Sequencing, disruption, and characterization of the Candida albicans sterol methyltransferase (ERG6) gene: drug susceptibility studies in erg6 mutants

Abstract

The rise in the frequency of fungal infections and the increased resistance noted to the widely employed azole antifungals make the development of new antifungals imperative for human health. The sterol biosynthetic pathway has been exploited for the development of several antifungal agents (allylamines, morpholines, azoles), but additional potential sites for antifungal agent development are yet to be fully investigated. The sterol methyltransferase gene (ERG6) catalyzes a biosynthetic step not found in humans and has been shown to result in several compromised phenotypes, most notably markedly increased permeability, when disrupted in Saccharomyces cerevisiae. The Candida albicans ERG6 gene was isolated by complementation of a S. cerevisiae erg6 mutant by using a C. albicans genomic library. Sequencing of the Candida ERG6 gene revealed high homology with the Saccharomyces version of ERG6. The first copy of the Candida ERG6 gene was disrupted by transforming with the URA3 blaster system, and the second copy was disrupted by both URA3 blaster transformation and mitotic recombination. The resulting erg6 strains were shown to be hypersusceptible to a number of sterol synthesis and metabolic inhibitors, including terbinafine, tridemorph, fenpropiomorph, fluphenazine, cycloheximide, cerulenin, and brefeldin A. No increase in susceptibility to azoles was noted. Inhibitors of the ERG6 gene product would make the cell increasingly susceptible to antifungal agents as well as to new agents which normally would be excluded and would allow for clinical treatment at lower dosages. In addition, the availability of ERG6 would allow for its use as a screen for new antifungals targeted specifically to the sterol methyltransferase.

FIG. 1

UV scan of nonsaponifiable sterols in which erg6 sterols containing a conjugated double bond in the sterol side chain show absorption maxima at 230 and 238 nm. Wild-type erg6 transformants containing the Candida ERG6 gene do not have the conjugated double-bond system in the sterol side chain.

FIG. 2

A C. albicans ERG6 genomic clone (pCERG6-20) with restriction sites and three complementing subclones, pIU880, pIU882, and pIU885. Deletion of a 0.7-kb HindIII fragment within pIU885, filling in of cohesive ends, addition of BamHI linkers (pIU886-L), and subsequent insertion of the URA3 blaster into this site as shown (pIU887-A) are represented.

FIG. 3

The DNA and amino acid sequences of the C. albicans ERG6 gene. The S-adenosylmethionine binding site is indicated by underlining.

FIG. 4

Alignment of the amino acid sequences of the sterol methyltransferases from C. albicans, S. cerevisiae, A. thaliana, and T. ativum. Shaded areas indicate regions of sequence identity.

FIG. 5

GC of the sterols of the wild type and an erg6 strain of C. albicans. Peak A, zymosterol; peak B, ergosterol; peak C, fecosterol; peak D, cholesta-5,7,24-trien-3β-ol; peak E, cholesta-7,24-dien-3β-ol; peak F, cholesta-5,7,22,24-tetraen-3β-ol.

FIG. 6

(A) URA3 blaster disruption of the ERG gene showing location of PCR primers; (B) agarose gel electrophoresis confirmation of heterozygote and homozygote disruptants of the ERG6 gene. Lanes (left to right): 1 and 2, CAI4 (wild type); 3 and 4, CA14-6-5 (heterozygote); 5 and 6, 5AB-15 (homozygote derived from URA3 blaster transformation followed by mitotic recombination); 7 and 8, HO11-A3 (homozygote derived from two rounds of URA3 blaster transformation). The PCR primer pairs used are indicated at the tops of the lanes (e.g., 1-2 is P1-P2). The image was captured on disc and the photograph was generated by using Photoshop on Macintosh.

FIG. 6

(A) URA3 blaster disruption of the ERG gene showing location of PCR primers; (B) agarose gel electrophoresis confirmation of heterozygote and homozygote disruptants of the ERG6 gene. Lanes (left to right): 1 and 2, CAI4 (wild type); 3 and 4, CA14-6-5 (heterozygote); 5 and 6, 5AB-15 (homozygote derived from URA3 blaster transformation followed by mitotic recombination); 7 and 8, HO11-A3 (homozygote derived from two rounds of URA3 blaster transformation). The PCR primer pairs used are indicated at the tops of the lanes (e.g., 1-2 is P1-P2). The image was captured on disc and the photograph was generated by using Photoshop on Macintosh.

FIG. 7

Growth responses of the wild type (CAI4), a homozygous erg6 strain derived from URA3 blaster transformation (5AB-15), and a homozygous erg6 strain derived from mitotic recombination (HO11-A3) in the presence of sterol biosynthesis inhibitors and metabolic inhibitors. Cells were grown at 37°C to a density of 10⁷ cells/ml, and 5 μl was inoculated at 10⁰, 10⁻¹, and 10⁻² dilutions. The image was captured on disc and the photograph was generated by using Photoshop on Macintosh.