Mapping of a Leishmania major gene/locus that confers pentamidine resistance by deletion and insertion of transposable element

Abstract

Pentamidine (PEN) is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets of partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK) into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.

High Resolution Melting Analysis in Leishmania Resistance: The Role of Multi Drug Resistance 1 Gene

“…MDR1 gene is 37% similar to human MDR gene and is located on chromosome 34. P-gp is an ATP-dependent drug e ux pump, which can reduce the drug's accumulation in resistant cells and often increase drug resistance (16). According to the importance of drug resistance in patients, modern molecular techniques can provide an opportunity for disease control and treatment for rapid identi cation of drug resistance.…”

The genetic toolbox forLeishmaniaparasites

“…8,[61][62][63][64] This system can potentially be used to study the regulation of gene expression and the localization of proteins, and may facilitate whole proteogenomic approaches. 8,65,66 Summary A substantial number of genetic tools are available to manipulate Leishmania, making this parasite an ideal model organism. Foremost, the ability to generate gene deletion mutants has facilitated an understanding of the function and importance of a number of targeted genes.…”

Antileishmanial activity of polycyclic derivatives

“…However, pentamidine and amphotericin B have shown a high toxicity at the effective therapeutic doses (Maddux & Barriere, 1980;Soto-Mancipe et al, 1994). Resistance to treatment with antimonybased agents occurs frequently, and resistance to other drugs like pentamidine or amphotericin B was mentioned (Sereno et al, 1997;Durand et al, 1998;Lira et al, 1999;Thakur et al, 2001;Basselin et al, 2002;Khan et al, 2002;Coelho et al, 2004). So, no drug is totally active and safe.…”