Resistance to pentamidine in Leishmania mexicana involves exclusion of the drug from the mitochondrion

Abstract

The uptake of [(3)H]pentamidine into wild-type and drug-resistant strains of Leishmania mexicana was compared. Uptake was carrier mediated. Pentamidine-resistant parasites showed cross-resistance to other toxic diamidine derivatives. A substantial decrease in accumulation of the drug accompanied the resistance phenotype, although the apparent affinity for pentamidine by its carrier was not altered when initial uptake velocity was measured. The apparent V(max), however, was reduced. An efflux of pentamidine could be measured in both wild-type and resistant cells. Only a relatively small proportion of the total accumulated pentamidine was available for efflux in wild-type cells, while in resistant cells the majority of loaded pentamidine was available for release. Pharmacological reagents which diminish the mitochondrial membrane potential reduced pentamidine uptake in wild-type parasites, and the mitochondrial membrane potential was shown to be reduced in resistant cells. A fluorescent analogue of pentamidine, 4',6'-diamidino-2-phenylindole, accumulated in the kinetoplast of wild-type but not resistant parasites. These data together indicate that diamidine drugs accumulate in the Leishmania mitochondrion and that the development of the resistance phenotype is accompanied by lack of mitochondrial accumulation of the drug and its exclusion from the parasites.

FIG. 1.

Time course of pentamidine uptake by wild-type and pentamidine-resistant L. mexicana promastigotes. Parasites were incubated with 0.25 μM pentamidine at 25°C, and pentamidine uptake was measured between 5 s and 3 h. Wild-type (solid circles) and resistant Pent^r2.5 (solid squares), Pent^r5 (open circles), Pent^r10 (open triangles), and Pent^r30 (open square) strains were tested. Results are the mean ± standard error of the mean of at least three experiments.

FIG. 2.

Efflux of pentamidine by L. mexicana promastigotes. Wild-type and pentamidine-resistant parasites were incubated with 0.25 μM pentamidine at 25°C (A) or on ice (B) for 10 min, centrifuged, washed three times with PBSG, and resuspended in drug-free PBSG. Retained pentamidine was measured between 1 and 60 min. Results are the mean ± standard error of the mean of at least three experiments.

FIG. 3.

Pentamidine efflux by pentamidine-resistant promastigotes under energy- and proton-depleting conditions. Retained pentamidine was measured after 30 min in the absence (control) and presence of inhibitors: 1 mM dinitrophenol (DNP), 10 μM carbonyl cyanide (CCCP), 50 μM N,N′-dicyclohexylcarbodiimide (DCCD), and 50 μM verapamil. Results are the mean ± standard error of the mean of at least three experiments.

FIG. 4.

Inhibition effects of different multidrug resistance-associated protein inhibitors on pentamidine efflux by pentamidine-resistant promastigotes. Time course of pentamidine efflux was measured in the presence and absence of 20 μM PCP and 20 μM TFP and after 24 h of treatment with 1 mM BSO. Results are the mean ± standard error of the mean of at least four experiments.

FIG. 5.

Effect of multidrug resistance-associated protein inhibitors on time course of pentamidine accumulation by wild-type and pentamidine-resistant promastigotes. The effect of 10 μM PCP and 10 μM TFP in sensitive wild-type cells and 20 μM PCP and 20 μM TFP in resistant cells on pentamidine influx was studied. The wild type (shaded circles), wild type plus 10 μM PCP (solid circles), wild type plus 10 μM TFP (open circles), resistant line Pent^r30 (shaded squares), resistant line Pent^r30 plus 20 μM PCP (solid squares), and resistant line Pent^r30 plus 20 μM TFP (open squares) were tested. Results are means of at least three experiments.

FIG. 6.

Flow cytometry analysis of wild-type and pentamidine-resistant L. mexicana promastigotes. Parasite fluorescence was measured immediately (black line) or 30 min (irregular dotted line) after addition of rhodamine-123. The level of fluorescence of the parasites in the absence of rhodamine-123 is represented for comparison (regular dotted line). Data from one representative experiment are presented.