Isolation of Genes Mediating Resistance to Inhibitors of Nucleoside and Ergosterol Metabolism in Leishmania by Overexpression/Selection

Abstract

We tested a general method for the identification of drug resistance loci in the trypanosomatid protozoan parasite Leishmania major. Genomic libraries in a multicopy episomal cosmid vector were transfected into susceptible parasites, and drug selections of these transfectant libraries yielded parasites bearing cosmids mediating resistance. Tests with two antifolates led to the recovery of cosmids encoding DHFR-TS or PTR1, two known resistance genes. Overexpression/selection using the toxic nucleoside tubercidin similarly yielded the TOR (toxic nucleoside resistance) locus, as well as a new locus (TUB2) conferring collateral hypersensitivity to allopurinol. Leishmania synthesize ergosterol rather than cholesterol, making this pathway attractive as a chemotherapeutic target. Overexpression/selection using the sterol synthesis inhibitors terbinafine (TBF, targeting squalene epoxidase) and itraconazole (ITZ, targeting lanosterol C 14 -demethylase) yielded nine new resistance loci. Several conferred resistance to both drugs; several were drug-specific, and two TBF-resistant cosmids induced hypersensitivity to ITZ. One TBFresistant cosmid encoded squalene synthase (SQS1), which is located upstream of the sites of TBF and ITZ action in the ergosterol biosynthetic pathway. This suggests that resistance to "downstream" inhibitors can be mediated by increased expression of ergosterol biosynthetic intermediates. Our studies establish the feasibility of overexpression/selection in parasites and suggest that many Leishmania drug resistance loci are amenable to identification in this manner.

Functional genetic identification of PRP1, an ABC transporter superfamily member conferring pentamidine resistance in Leishmania major

“…Promastigotes were grown in M199 medium supplemented as described [11,22]. Parasites from late log phase cultures were transfected by electroporation (500 F, 2.25 kV/cm) using 20-40 g of cosmid DNA.…”

“…Parasites from late log phase cultures were transfected by electroporation (500 F, 2.25 kV/cm) using 20-40 g of cosmid DNA. Typically, the PEN IC 50 for inhibition of LmFA1 cells was 0.65 g/ml in liquid media, however, higher concentrations were used in PEN selections as described below and previously [11,23]. Parasite cell determinations, IC 50 calculations, and statistical tests for PEN r were carried out as described [11].…”

“…In this work we began with two transfected L. major cell populations, each bearing a separate cosmid from an L. major library constructed in the shuttle vector cLHYG [11,24]. Library or control cultures were plated on M199 semisolid medium containing increasing concentrations of PEN, 4.8-15.6 g/ml of PEN, as described [11].…”

“…Library or control cultures were plated on M199 semisolid medium containing increasing concentrations of PEN, 4.8-15.6 g/ml of PEN, as described [11]. Cosmid DNA from the primary PEN-resistant transfectants was recovered by transformation of Escherichia coli DH5␣ strain and analyzed by restriction enzyme digestion [24].…”

“…In contrast, a stepwise-selected PEN-resistant L. major line (PT-R20) did not show H region or other amplifications [8]. Recently, we described a method exploiting libraries of Leishmania transfected with episomal vectors bearing random DNA fragments for the identification of genes whose overexpression leads to drug resistance in Leishmania [11]. In this work it has been applied to the identification of loci implicated in PEN r .…”