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 theCandida ERG6 gene revealed high homology with theSaccharomyces 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 ERG6gene 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.

Steroid-transforming enzymes in fungi

“…ERG6 have also been identified at the gene level in C. albicans, C. neoformans and Pnumocystis carinii [55,57]. Sequencing of the Candida erg6 gene has revealed high homology with the Saccharomyces version of ERG6 [55].…”

“…Sequencing of the Candida erg6 gene has revealed high homology with the Saccharomyces version of ERG6 [55]. Recently, ERG6 was purified from C. albicans: it has a monomer mass of 43 kDa and an apparent mass of 178 kDa as a native enzyme, and it converts zymosterol to fecosterol with a K M of 50 M and a k cat of 0.01 s −1 [58].…”

“…Although the absence of the erg6 gene product is not lethal, it has been shown to result in several severely compromised phenotypes [55]. All of the ERG6 have four regions that show particular similarities, and which are referred to as: the sterol-binding segment region I; the S-adenosylmethionine binding segment II; the sterol-binding segment III; and the sterol-binding segment IV [56][57][58].…”

Cloning and sequencing of the Candida albicans C‐4 sterol methyl oxidase gene (ERG25) and expression of an ERG25 conditional lethal mutation in Saccharomyces cerevisiae

“…Although the ERG6 gene is not essential in S. cerevisiae, its disruption results in a cell that is severely compromised in several functions associated with membrane permeability (5,6). The disruption of both copies of the ERG6 gene in C. albicans likewise does not render the cell inviable but does make it hypersusceptible to a number of antifungal compounds and cellular inhibitors (4).…”

“…The complementation of S. cerevisiae sterol mutants with a C. albicans genomic library to isolate sterol genes in C. albicans is a first step in verifying gene essentiality in this organism and the suitability of that step for seeking inhibitors that might serve as antifungal agents. This approach has recently been used to clone and disrupt the ERG6 gene of C. albicans (4). Although the ERG6 gene is not essential in S. cerevisiae, its disruption results in a cell that is severely compromised in several functions associated with membrane permeability (5,6).…”