Mechanisms of drug-resistance and analysis of drug-resistant genes in Leishmania

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A protozoan parasite, Leishmania, contains several P-glycoprotein genes as a multigene family. Two types of the gene (Ldmdrl and LtpgpA) have been isolated from different Leishmania species, and found to play a role of drug-resistance. In this study, we aimed to isolate and characterize P-glycoprotein genes in L.amazonensis, one of the causative agents of cutaneous leishmaniasis in the New World. Polymerase chain reaction (PCR) was performed to amplify ATP binding sites within P-glycoproteins of L.amazonensis by using primers specific to conserved sequences in mammalian and leishmanial P-glycoprotein genes. We detected a 408-bp PCR product which showed 68% and 37% identity to Ldmdrl and LtpgpA,respectively, at the amino acid level, suggesting that this DNA fragment is in part of new type of leishmanial P-glycoprotein gene. To isolate the putative gene in whole, a genomic library of L.amazonensis DNA was constructed into the lambda-GEM-11 vector and screened with the PCR product. One of the positive lambda clones was digested with Sac I restriction endonuclease, and resultant DNA fragments were subcloned into pUC19 vector. At present, nucleotide sequencing of a 1005-bp Sac I fragment was completed, and the rest of the fragments is ready to be sequenced. In addition, pulsed field gel electrophoresis and Southern hybridization analysis strongly suggest that the L.amazonensis contains at least three different types of P-glycoprotein gene, the Ldmdrl homologue, LtpgpA homologue and new type gene found in this study, but that chromosomal location of the LtpgpA is different from those of the other two genes.