Heather L. Callahan a 1, William L. Roberts b, Petrie M. Rainey b,. Stephen M. ..... [8] Papadopoulou, B., Roy, G. and Ouellette, M. (1992) A novel antifolate ...
MOLF_L-ULAR AND ELSEVIER
Molecular and Biochemical Parasitology 68 (1994) 145-149
BIOCHEMICAL PARASITOIZ)GY
Short communication
The PGPA gene of Leishmania major mediates antimony (SblII) resistance by decreasing influx and not by increasing efflux Heather L. Callahan a 1, William L. Roberts b, Petrie M. Rainey b, Stephen M. Beverley a,, " Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA b Department of Laboratory Medicine, Yale University School of Medicine, 333 Cedar St., New Haven CT 06510, USA Received 8 June 1994; accepted 28 July 1994
Keywords: DNA transfection; Trypanosomatid protozoa; Gene amplification; Atomic absorption spectrometry
Two types of proteins belonging to the P-glycoprotein family but differing greatly in sequence and function have been described in the trypanosomatid parasite Leishmania. One includes the MDR1 genes of L. donovani and L. enriettii [1-3]. Overexpression of MDR1 by either transfection or gene amplification confers resistance to hydrophobic drugs including vinblastine and puromycin. This phenotype is similar to that conferred by overexpression of mammalian MDR1 genes, where these P-glycoproteins mediate decreased drug accumulation by increasing ATP-dependent drug efflux [19,20]. Decreased puromycin accumulation was also found in Leishmania overexpressing MDR1, although the relative contribution of efflux and influx could not be determined [1]. Another class of P-glycoproteins does not confer resistance to hydrophobic drugs and is exemplified
* Corresponding author. 1 Present address: Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Division of Experimental Therapeutics, Washington DC 20307, USA.
by PGPA and related genes in L. tarentolae and L. major [4,5]. PGPA was originally detected as a gene located within the prototypic H region amplification [4,6], a 40-kb region encompassing a number of genes including one conferring methotrexate resistance (ltdh or lmhmtxr; this gene has recently been renamed PTR1 [7-9]). The H region amplification occurs frequently in Leishmania selected for resistance to methotrexate, arsenite, primaquine, or terbinafine, or even occasionally in unselected laboratory lines [6,10-17]. Although the presence of multiple gene products on the H region amplification complicates the assignment of drug resistance to individual gene products, transfectional analysis shows that PGPA alone can confer resistance to heavy metals including arsenite and antimonials but not cadmium or zinc [5,17]. PGPA-mediated resistance is not reversed by agents such as verapamil, which are effective against mammalian multidrug or malarial chloroquine resistance [18]. Many workers have suggested that pentavalent antimonial action is mediated through reduction to active SblII, which suggests that PGPA could be involved in the susceptibility of Leishmania to the pentavalent antimonial
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compounds used currently as the primary anti-leishmanial treatment. Moreover, certain PGPA alleles confer resistance to pentavalent antimonials directly [17]. Thus, understanding the mechanism and occurrence of PGPA-mediated antimonial resistance is relevant to current anti-leishmanial chemotherapy. To study antimonial accumulation in PGPA-overexpressing Leishmania, we used the sensitive method of electrothermal atomic absorption spectrophotometry [21]. Elemental Sb is measured after atomization, eliminating the need for expensive custom synthesis of radiolabeled antimony derivatives in different chemical states. Elemental Sb accumulation was measured after pelleting cells through dibutylphthalate as described previously for MTX [23], with Leishmania grown in antimony potassium tartrate (SblII tartrate). It should be noted that the chemical state of the antimony following influx is unknown. We studied three different lines of L. major: the wild-type CC-1 line, and CC-1 transfected either with an empty shuttle vector (pSNAR) or the L. major PGPA gene (pSNAR-H3BAHindIII). The PGPA transfectant shows 12-fold resistance to trivalent antimonials [5]. In most studies we examined antimony accumulation at an external SbIII tartrate concentration of 10 /xg ml-1, about twice that which inhibits wild-type Leishmania growth by 50% [5]. Under these conditions, the PGPA-transfectant accumulated 2 - 4 fold less Sb, during incubations of up to 24 h (Fig. 1 or
not shown). Accumulation was dependent upon the external SbIII tartrate concentration over the range of 10-40 /.tg m1-1, with the PGPA-transfectant accumulating less Sb at all concentrations (not shown). Although modest, the observed 3-fold decrease may account entirely for the 12-fold SblII resistance. For example, strong cadmium resistance in prokaryotes is associated with comparably modest reductions in cadmium accumulation [24-27]. Even when measured at very early time points (5 min), Sb accumulation was reduced 4-fold in the PGPA-transfectants (Fig. 1A). Differences in the linear initial rates of accumulation (up to about 40 min) suggest that PGPA overexpression leads to a reduced influx of antimony. PGPA transfectants accumulated enough Sb to permit measurement of the rate of efflux (Fig. 1). Parasites were incubated 8-14 h in 10 /zg ml -x SblII tartrate, rapidly washed, and placed in drug-free medium. Sb levels in the drug-grown cells were unchanged by washing, and changed very little over the next 10 minutes (not shown). Over a period of 8 h, Sb levels declined similarly in both control and PGPA transfectants. The rates of loss were similar in all lines, with a half-life of 3 h (Fig. 2). Sb levels continued to decline up to 8 h, suggesting that antimony is not irreversibly bound or sequestered within the cell. Thus, the SblII-resistant PGPA transfectants show unaltered, wild-type rates of Sb efflux.
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Fig. 1. Sb accumulation in control and PGPA-transfected L. major. Accumulation was measured following incubation in M199 medium [22] containing 10 /~g ml-1 SbIII tartrate over intervals of 5 - 6 0 min (A) or 1.25-8 h (B). Elemental Sb levels were determined by electrothermal atomic absorption spectrophotometry [21] using SbIII tartrate in aqueous nitric acid as a calibration standard. Data are given as ng Sb/107 cells and standard deviations are shown. Parasite lines were L. major strain CC-1 (m), and CC-1 transfected with pSNAR ( [] ) or pSNAR-H3BAHindIII ( ,x ; PGPA [5]).
H.L. CaUahan et aL / Molecular and Biochemical Parasitology 68 (1994) 145-149 12 ¸
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Fig. 2. Antimony efflux from control and PGPA-transfected L. major. Cells were incubated 14 h in medium containing 10 p,g ml-1 SblII tartrate and rapidly placed into drug-free medium for 0 - 8 h (panel A). Elemental Sb accumulation was normalized to 107 cells/cell pellet, counted at the first data point (T = 0) which was taken immediately prior to resuspension in drug-free medium. The results are expressed as the percent of the initial Sb accumulation to compensate for the different starting levels (Fig. 1). Cell lines and symbols are described in Fig. 1.
At 4°C, Sb accumulation in wild-type Leishmania was reduced to the same levels seen in the PGPA transfectant, which showed no temperature dependence (Fig. 3B). In contrast, effiux in both the wild-type and PGPA transfectant was comparably temperature-dependent, showing approximately a 3fold decrease in rate at 4°C (Fig. 3A). These data
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147
suggest that both SblII tartrate uptake and Sb loss are active processes in wild-type cells. However, PGPA transfectants lack the temperature-dependent uptake seen in wild-type cells, providing further support for the conclusion that influx is specifically altered in the PGPA-transfectants. Previous studies of L. tarentolae transfected with PGPA did reveal reduced accumulation of radiolabeled arsenite [17], although a more complex protocol involving energy starvation and replenishment was used in contrast to the non-perturbative method employed here. However, these lines were only 2-fold resistant to arsenite, and the situation with antimony may differ as they showed 8-fold resistance to SblII. Although decreased accumulation mediated by increased efflux is a hallmark of drug resistance caused by the prototypic P-glycoprotein MDR1, we were unable to detect any changes in temperature-dependent antimony effiux in the PGPA transfectants. Instead, a 3 - 4 fold decrease in SblII tartrate uptake was observed, essentially constituting the entire temperature-dependent component. Thus, PGPA-mediated SblII resistance differs in some fundamental respect from the classic MDR paradigm of increased efflux across the plasma membrane observed in both Leishmania and other organisms. One possible explanation is that PGPA indeed functions as an SblII transporter analogous to drug-exporting MDR P-glycoproteins, but is localized in a functional compart-
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Fig. 3. Temperature Dependence of antimony accumulation and efflux. Sb efflux at 26°C or 4°C was measured as described in Fig. 2 after incubation in 10 p.g m1-1 SbIII tartrate for 14 h at 26°C (panel A). Elemental Sb accumulation was measured as described in Fig. 1 at 4°C and 26°C with an external SbIII tartrate concentration of 10 p~g m1-1 (panel B). Open Symbols, 26°C; filled symbols, 4°C; CC-1 (squares), CC-1 transfected with pSNAR (circles) or the PGPA construct (triangles).
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H.L. Callahan et al. / Molecular and Biochemical Parasitology 68 (1994) 145-149
ment distinct from the general plasma membrane. Another possibility is that PGPA overexpression mediates decreased SblII influx by non-classical mechanisms, such as the original model proposed for P-glycoproteins [28] which invoked general membrane perturbations. This seems unlikely as amplification of the PGPA alleles in lines selected for resistance to organic drugs does not always lead to SblII resistance (unpublished data; [14]), suggesting that specific interactions are required. More likely is the possibility that overexpression of PGPA confers a dominant-negative phenotype through interactions with another Leishmania protein, possibly belonging to the large and diverse P-glycoprotein superfamily. To resolve these questions, it wilt be necessary in the future to determine the cellular localization of the PGPA protein and whether it interacts with other Leishmania proteins. Acknowledgements We thank D.E. Dobson, F.J. Gueiros-Filho and B. Nare for discussions. Supported by a postdoctoral fellowships to HLC (NIH) and WLR (Merck/American Federation for Clinical Research Foundation) and NIH grants to SMB (AI21903) and PMR (AI27382). References [1] Henderson, D.M., Sifri, C.D., Rodgers, M., Wirth, D.F., Hendrickson, N. and Ullman, B. (1992) Multidrug resistance in Leishmania donovani is conferred by amplification of a gene homologous to the mammalian mdrl gene. Mol. Cell. Biol. 12, 2855-2865. [2] Hendrickson, N., Sifri, C.D., Henderson, D.M., Allen, T., Wirth, D.F. and Ullman, B. (1993) Molecular characterization of the ldmdrl multidrug resistance gene from Leishmania donovani. Mol. Biochem. Parasitol. 60, 53-64. [3] Chow, L.M.C., Wong, A.K.C., UUman, B. and Wirth, B.F. (1993) Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii. Mol. Biochem. Parasitol. 60, 195-208. [4] Ouellette, M., Fase-Fowler, F. and Borst, P. (1990) The amplified H circle of methotrexate-resistant Leishmania tarentolae contains a novel P-glycoprotein gene. EMBO J. 9, 1027-1033. [5] Callahan, H.L. and Beverley, S.M. (1991) Heavy metal resistance: a new role for P-glycoproteins in Leishmania. J. Biol. Chem. 266, 18427-18430.
[6] Beverley, S.M., Coderre, J.A., Santi, D.V. and Schimke, R.T. (1984) Unstable DNA amplifications in methotrexateresistant Leishmania consist of extra-chromosomal circles which relocalize during stabilization. Cell 38, 431-439. [7] Callahan, H.L. and Beverley, S.M. (1992) A member of the aldoketo-reductase family confers methotrexate resistance in Leishmania. J. Biol. Chem. 267, 24165-24168. [8] Papadopoulou, B., Roy, G. and Ouellette, M. (1992) A novel antifolate resistance gene on the amplified H circle of Leishmania. EMBO J. 11, 3601-3608. [9] Bello, A., Nare, B., Freedman, D., Hardy, L. and Beverley, S.M. (In Press) PTRI: a new pteridine reductase responsible for methotrexate resistance and pteridine salvage in Leishmania, Proc. Natl. Acad. Sci. USA. [10] White, T.C., Fase-Fowler, F., Luenen, H. va, Calafat, J. and Borst, P. (1988) The H circles of Leishmania tarentolae are a unique amplifiable system of oligomeric DNAs associated with drug resistance. J. Biol. Chem. 263, 16977-16983. [11] Hightower, R.C., Ruiz-Perez, L.M., Wong, M.L. and Santi, D.V. (1988) Extrachromosomal elements in the lower eukaryote Leishmania. J. Biol. Chem. 263, 16970-16976. [12] Petrillo-Peixoto, M.L. and Beverley, S.M. (1988) Amplified DNAs in laboratory stocks of L. tarentolae: extrachromosoreal circles structurally and functionally similar to the inverted H region amplification of methotrexate-resistant L. major. Mol. Cell. Biol. 8, 5188-5199. [13] Detke, S., Katakura, K. and Chang, K.P. (1989) DNA amplification in arsenite-resistant Leishmania. Exp. Cell Res. 180, 161-170. [14] Ellenberger, T.E. and Beverley, S.M. (1989) Multiple drug resistance and conservative amplification of the H region in Leishmania major. J. Biol. Chem. 264, 15094-15103. [15] Ouellette, M., Hettema, E., Wust, D., Fase-Fowler, F. and Borst, P. (1991) Direct and inverted DNA repeats associated with P-glycoprotein gene amplification in drug resistant Leishmania. EMBO J. 10, 1009-1016. [16] Papadopoulou, B., Roy, G. and Ouellette, M. (1993) Frequent amplification of a short chain dehydrogenase gene as part of circular and linear amplicons in methotrexate-resistant Leishmania. Nucleic Acids Res. 21, 4305-4312. [17] Papadopoulou, B., Roy, G., Dey, S., Rosen, B.P. and Oucllette, M. (1994) Contribution of the Leishmania P-glycoprotein-related gene ltpgpA to oxyanion resistance. J. Biol. Chem. 269, 11980-11986. [18] Martin, S.K., Oduola, A.M.J. and Milhous, W.K. (1987) Reversal of chloroquine resistance in Plasmodium falciparum by verapamil. Science 235, 899-901. [19] Gottesman, M.M. and Pastan, I. (1993) Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu. Rev. Biochem. 62, 385-427. [20] Endicott, J.A. and Ling, V. (1989) The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu. Rev. Biochem. 58, 137-171. [21] Roberts, W.L. and Rainey, P.M. (1993) Antimony quantification in Leishmania by electrothermal atomic absorption spectroscopy. Anal. Biochem. 211, 1-6.
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[22] Kapler, G.M., Coburn, C.M. and Beverley, S.M. (1990) Stable transfection of the human parasite Leishmania delineates a 30 kb region sufficient for extra-chromosomal replication and expression. Mol. Cell. Biol. 10, 1084-1094. [23] Ellenberger, T.E. and Beverley, S.M. (1987) Biochemistry and regulation of folate and methotrexate transport in Leishmania major. J. Biol. Chem. 262, 10053-10058. [24] Tynecka, Z., Zajac, J. and Gos, Z. (1975) Plasmid dependent impermeability barrier to cadmium ions in Staphylococcus aureus. Acta Microbiol. Pol. 7, 11-20. [25] Tynecka, Z., Gos, Z. and Zajac, J. (1981) Energy-dependent efflux of cadmium coded by a plasmid resistance determinant in Staphylococcus aureus. J. Bacteriol. 147, 313-319.
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