Functional genetic identification of PRP1, an ABC transporter superfamily member conferring pentamidine resistance in Leishmania major.
Pentamidine (PEN) is a second-line agent in the treatment of leishmaniasis whose mode of action and resistance is not well understood. Here, we used a genetic strategy to search for loci able to mediate PEN resistance (PENr) when overexpressed in Leishmania major. A shuttle cosmid library containing genomic DNA inserts was transfected into wild-type promastigotes and screened for PEN-resistant transfectants. Two different cosmids identifying the same locus were found, which differed from other known Leishmania drug resistance genes. The PENr gene was mapped by deletion and transposon mutagenesis to an open reading frame (ORF) belonging to the P-glycoprotein (PGP)/MRP ATP-binding cassette (ABC) transporter superfamily that we named pentamidine resistance protein 1 (PRP1). The predicted PRP1 protein encodes 1,807 amino acids with the typical dimeric structure involving 10 transmembrane domains and two nucleotide-binding domains (NBDs). PRP1-mediated PENr could be reversed by verapamil and PRP1 overexpressors showed cross-resistance to trivalent antimony but not to pentavalent antimony (glucantime). Although the degree of PENr was modest (1.7- to 3.7-fold), this may be significant in clinical drug resistance given the marginal efficacy of PEN against Leishmania.
Coelho
Adriano C
AC
Instituto de Medicina Tropical, São Paulo University Medical School, Av. Dr. Enéas Carvalho Aguiar, 470; 4o andar, São Paulo-SP, 05403-900, Brazil.
Beverley
Stephen M
SM
Cotrim
Paulo C
PC
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000965:Antimony / Q000494:pharmacology; D000981:Antiprotozoal Agents; D004251:DNA Transposable Elements; D004351:Drug Resistance / Q000235:genetics*; D017353:Gene Deletion; D017125:Genes, Protozoan; D015698:Genomic Library; D018320:Leishmania major / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010419:Pentamidine / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015183:Restriction Mapping; D014170:Transformation, Genetic; D014700:Verapamil / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D000981:Antiprotozoal Agents; D004251:DNA Transposable Elements; D015800:Protozoan Proteins; C480121:pentamidine resistance protein 1, Leishmania major; D010419:Pentamidine; D000965:Antimony; D014700:Verapamil