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

• Published in: The Journal of biological chemistry 1999-12, Vol.274 (53), p.37723-37730
• Main Authors: Cotrim, P C, Garrity, L K, Beverley, S M
• Format: Article
• Language: English
• Subjects: allopurinol; Amino Acid Sequence; Animals; antifolate; Base Sequence; DHFR-TS gene; DNA Primers; Drug Resistance - genetics; ergosterol; Ergosterol - antagonists & inhibitors; Ergosterol - metabolism; Farnesyl-Diphosphate Farnesyltransferase - chemistry; Farnesyl-Diphosphate Farnesyltransferase - genetics; Humans; lanosterol C sub-demethylase; lanosterol C14-demethylase; Leishmania major; Leishmania major - drug effects; Leishmania major - enzymology; Leishmania major - genetics; Molecular Sequence Data; Ptr1 gene; Selection, Genetic; Sequence Homology, Amino Acid; squalene synthase; TOR gene; TUB2 gene; tubercidin; Tubercidin - antagonists & inhibitors; Tubercidin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.53.37723

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 TBF-resistant 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.