Role of the ABC transporter PRP1 (ABCC7) in pentamidine resistance in Leishmania amastigotes

Abstract

Pentamidine is a second-line agent in the treatment of leishmaniasis whose mode of action and resistance mechanism are not well understood. In this work, we show that the intracellular ABC protein PRP1 (pentamidine resistance protein 1) (ABCC7) can confer resistance to pentamidine in Leishmania sp. parasites in the intracellular stage.

FIG. 1.

PRP1 confers pentamidine resistance in Leishmania infantum (Li) axenic amastigotes. A growth curve is shown for L. infantum axenic amastigotes in MAA/20 medium (24) in the presence of pentamidine. Promastigote parasites were transfected with the plasmids indicated (the control plasmid, pSNBR [22]; PRP1 [pSNBR/8kb SmaI-A] [3]; and PRP1-GFP [4]) and induced for differentiation to axenic amastigotes (24). Experiments were done in triplicate.

FIG. 2.

Cellular localization of the construct PRP1-GFP. The fusion protein was studied by fluorescence microscopy with L. infantum axenic amastigotes. Transfectant parasites were immobilized in 1% (wt/vol) low-melting-point agarose in phosphate-buffered saline and observed with a Nikon Eclipse TE300 microscope (100× objective) using appropriate GFP excitation/emission filters (HYQ filter cube; Nikon). A, phase contrast; B, GFP fluorescence; C, panels A and B merged.

FIG. 3.

PRP1 mediates pentamidine resistance in intracellular Leishmania amastigotes. Luciferase-expressing amastigotes of Leishmania spp. were grown in a human leukemia monocyte cell line (THP-1 cells) as described previously (26). THP-1 cells were infected with stationary-phase L. amazonensis (La) and L. major (Lm) promastigotes in 24-microwell plates at a parasite/macrophage ratio of 15:1. Noninternalized parasites were removed by three washes, and pentamidine was added to the infected macrophages. After 4 days of infection with the drug, cells were washed and the luciferase activities of the luciferase gene-expressing recombinant parasites were determined as described elsewhere (21). Promastigote parasites were cotransfected with the plasmid pSNBR (22) or PRP1 (pSNBR/8kb SmaI-A) (3) and with the vector pSP1.2LUCαHYGα (27) to quantify intracellular parasites (21, 26). Experiments were done at least three times in triplicate. Values are represented as numbers of relative light units (RLU).