Candida glabrata erg1 Mutant with Increased Sensitivity to Azoles and to Low Oxygen Tension

Abstract

A Candida glabrata erg1 (Cgerg1) mutant, CgTn201S, was identified by transposon mutagenesis and by increased fluconazole susceptibility. CgERG1 encodes a 489-amino-acid protein which, on the basis of its homology with Saccharomyces cerevisiae ERG1, is a squalene epoxidase essential for ergosterol synthesis. Interruption following codon 475 of CgErg1p decreased the ergosterol content by 50%; caused accumulation of the squalene precursor; increased the levels of susceptibility to fluconazole, itraconazole, and terbinafine; increased the level of resistance to amphotericin B; increased the levels of rhodamine 6G and [ 3 H]-fluconazole uptake; reduced the level of growth; and blocked growth under conditions of low oxygen tension. In addition, CgTn201S efficiently took up exogenous cholesterol from cholesterol-containing serum. Cholesterol constituted 34% of the extractable sterols in CgTn201S when it was grown aerobically on serum-containing medium. Under the same conditions, C. albicans contained only 0.1 to 1.2% cholesterol. Exogenous sterols also restored growth under conditions of low oxygen tension. Finally, complementation of the Cgerg1 mutation restored the levels of [ 3 H]fluconazole uptake and drug susceptibility to wild-type levels.Transposon-based insertion into Candida glabrata has been used effectively for mutational analysis (3). We adapted a commercial Tn5-based system for insertion of Tn5 into C. glabrata genomic DNA. After rescue cloning of the Tn5 DNA from the C. glabrata transformants with increased fluconazole susceptibilities, we found that one of the fluconazole-susceptible transformants, CgTn201S, had an insertion in the 3Ј end of the C. glabrata ERG1 (CgERG1) open reading frame (ORF). We explored the mechanism by which this mutation causes fluconazole susceptibility because of our interest in azole resistance in this species. Prolonged treatment of patients with fluconazole causes substantial increases in the levels of azole resistance in C. glabrata. This species is naturally about 8-fold more resistant to fluconazole than Candida albicans, and following drug exposure, the levels of resistance can increase 16-fold more, leading to clinically significant fluconazole resistance (1, 18). The major mechanisms for clinical resistance described to date have been increased drug efflux and alteration of the azole-binding site, the C-14 sterol demethylase, coded for by ERG11. Recently, an erg1 C1228G Saccharomyces cerevisiae mutant was reported to be aerobically viable and to have at least a 10-fold increase in the level of terbinafine resistance but no change in the level of itraconazole resistance (9). Null erg1 mutants of S. cerevisiae are not aerobically viable but are anaerobically viable in the presence of exogenous ergosterol (11). In the absence of data about Cgerg1 mutants, we studied the phenotype of strain CgTn201S, including its azole susceptibility, its ability to grow under conditions of low oxygen tension, and its uptake of exogenous sterols. MATERIALS AND METHODSStrains and culture co...

Characterization of Squalene Epoxidase ofSaccharomyces cerevisiaeby Applying Terbinafine-Sensitive Variants

“…These results compare well to those obtained for the TERBINAFINE-SENSITIVE SQUALENE EPOXIDASE 281 recently described mutant carrying the erg1-1 allele, which encodes an SE variant with reduced enzymatic activity and which confers terbinafine sensitivity (8). In addition, the decrease in ergosterol content caused by the interruption of the Candida glabrata ERG1 gene also increased the sensitivity of this organism to terbinafine and azoles (35). Similar effects were observed in Candida albicans when ergosterol levels were reduced by the limited expression of SE (22), but this mutant also became more susceptible to other inhibitors not involved in sterol synthesis due to reduced drug efflux activity.…”

Comparison of Sterol Import under Aerobic and Anaerobic Conditions in Three Fungal Species, Candida albicans, Candida glabrata, and Saccharomyces cerevisiae

“…Previous work suggests that C. albicans, unlike the two species mentioned above, depends solely on the synthesis of endogenous ergosterol. No mechanism of sterol uptake was identified in previous work (27,31,32). Unlike S. cerevisiae and C. glabrata, there is lack of response to exogenous sterols by C. albicans wild-type, CaHEM1-null mutant, or sterol auxotrophs (27).…”

Candida glabrata PDR1 , a Transcriptional Regulator of a Pleiotropic Drug Resistance Network, Mediates Azole Resistance in Clinical Isolates and Petite Mutants

“…A fosmid genomic library of Cg13928, a fluconazole-resistant clinical isolate, was constructed in copy control fosmid vector pCC1URA3 as described in Tsai et al (36). The average insert size of the genomic library was about 40 kb.…”

“…The Cgpdr1 mutant, CgTn173S, was generated by transposon mutagenesis using the custom transposon Tn5ϽCm URA3Ͼ, as described by Tsai et al (36). The ura3 mutants were selected on a MIN plus uracil agar plate containing 0.1% 5-fluoroorotic acid (FOA) (Lancaster, Pelham, NH).…”

“…We studied Cgpdr1 mutants obtained by transposon mutagenesis (36) and by targeted gene disruption, including Cgpdr1 mutants in a petite background. We also sequenced CgPDR1 and its 5Ј flanking region in clinical isolates obtained before and after the appearance of fluconazole resistance (2).…”