Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate

Abstract

The pathogenic yeast, Candida albicans, is insensitive to the anti-mitotic drug, benomyl, and to the dihydrofolate reductase inhibitor, methotrexate. Genes responsible for the intrinsic drug resistance were sought by transforming Saccharomyces cerevisiae, a yeast sensitive to both drugs, with genomic C. albicans libraries and screening on benomyl or methotrexate. Restriction analysis of plasmids isolated from benomyl- and methotrexate-resistant colonies indicated that both phenotypes were encoded by the same DNA fragment. Sequence analysis showed that the fragments were nearly identical and contained a long open reading frame of 1694 bp (ORF1) and a small ORF of 446 bp (ORF2) within ORF1 on the opposite strand. By site-directed mutagenesis, it was shown that ORF1 encoded both phenotypes. The protein had no sequence similarity to any known proteins, including beta-tubulin, dihydrofolate reductase, and the P-glycoprotein of the multi-drug resistance family. The resistance gene was detected in several C. albicans strains and in C. stellatoidea by DNA hybridization and by the polymerase chain reaction.

Transcriptional Regulation of MDR1 , Encoding a Drug Efflux Determinant, in Fluconazole-Resistant Candida albicans Strains through an Mcm1p Binding Site

“…In some strains of C. albicans, e.g., clinical strain B792, this sequence is identical to the consensus Ste12p binding site (16). These observations suggest that C. albicans Cph1p, the Ste12p homolog, participates in regulating the expression of MDR1.…”

Activation of the Multiple Drug Resistance Gene MDR1 in Fluconazole-Resistant, Clinical Candida albicans Strains Is Caused by Mutations in a trans -Regulatory Factor

“…The MDR1 promoter regions from the clinical C. albicans isolates were amplified with the primers MDR1p1, 5Ј-CGATAAATGATAAG TCACTCTACC-3Ј (positions 57 to 34 within the coding region), and MDR1p2, 5Ј-CAACTCTACTGGTAACTATTGGCG-3Ј (positions Ϫ561 to Ϫ538 with respect to the start codon), deduced from the published sequence of the MDR1 gene (6). The PCR products were phosphorylated and cloned into the SmaI site of the vector pUC18.…”

Citridones, New Potentiators of Antifungal Miconazole Activity, Produced by Penicillium sp. FKI-1938

“…Recently, resistance to azole antifungals became a significant problem. Several resistant mechanisms have been proposed; 1) overexpression of transporters like ATP binding cassette (ABC) transporter [2], 2) mutation of cytochrome P-450 14-a demethylase (P-450 14DM) [3], 3) overexpression of P450 14DM [4], and so on. Therefore, the resistant mechanisms will be a new target of inhibition for overcoming infections by azole-resistant C. albicans.…”