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Antimicrobial Agents and Chemotherapy, November 2005, p. 4807-4808, Vol. 49, No. 11
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.11.4807-4808.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

LETTERS TO THE EDITOR

Mutations Conferring Drug Resistance in Malaria Parasite Drug Transporters Pgh1 and PfCRT Do Not Affect Steady-State Vacuolar Ca²⁺

Top Letter References

 
Resistance to chloroquine (CQ) by the malaria parasite Plasmodium falciparum has been observed in every region where P. falciparum occurs (20). The exact mode of action of CQ has not been fully elucidated, but it is generally accepted that a crucial step in this process is the binding of the drug to ferriprotoporphyrin IX (heme), a by-product of hemoglobin degradation which occurs in the parasite digestive food vacuole (DV).

A number of studies have contributed to pinpointing the pfcrt gene as the major determinant of CQ resistance (1, 3, 4, 6, 14, 15, 17-19). In addition, mutations of the pfmdr1 gene (expressing Pgh1) have been shown to modulate the level of CQ resistance (11), as well as being partially responsible for the acquired resistance to other drugs such as mefloquine (10). Currently there are two hypotheses as to the function of PfCRT in CQ resistance. The first of these proposes that PfCRT actively removes CQ from the DV, either as an ATP-dependent pump or as a secondary active transporter (12, 13). Alternatively, the "charged drug leak model" proposes that diprotonated CQ (CQ++) leaves the DV via mutated PfCRT passively down its concentration gradient (5). Both theories are in agreement, however, that CQ is transported out of the DV and that this is the key mechanism of CQ resistance.

Little is known of the functional role of the PfCRT transporter in P. falciparum physiology. PfCRT is localized to the DV membrane (4), and bioinformatic studies indicate that PfCRT is a member of the drug/metabolite transporter superfamily (7, 16), other members of which are known to transport a variety of substrates, including amino acids, weak bases, and organic cations. Studies which have heterologously expressed PfCRT into yeast (Pichia pastoris) (21) and Xenopus oocytes (9) have suggested that PfCRT is able to modulate host transport systems. In yeast, PfCRT is reported to function in the passive movement of Cl^– (21), while in the Xenopus system, PfCRT-expressing oocytes exhibit a depolarized membrane potential (_m) and a higher intracellular pH (pH_i) compared to control oocytes (9). One possibility is that PfCRT interferes with second messengers such as Ca²⁺ (9). In addition, Ca²⁺ channel blockers such as verapamil are believed to block PfCRT, resulting in a chemosensitization of CQ-resistant parasites (for examples, see references 5 and 8). Given that the DV of P. falciparum has been shown to contain elevated levels of free Ca²⁺ relative to the cytosol (2), this study was undertaken to determine whether PfCRT has a role in DV Ca²⁺ homeostasis.

The resting concentrations of free DV Ca²⁺ ([Ca²⁺]_DV) from a number of P. falciparum isolates and allelically exchanged pfcrt and pfmdr1 strains (14) were measured. Measurements were carried out using confocal laser-scanning single-cell imaging as described before (2), with the exception that Fluo 5AM was used in preference to Fluo 4AM due to its higher K_d for Ca²⁺. In addition, in situ [Ca²⁺]_DV calibrations were carried out on erythrocyte-free parasites, as nigericin induces parasites to exit from their host cell. Results shown in Table 1 show that the measured [Ca²⁺]_DV of all the strains was in the region of 2 µM. These values are a little higher than those reported previously (0.4 µM) and probably reflect the higher external [Ca²⁺] experienced by free parasites (in RPMI medium) compared to the lower [Ca²⁺] experienced by the intraerythrocytic parasites measured previously (2). There was, however, no correlation between the steady-state [Ca²⁺]_DV values and the CQ sensitivity status of the various Plasmodium strains.

View this table: [in this window] [in a new window]

TABLE 1. Resting vacuolar Ca2+ concentration of P. falciparum strains with various chloroquine sensitivities

We conclude that mutations in pfcrt or pfmdr1, conferring drug resistance, do not affect the [Ca²⁺]_DV. We further propose that it is unlikely therefore that the functional role of PfCRT is connected to Ca²⁺ homeostasis, unless redundancy in the pathways maintaining DV Ca²⁺ homeostasis masked any effect on this process conferred by PfCRT.

 
G.A.B. is supported by an Early Career Leverhulme Trust Fellowship. P.G.B. and S.A.W. are supported by BBSRC, MRC, and Wellcome Trust grants.

We thank staff and patients of Ward 7Y and the Gastroenteritis Unit, Royal Liverpool Hospital, for their generous donation of blood.

Top Letter References

 

1
1. Basco, L. K., and P. Ringwald. 2001. Analysis of the key pfcrt point mutation and in vitro and in vivo response to chloroquine in Yaounde, Cameroon. J. Infect. Dis. 183:1828-1831.[CrossRef][Medline]
2
2. Biagini, G. A., P. G. Bray, D. G. Spiller, M. R. White, and S. A. Ward. 2003. The digestive food vacuole of the malaria parasite is a dynamic intracellular Ca2+ store. J. Biol. Chem. 278:27910-27915.[Abstract/Free Full Text]
3
3. Chen, N., B. Russell, J. Staley, B. Kotecka, P. Nasveld, and Q. Cheng. 2001. Sequence polymorphisms in pfcrt are strongly associated with chloroquine resistance in Plasmodium falciparum. J. Infect. Dis. 183:1543-1545.[Medline]
4
4. Fidock, D. A., T. Nomura, A. K. Talley, R. A. Cooper, S. M. Dzekunov, M. T. Ferdig, L. M. Ursos, A. B. Sidhu, B. Naude, K. W. Deitsch, X. Z. Su, J. C. Wootton, P. D. Roepe, and T. E. Wellems. 2000. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol. Cell 6:861-871.[CrossRef][Medline]
5
5. Johnson, D. J., D. A. Fidock, M. Mungthin, V. Lakshmanan, A. B. Sidhu, P. G. Bray, and S. A. Ward. 2004. Evidence for a central role for PfCRT in conferring Plasmodium falciparum resistance to diverse antimalarial agents. Mol. Cell 15:867-877.[CrossRef][Medline]
6
6. Lakshmanan, V., P. G. Bray, D. Verdier-Pinard, D. J. Johnson, P. Horrocks, R. A. Muhle, G. E. Alakpa, R. H. Hughes, S. A. Ward, D. J. Krogstad, A. B. Sidhu, and D. A. Fidock. 2005. A critical role for PfCRT K76T in Plasmodium falciparum verapamil-reversible chloroquine resistance. EMBO J. 24:2294-2305. (First published 9 June 2005; doi:10.1038/sj.emboj.7600681.)[CrossRef][Medline]
7
7. Martin, R. E., and K. Kirk. 2004. The malaria parasite's chloroquine resistance transporter is a member of the drug/metabolite transporter superfamily. Mol. Biol. Evol. 21:1938-1949.[Abstract/Free Full Text]
8
8. Martin, S. K., A. M. Oduola, and W. K. Milhous. 1987. Reversal of chloroquine resistance in Plasmodium falciparum by verapamil. Science 235:899-901.[Abstract/Free Full Text]
9
9. Nessler, S., O. Friedrich, N. Bakouh, R. H. Fink, C. P. Sanchez, G. Planelles, and M. Lanzer. 2004. Evidence for activation of endogenous transporters in Xenopus laevis oocytes expressing the Plasmodium falciparum chloroquine resistance transporter, PfCRT. J. Biol. Chem. 279:39438-39446.[Abstract/Free Full Text]
10
10. Price, R. N., A. C. Uhlemann, A. Brockman, R. McGready, E. Ashley, L. Phaipun, R. Patel, K. Laing, S. Looareesuwan, N. J. White, F. Nosten, and S. Krishna. 2004. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet 364:438-447.[CrossRef][Medline]
11
11. Reed, M. B., K. J. Saliba, S. R. Caruana, K. Kirk, and A. F. Cowman. 2000. Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature 403:906-909.[CrossRef][Medline]
12
12. Sanchez, C. P., J. E. McLean, W. Stein, and M. Lanzer. 2004. Evidence for a substrate specific and inhibitable drug efflux system in chloroquine resistant Plasmodium falciparum strains. Biochemistry 43:16365-16373.[CrossRef][Medline]
13
13. Sanchez, C. P., W. Stein, and M. Lanzer. 2003. Trans stimulation provides evidence for a drug efflux carrier as the mechanism of chloroquine resistance in Plasmodium falciparum. Biochemistry 42:9383-9394.[CrossRef][Medline]
14
14. Sidhu, A. B., D. Verdier-Pinard, and D. A. Fidock. 2002. Chloroquine resistance in Plasmodium falciparum malaria parasites conferred by pfcrt mutations. Science 298:210-213.[Abstract/Free Full Text]
15
15. Su, X., M. T. Ferdig, Y. Huang, C. Q. Huynh, A. Liu, J. You, J. C. Wootton, and T. E. Wellems. 1999. A genetic map and recombination parameters of the human malaria parasite Plasmodium falciparum. Science 286:1351-1353.[Abstract/Free Full Text]
16
16. Tran, C. V., and M. H. Saier, Jr. 2004. The principal chloroquine resistance protein of Plasmodium falciparum is a member of the drug/metabolite transporter superfamily. Microbiology 150:1-3.[Free Full Text]
17
17. Walker-Jonah, A., S. A. Dolan, R. W. Gwadz, L. J. Panton, and T. E. Wellems. 1992. An RFLP map of the Plasmodium falciparum genome, recombination rates and favored linkage groups in a genetic cross. Mol. Biochem. Parasitol. 51:313-320.[CrossRef][Medline]
18
18. Wellems, T. E., L. J. Panton, I. Y. Gluzman, V. E. do Rosario, R. W. Gwadz, A. Walker-Jonah, and D. J. Krogstad. 1990. Chloroquine resistance not linked to mdr-like genes in a Plasmodium falciparum cross. Nature 345:253-255.[CrossRef][Medline]
19
19. Wellems, T. E., A. Walker-Jonah, and L. J. Panton. 1991. Genetic mapping of the chloroquine-resistance locus on Plasmodium falciparum chromosome 7. Proc. Natl. Acad. Sci. USA 88:3382-3386.[Abstract/Free Full Text]
20
20. Wongsrichanalai, C., A. L. Pickard, W. H. Wernsdorfer, and S. R. Meshnick. 2002. Epidemiology of drug-resistant malaria. Lancet Infect. Dis. 2:209-218.[CrossRef][Medline]
21
21. Zhang, D., W. Pan, D. Lu, and L. Jiang. 2002. Synthesis and expression of 42 kD C-terminal region of the major merozoite surface protein (MSP1-42) of P. falciparum 3D7 strain in Pichia pastoris. Zhonghua Yixue Zazhi 82:198-202. (In Chinese.)

						Giancarlo A. Biagini* Liverpool School of Tropical Medicine Pembroke Place Liverpool L35 QA, United Kingdom David A. Fidock Department of Microbiology and Immunology Albert Einstein College of Medicine Bronx, New York 10461 Patrick G. Bray Stephen A. Ward Liverpool School of Tropical Medicine Pembroke Place Liverpool L35 QA, United Kingdom
						* Phone: 4401517053151, Fax: 4401517053371, E-mail: biagini{at}liv.ac.uk

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Antimicrobial Agents and Chemotherapy, November 2005, p. 4807-4808, Vol. 49, No. 11
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.11.4807-4808.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Biagini, G. A. Articles by Ward, S. A. Search for Related Content PubMed PubMed Citation Articles by Biagini, G. A. Articles by Ward, S. A.