protein; WT, wild type; RBC, red blood cell; PBS, phosphate-buffered saline; AMQ, amodiaquine; PG, proguanil; ROS, reactive oxygen species; FV, food vacuole. transporters belong to a supergene family present in organisms from prokaryotes to mammals (1Dean M. Annilo T. Annu. Re.v Genomics Hum. Genet. 2005; 6: 123-142Crossref PubMed Scopus (489) Google Scholar, 2Igarashi Y. Aoki K.F. Mamitsuka H. Kuma K. Kanehisa M. Mol. Biol. Evol. 2004; 21: 2149-2160Crossref PubMed Scopus (26) Google Scholar). These genes encode transmembrane proteins that can transport a wide variety of substrates across extra- and intracellular membranes, including metabolic products, lipids, sterols, and drugs; changes in the protein sequences or expressional levels of ABC transporters have been linked to alteration in stress response, cellular detoxification, and various diseases and disorders (3Stefkova J. Poledne R. Hubacek J.A. Physiol. Res. 2004; 53: 235-243PubMed Google Scholar, 4Dean M. Methods Enzymol. 2005; 400: 409-429Crossref PubMed Scopus (130) Google Scholar, 5Ernst R. Klemm R. Schmitt L. Kuchler K. Methods Enzymol. 2005; 400: 460-484Crossref PubMed Scopus (64) Google Scholar, 6Yazaki K. FEBS Lett. 2006; 580: 1183-1191Crossref PubMed Scopus (288) Google Scholar, 7Jungwirth H. Kuchler K. FEBS Lett. 2006; 580: 1131-1138Crossref PubMed Scopus (167) Google Scholar). Mutations and/or overexpression of many ABC transporters can also lead to drug resistance in disease-causing microbes and to treatment failure of human cancer and other diseases (8Ouellette M. Legare D. Papadopoulou B. Trends Microbiol. 1994; 2: 407-411Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 9Chakraborti P.K. Bhatt K. Banerjee S.K. Misra P. Biosci. Rep. 1999; 19: 293-300Crossref PubMed Scopus (13) Google Scholar, 10Allen J.D. Brinkhuis R.F. van Deemter L. Wijnholds J. Schinkel A.H. Cancer Res. 2000; 60: 5761-5766PubMed Google Scholar, 11Cervenak J. Andrikovics H. Ozvegy-Laczka C. Tordai A. Nemet K. Varadi A. Sarkadi B. Cancer Lett. 2006; 234: 62-72Crossref PubMed Scopus (36) Google Scholar). The genome of the human malaria parasite Plasmodium falciparum contains at least 15 predicted genes encoding putative ABC transporters (12Gardner M.J. Hall N. Fung E. White O. Berriman M. Hyman R.W. Carlton J.M. Pain A. Nelson K.E. Bowman S. Paulsen I.T. James K. Eisen J.A. Rutherford K. Salzberg S.L. Craig A. Kyes S. Chan M.S. Nene V. Shallom S.J. Suh B. Peterson J. Angiuoli S. Pertea M. Allen J. Selengut J. Haft D. Mather M.W. Vaidya A.B. Martin D.M. Fairlamb A.H. Fraunholz M.J. Roos D.S. Ralph S.A. McFadden G.I. Cummings L.M. Subramanian G.M. Mungall C. Venter J.C. Carucci D.J. Hoffman S.L. Newbold C. Davis R.W. Fraser C.M. Barrell B. Nature. 2002; 419: 498-511Crossref PubMed Scopus (3473) Google Scholar). In particular, mutations and amplification of the gene encoding a homolog of the human P-glycoprotein (PfPgh-1) has been shown to contribute to or associated with parasite responses to chloroquine (CQ), mefloquine (MQ), and quinine (QN) (13Foote S.J. Thompson J.K. Cowman A.F. Kemp D.J. Cell. 1989; 57: 921-930Abstract Full Text PDF PubMed Scopus (516) Google Scholar, 14Reed M.B. Saliba K.J. Caruana S.R. Kirk K. Cowman A.F. Nature. 2000; 403: 906-909Crossref PubMed Scopus (722) Google Scholar, 15Duraisingh M.T. Roper C. Walliker D. Warhurst D.C. Mol. Microbiol. 2000; 36: 955-961Crossref PubMed Scopus (160) Google Scholar, 16Ferdig M.T. Cooper R.A. Mu J. Deng B. Joy D.A. Su X.-Z. Wellems T.E. Mol. Microbiol. 2004; 52: 985-997Crossref PubMed Scopus (208) Google Scholar, 17Price R.N. Uhlemann A.C. Brockman A. McGready R. Ashley E. Phaipun L. Patel R. Laing K. Looareesuwan S. White N.J. Nosten F. Krishna S. Lancet. 2004; 364: 438-447Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 18Sidhu A.B. Valderramos S.G. Fidock D.A. Mol. Microbiol. 2005; 57: 913-926Crossref PubMed Scopus (282) Google Scholar). Overexpression of another putative ABC transporter (PfMDR2) was also linked to parasite responses to CQ and MQ, but no additional evidence has been obtained to support a role for PfMDR2 in CQ and MQ response since the earliest descriptions (19Wilson C.M. Serrano A.E. Wasley A. Bogenschutz M.P. Shankar A.H. Wirth D.F. Science. 1989; 244: 1184-1186Crossref PubMed Scopus (396) Google Scholar, 20Rubio J.P. Cowman A.F. Exp. Parasitol. 1994; 79: 137-147Crossref PubMed Scopus (49) Google Scholar). Recently, PfMDR2 was found to transport heavy metal and to play a role in heavy metal resistance in P. falciparum (21Rosenberg E. Litus I. Schwarzfuchs N. Sinay R. Schlesinger P. Golenser J. Baumeister S. Lingelbach K. Pollack Y. J. Biol. Chem. 2006; 281: 27039-27045Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Many drug resistances are complex phenotypes involving multiple genes (16Ferdig M.T. Cooper R.A. Mu J. Deng B. Joy D.A. Su X.-Z. Wellems T.E. Mol. Microbiol. 2004; 52: 985-997Crossref PubMed Scopus (208) Google Scholar, 22Dent J.A. Smith M.M. Vassilatis D.K. Avery L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2674-2679Crossref PubMed Scopus (321) Google Scholar); however, a limited number of genes usually play a key role in determining a drug phenotype. One example is that mutations in the gene encoding a putative P. falciparum chloroquine resistance transporter (PfCRT) can convert a clinical CQ-sensitive (CQS) P. falciparum parasite into a CQ-resistant (CQR) one (23Fidock D.A. Nomura T. Talley A.K. Cooper R.A. Dzekunov S.M. Ferdig M.T. Ursos L.M. bir Singh Sidhu A. Naude B. Deitsch K.W. Su X. Wootton J.C. Roepe P.D. Wellems T.E. Mol. Cell. 2000; 6: 861-871Abstract Full Text Full Text PDF PubMed Scopus (1163) Google Scholar); however, parasites with the same mutant PfCRT allele can display very different levels of resistance to CQ, suggesting contribution from proteins such as PfPgh-1 and other molecules (24Mu J. Ferdig M.T. Feng X. Joy D.A. Duan J. Furuya T. Subramanian G. Aravind L. Cooper R.A. Wootton J.C. Xiong M. Su X.-Z. Mol. Microbiol. 2003; 49: 977-989Crossref PubMed Scopus (219) Google Scholar, 25Duraisingh M.T. von Seidlein L.V. Jepson A. Jones P. Sambou I. Pinder M. Warhurst D.C. Parasitology. 2000; 121: 1-7Crossref PubMed Scopus (42) Google Scholar). Indeed, mutations in genes encoding several putative transporters (other than PfPgh-1 and PfMDR2) were found to be associated with higher CQ and/or QN half-maximal inhibitory concentration (IC50) among P. falciparum isolates (16Ferdig M.T. Cooper R.A. Mu J. Deng B. Joy D.A. Su X.-Z. Wellems T.E. Mol. Microbiol. 2004; 52: 985-997Crossref PubMed Scopus (208) Google Scholar, 24Mu J. Ferdig M.T. Feng X. Joy D.A. Duan J. Furuya T. Subramanian G. Aravind L. Cooper R.A. Wootton J.C. Xiong M. Su X.-Z. Mol. Microbiol. 2003; 49: 977-989Crossref PubMed Scopus (219) Google Scholar). These transporters may play a role in modulating the levels of parasite response to antimalarial drugs. One of the putative transporters (PFA0590w, also called G2 in Ref. 24Mu J. Ferdig M.T. Feng X. Joy D.A. Duan J. Furuya T. Subramanian G. Aravind L. Cooper R.A. Wootton J.C. Xiong M. Su X.-Z. Mol. Microbiol. 2003; 49: 977-989Crossref PubMed Scopus (219) Google Scholar) associated with responses to CQ and QN is a member of the ABC transporter C subfamily. ABC transporters in this subfamily are also known as multidrug resistance-associated proteins (MRPs) that can also transport substrates such as glutathione (GSH), glucuronate, and sulfate conjugates as well as various drugs (26Rebbeor J.F. Connolly G.C. Ballatori N. Biochim. Biophys. Acta. 2002; 1559: 171-178Crossref PubMed Scopus (46) Google Scholar). The gene (pfmrp) was reported from P. falciparum previously, and the expression of its mRNA and protein were investigated (27Klokouzas A. Tiffert T. van Schalkwyk D. Wu C.P. van Veen H.W. Barrand M.A. Hladky S.B. Biochem. Biophys. Res. Commun. 2004; 321: 197-201Crossref PubMed Scopus (53) Google Scholar). PfMRP is encoded by a single exon of 5469 bp with 11 predicted transmembrane α-helix segments and is expressed from early trophozoite to late schizont, according to microarray analyses (28Le Roch K.G. Zhou Y. Blair P.L. Grainger M. Moch J.K. Haynes J.D. De La Vega P. Holder A.A. Batalov S. Carucci D.J. Winzeler E.A. Science. 2003; 301: 1503-1508Crossref PubMed Scopus (1030) Google Scholar, 29Bozdech Z. Llinas M. Pulliam B.L. Wong E.D. Zhu J. DeRisi J.L. PLoS Biol. 2003; 1: E5Crossref PubMed Scopus (1272) Google Scholar, 30Young J.A. Fivelman Q.L. Blair P.L. de la Vega P. Le Roch K.G. Zhou Y. Carucci D.J. Baker D.A. Winzeler E.A. Mol. Biochem. Parasitol. 2005; 143: 67-79Crossref PubMed Scopus (255) Google Scholar). Two mutations in the PfMRP were associated with higher levels of IC50 to CQ and QN in P. falciparum field isolates, with good correlation of the mutations in the gene and higher IC50 (24Mu J. Ferdig M.T. Feng X. Joy D.A. Duan J. Furuya T. Subramanian G. Aravind L. Cooper R.A. Wootton J.C. Xiong M. Su X.-Z. Mol. Microbiol. 2003; 49: 977-989Crossref PubMed Scopus (219) Google Scholar); however, whether PfMRP contributes to parasite drug responses remains controversial (31Anderson T.J. Nair S. Qin H. Singlam S. Brockman A. Paiphun L. Nosten F. Antimicrob. Agents Chemother. 2005; 49: 2180-2188Crossref PubMed Scopus (98) Google Scholar, 32Cojean S. Noel A. Garnier D. Hubert V. Le Bras J. Durand R. Malaria J. 2006; 5: 24Crossref PubMed Scopus (17) Google Scholar) and requires additional functional studies. To further investigate the functions of PfMRP in metabolite transport and drug resistance in malaria parasites and to resolve the discrepancies among different association studies, we have disrupted the putative transporter PfMRP in the P. falciparum parasite. We showed that when the gene encoding the PfMRP in a CQR parasite (W2) was disrupted, the parasite growth was affected and became more sensitive to multiple antimalarial drugs. The pfmrp knock-out parasite also accumulated more CQ and QN compared with its wild type (WT) parasite W2. Our study showed that PfMRP played a role in parasite response to CQ, QN, and other drugs and provided information for better understanding the functions of the PfMRP in transporting antimalarial drugs and other metabolites. Parasite and Parasite Culture-P. falciparum parasites used in this study have been described (24Mu J. Ferdig M.T. Feng X. Joy D.A. Duan J. Furuya T. Subramanian G. Aravind L. Cooper R.A. Wootton J.C. Xiong M. Su X.-Z. Mol. Microbiol. 2003; 49: 977-989Crossref PubMed Scopus (219) Google Scholar, 33Furuya T. Mu J. Hayton K. Liu A. Duan J. Nkrumah L. Joy D.A. Fidock D.A. Fujioka H. Vaidya A.B. Wellems T.E. Su X.-Z. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 16813-16818Crossref PubMed Scopus (64) Google Scholar). The parasites were cultured in vitro according to the methods of Trager and Jensen (34Trager W. Jensen J.B. Science. 1976; 193: 673-675Crossref PubMed Scopus (6219) Google Scholar). Briefly, parasites were maintained in RPMI 1640 medium containing 25 mm HEPES, 5% human O+ erythrocytes (5% hematocrit), 0.5% Albumax (Invitrogen), 24 mm sodium bicarbonate, and 10 μg/ml gentamycin at 37 °C with 5% CO2, 5% O2, and 90% N2 with daily medium changes. Antibody Production and Western Blotting-Polyclonal antibodies against PfMRP were obtained using DNA vaccination. DNA segments encoding two relatively hydrophilic peptides were amplified and cloned separately into VR2001/MRP plasmid DNA (35Oliveira F. Kamhawi S. Seitz A.E. Pham V.M. Guigal P.M. Fischer L. Ward J. Valenzuela J.G. Vaccine. 2006; 24: 374-390Crossref PubMed Scopus (99) Google Scholar) for immunization (Fig. 1A). Five-month-old female Swiss Webster mice were injected three times subcutaneously into the tails with 40 μg of purified plasmid DNA (35Oliveira F. Kamhawi S. Seitz A.E. Pham V.M. Guigal P.M. Fischer L. Ward J. Valenzuela J.G. Vaccine. 2006; 24: 374-390Crossref PubMed Scopus (99) Google Scholar) at 3-week intervals under National Institutes of Health animal protocol LMVR85E. Blood was collected from the tail vein of the injected animals, and sera were tested for binding to PfMRP on Western blot. Preimmune sera were collected 1 day prior to the first plasmid injection. Antibodies against PfMSP-1, PfCRT, and PfMDV-1 were described previously (23Fidock D.A. Nomura T. Talley A.K. Cooper R.A. Dzekunov S.M. Ferdig M.T. Ursos L.M. bir Singh Sidhu A. Naude B. Deitsch K.W. Su X. Wootton J.C. Roepe P.D. Wellems T.E. Mol. Cell. 2000; 6: 861-871Abstract Full Text Full Text PDF PubMed Scopus (1163) Google Scholar, 33Furuya T. Mu J. Hayton K. Liu A. Duan J. Nkrumah L. Joy D.A. Fidock D.A. Fujioka H. Vaidya A.B. Wellems T.E. Su X.-Z. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 16813-16818Crossref PubMed Scopus (64) Google Scholar, 36Singh S. Plassmeyer M. Gaur D. Miller L.H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 20043-20048Crossref PubMed Scopus (51) Google Scholar). Parasite proteins were prepared by treatment of parasitized RBCs with 0.1% saponin in PBS on ice for 10 min followed by a cold PBS wash, dissolved in SDS sample loading buffer (50 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mm EDTA, 0.02% bromphenol blue) and were separated in 12% SDS-PAGE gels after boiling in sample loading buffer for 5 min. Separated proteins were transferred to a Protran nitrocellulose membrane and probed with antibodies from immunized mice. The signals were developed using horseradish peroxidase-conjugated anti-mouse IgG (1:3000), secondary antibodies diluted in blocking buffer (5% nonfat milk and 0.1% Tween 20 in PBS), and ECL Western blotting detection reagents (Amersham Biosciences) after incubation of the membrane with anti-PfMRP antibodies (1:1000) in blocking buffer at room temperature for 2 h. Construction of Transfection Vectors-The plasmid pHD22Y was obtained from Dr. Thomas E. Wellems (37Fidock D.A. Wellems T.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10931-10936Crossref PubMed Scopus (414) Google Scholar). A segment of 956-bp DNA (132–1068 bp from the 5′ end of the coding region) of the gene was amplified from W2 genomic DNA using PCR primers 5′-GCACTGCAGGGAGATATTCAAGAACT-3′ and 5′-GCAGCGGCCGCGGACAACCATATAGC-3′. The amplified DNA was digested with the restriction enzymes PstI and NotI and cloned into the pHD22Y vector (Fig. 1B). The DNA sequences of all inserts were confirmed by DNA sequencing. Parasite Transformation and Selection-Asexual stages of W2 parasite were cultured as described above. The parasites were synchronized using 5% d-sorbitol, and schizont stages at 8–10% parasitemia were purified using a Percoll-sorbitol separation method (38Lambros C. Vanderberg J.P. J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2855) Google Scholar, 39Fernandez V. Treutiger C.J. Nash G.B. Wahlgren M. Infect. Immun. 1998; 66: 2969-2975Crossref PubMed Google Scholar). Uninfected RBCs were electroporated with 200 μg of supercoiled pHD22Y containing DNA inserts as described (33Furuya T. Mu J. Hayton K. Liu A. Duan J. Nkrumah L. Joy D.A. Fidock D.A. Fujioka H. Vaidya A.B. Wellems T.E. Su X.-Z. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 16813-16818Crossref PubMed Scopus (64) Google Scholar, 40Wu Y. Wellems T.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1130-1134Crossref PubMed Scopus (339) Google Scholar). Following transformation, the parasites were maintained in drug-free medium for 48 h before the addition of WR99210 (Jacobus Pharmaceuticals) to a final concentration of 10 nm. Drug-resistant parasites appeared in 3–4 weeks after transfection. DNA samples from the parasite cultures 6–8 weeks after transfection were tested for the presence of integration of plasmid DNA into chromosomes using PCR (forward primer F 5′-GATTGGATAAGACCGTTAATA-3′ and reverse primer R 5′-TAAACTTGGTAAAAATTCAAATAG-3′) (Fig. 1B). When evidence of integration was detected (see Fig. 1C), the parasites were grown without the drug for 2 weeks and then with drug selection for 2 weeks. The procedure was done twice before limiting dilution to clone parasites with disrupted pfmrp. Parasite Growth and Invasion Rate Assays-A culture of synchronous ring stage was diluted to 0.1% parasitemia and 2% hematocrit. Blood smears were prepared every 24 h over a period of 10 days, and parasitemia were counted under a light microscope after Giemsa staining. Culture media were changed daily or twice a day without the addition of red blood cells until the cultures crashed because of high parasitemia. The invasion rates were calculated by dividing the number of successfully invaded parasites with the initial parasitemia after one cycle (48 h). The experiment was repeated four times. Immunofluorescence Assay-Glass slides with parasite smear were fixed in cold methanol (in dry ice) for 15 min and dried at room temperature for 15 min. The samples were blocked with blocking buffer (5% nonfat milk in PBS) at room temperature for 2 h. The slides were incubated with mouse primary antibody against PfMRP (diluted 1:200) at room temperature for 2 h or 4 °C overnight and then with diluted goat anti-mouse or anti-rabbit antibodies (1:1000) at room temperature for 30 min after washing the slides three times with blocking buffer. The slides were mounted with ProLong Gold antifade reagent with 4′,6′-diamino-2-phenylindole (Invitrogen) after washing five times with buffer and were observed under a confocal microscope (Leica SP2, Leica Microsystems, Exton, PA) using a 100× oil immersion objective NA 1.4. The images were deconvolved with Huygens Essential software (version 3.1; Scientific Volume Imaging BV, Hilversum, The Netherlands). Sequential Z-sections of stained cells were also collected for three-dimensional reconstruction and iso-surface modeling of representative cells with Imaris software (version 6.0; Bitplane AG, Zurich, Switzerland). Drug Assays-CQ, QN, MQ, artemisinin (ART), and primaquine (PRQ) were purchased from Sigma-Aldrich; amodiaquine (AMQ) was bought from LGC Promochem; piperaquine (PQ) was obtained as a gift from Dr. Xinhua Wang (Guanzhou University of Traditional Chinese Medicine, China); and proguanil (PG) was obtained from the National Institutes of Health pharmacy. CQ, QN, MQ, ART, PG, and AMQ were dissolved in 70% ethanol and stored at -80 °C until use. PRQ was dissolved in water, stored at 4 °C, and used within a week. PQ was dissolved in 0.5% lactic acid and stored at -80 °C until use. Drug assays were performed using a SYBR green staining method modified from that described previously (41Smilkstein M. Sriwilaijaroen N. Kelly J.X. Wilairat P. Riscoe M. Antimicrob. Agents Chemother. 2004; 48: 1803-1806Crossref PubMed Scopus (837) Google Scholar). Briefly, the parasites were diluted to 1% parasitemia with 1% hematocrit, and diluted parasites (150 μl) were added to wells in triplicate in a 96-well plate containing 50 μl of 2-fold serially diluted drugs. The parasites were incubated with the drugs at 37 °C for 72 h. After incubation, DNA were released and stained with lysis buffer containing SYBR green dye. The plate was kept in darkness for 30 min and read in a FLUOstar Optima microplate reader (BMG Labtech). All of the data points from each parasite and dilution were independently repeated at least three times. Drug Accumulation Assay-Highly synchronized trophozoite parasites (parasitemia of 4–6% and hematocrits of 5.0%) were washed with PBS and then incubated in RPMI 1640 medium supplemented by 25 mm HEPES (pH 7.35), 0.2% NaHCO3, 0.2% d-glucose, 1% human serum, and radioactive-labeled CQ (20 nm [3-3H]), QN (2 nm [9-3H]), and reduced GSH ([glycine-2-3H] at 1, 3, and 6 nm) (American Radiolabeled Chemicals) at 37 °C for 5–60 min under continuous stirring. The parasites were centrifuged through silicon oil (Fulka Chemical Corp.) (density, 1.049) in 1.5-ml Eppendorf tubes, separating the RBC pellet from unincorporated label. The bottom tips of the tubes containing the cell pellets were cut off and placed in scintillation vials containing 100 μl of ethanol and 50 μl of 0.5 n NCS-II tissue solubilizer solution (Amersham Biosciences). The lysates were incubated at 37 °C overnight and were decolorized by the addition of 25 μl of 30% H2O2. Luminescence was blocked by acidification with 25 μl of glacial acetic acid. Radioactive levels were measured in a liquid scintillation counter (1450 MicroBeta TriLux; PerkinElmer Life Sciences). Uninfected RBCs and controls were subjected to identical protocols. Data from different experiments were normalized for differences in hematocrit, parasitemia, and radioactive labels. Data Analysis and IC50 Calculation-Data from microplate reader were analyzed using GraphPad Prism (La Jolla, CA) or R software (version 2.7.0) (42R Development Core Team R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria2008Google Scholar). The IC50 values were estimated using 4 parameter logistic models, where for the computations the control (i.e. drug concentration = 0) and uninfected RBC (acting like an extremely large drug concentration that stops all growth) were set to 10-20 m and 1010 m, respectively. The model for the percentage of inhibition is b + (100 - b)/[1 + 10(d-X)*H], where X is the log transformed drug concentration, and the parameters b (background inhibition), d (logIC50), and H (Hill coefficient) are estimated by least squares. A separate IC50 was calculated for W2 and W2/MRPΔ for each experiment, and a paired t test on the log(IC50) values was used together with the associated confidence intervals. Generation of Antibodies against PfMRP-The predicted DNA sequence encoding PfMRP was downloaded from PlasmoDB, and two DNA segments encoding regions of the protein likely to be exposed on membrane surface (hydrophilic) were selected (codons 475–705 and 806–1120) as targets of DNA vaccination (Fig. 1A). The DNA segments were cloned into the VR2001 plasmid vector (35Oliveira F. Kamhawi S. Seitz A.E. Pham V.M. Guigal P.M. Fischer L. Ward J. Valenzuela J.G. Vaccine. 2006; 24: 374-390Crossref PubMed Scopus (99) Google Scholar) and were used to immunize mice separately. After three injections of plasmid constructs containing the target sequences, sera from five mice for each construct were collected and tested for antibodies against PfMRP in parasite lysates. All of the mice produced antibodies against PfMRP when tested on Western blots (data not shown). The antibodies from the region of codon 806–1120 gave stronger signals in Western blot and were used in all the experiments. It appeared to be specific for PfMRP because only one major band with predicted molecular weight was detected on the Western blot when tested using pooled antisera against PfMRP (Fig. 1D). Genetic Knock-out Gene Encoding PfMRP-To investigate the biological functions of PfMRP and whether it plays a role in transporting drugs and in parasite resistance to antimalarial drugs, we disrupted the coding region of PfMRP by inserting a plasmid (pHD22Y) cassette containing a gene encoding human dihydrofolate reductase into the 5′ PfMRP coding region (37Fidock D.A. Wellems T.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10931-10936Crossref PubMed Scopus (414) Google Scholar). Amplification of DNA segment using primers flanking the targeted sequence showed the presence of parasites with integration of the plasmid sequence in the transfected parasites. Insertion of the plasmid construct produced a PCR product of 8.68 kilobase pairs from the parasite with disrupted pfmrp, whereas the WT parasite had a PCR product of 1020 bp (Fig. 1C). Limiting dilution was performed to identify parasite clones with integration of the plasmid construct into the chromosome that disrupt the coding region of PfMRP. Five weeks after cloning, two clones of parasites with disrupted PfMRP were obtained. The absence of protein expression in the W2/MRPΔ parasites was confirmed using antibodies against PfMRP from DNA vaccination described above (Fig. 1D). No Changes in pfcrt, pfmdr1, and a Gene Encoding a Putative Sodium Hydrogen Exchanger (PfNHE) in the W2/MRPΔ Parasite-Cross-contamination of parasites during in vitro culture occurs frequently. To confirm the identities of the knock-out parasite clones, we genotyped DNA from W2 and W2/MRPΔ clones using 10 highly polymorphic microsatellite markers (data not shown and supplemental Table 1) (43Su X.-Z. Ferdig M.T. Huang Y. Huynh C.Q. Liu A. You J. Wootton J.C. Wellems T.E. Science. 1999; 286: 1351-1353Crossref PubMed Scopus (289) Google Scholar) and a multicopy molecular fingerprinting marker PfRRM (supplemental Fig. 1) (44Su X.-Z. Carucci D.J. Wellems T.E. Exp. Parasitol. 1998; 89: 262-265Crossref PubMed Scopus (42) Google Scholar). No differences were observed between W2 and two W2/MRPΔ clones, confirming that the W2/MRPΔ clones indeed derived from W2. Various putative parasite transporters have been associated with drug resistances. In P. falciparum, PfCRT, PfPgh-1, and PfNHE have been associated with responses to CQ, QN, MQ, and other drugs (16Ferdig M.T. Cooper R.A. Mu J. Deng B. Joy D.A. Su X.-Z. Wellems T.E. Mol. Microbiol. 2004; 52: 985-997Crossref PubMed Scopus (208) Google Scholar, 45Su X.-Z. Hayton K. Wellems T.E. Nat. Rev. Genet. 2007; 8: 497-506Crossref PubMed Scopus (65) Google Scholar). To rule out the possibility that the observed changes in drug responses and impaired growth at high parasitemia in the W2/MRPΔ were caused by changes in these genes, we compared DNA sequences encoding PfCRT, PfPgh-1, and PfNHE from W2 and W2/MRPΔ. No changes in the genes were found (data not shown). Impaired Parasite Growth in Vitro after Disruption of PfMRP- Although the W2/MRPΔ parasite appeared to grow normally, it could not grow to a density higher than 5% parasitemia if culture medium was changed once a day, whereas the WT parasite W2 could be routinely cultured up to 15% parasitemia under the same conditions (Fig. 2A). The impaired growth capability of the W2/MRPΔ was not due to lower efficiency in invading RBCs. We counted the numbers of newly invaded parasites for the first invasion cycle and found that the ratios of the numbers of ring stage over the initial numbers of parasites were similar for both W2 and W2/MRPΔ (4.85 ± 0.40 S.D. for W2/MRPΔ versus 5.27 ± 0.51 S.D. for W2). The growth impairment could be due to lower efficiency in removing toxic metabolites from inside the parasite cells or due to reduced ability in acquisition of nutrients from the culture medium. Indeed, change of culture medium twice a day (but no addition of red blood cells) increased the maximum parasitemia of the W2/MRPΔ to ∼7% (Fig. 2A). Because the same culture medium has been routinely used to grow variou
Referência(s)