Fluconazole Treatment Is Effective against a Candida albicans erg3/erg3 Mutant In Vivo Despite In Vitro Resistance

Abstract

Candida albicans ERG3 encodes a sterol C5,6-desaturase which is essential for synthesis of ergosterol. Defective sterol C5,6 desaturation has been considered to be one of the azole resistance mechanisms in this species. However, the clinical relevance of this resistance mechanism is still unclear. In this study, we created a C. albicans erg3/erg3 mutant by the "Ura-blaster" method and confirmed the expected azole resistance using standard in vitro testing and the presence of ergosta-7,22-dien-3␤-ol instead of ergosterol. For in vivo studies, a wild-type URA3 was placed back into its native locus in the erg3 homozygote to avoid positional effects on URA3 expression. Defective hyphal formation of the erg3 homozygote was observed not only in vitro but in kidney tissues. A marked attenuation of virulence was shown by the longer survival and the lower kidney burdens of mice inoculated with the reconstituted Ura ؉ erg3 homozygote relative to the control. To assess fluconazole efficacy in a murine model of disseminated candidiasis, inoculum sizes of the control and the erg3 homozygote were chosen which provided a similar organ burden. Under these conditions, fluconazole was highly effective in reducing the organ burden in both groups. This study demonstrates that an ERG3 mutation causing inactivation of sterol C5,6-desaturase cannot confer fluconazole resistance in vivo by itself regardless of resistance measured by standard in vitro testing. The finding questions the clinical significance of this resistance mechanism.Candida albicans is the most common cause of deep mycoses in humans. Azole therapy has been well tolerated and effective for many forms of candidiasis. Of concern is that long-term azole treatment of oropharyngeal candidiasis in human immunodeficiency virus-infected patients has encountered progressive azole resistance (14,40). Azole antifungals inhibit the biosynthesis of ergosterol, the major sterol of cell membrane, by targeting lanosterol 14␣-demethylase encoded by ERG11 (38). Alteration of amino acid composition of lanosterol 14␣-demethylase (37), increased drug efflux (32, 34), and altered ergosterol synthetic pathways due to blockage of sterol C5,6-desaturase encoded by ERG3 (17, 33) have been known as factors contributing to azole resistance in C. albicans and Saccharomyces cerevisiae (for reviews, see references 1 and 35). However, no relation between defective sterol C5,6-desaturase and azole resistance was found in Candida glabrata (9). The relevance of azole resistance in C. albicans erg3 mutants is still unclear. Although it has been reported that a few azole-resistant clinical isolates of C. albicans exhibited a sterol profile indicative of defective sterol C5,6 desaturation (4, 18, 26), the possibility remains that another mechanism(s) of azole resistance might have been present in those isolates. For instance, the Darlington strain, an erg3/erg3 mutant isolated from the oral cavity, was also azole resistant due to mutations in ERG11 (15,24).A mechanism by which erg3 mutations cause azole r...

Pathway analysis of Candida albicans survival and virulence determinants in a murine infection model

“…Further, the pTET-ERG2 and pTET-ERG6 conditional mutants demonstrated similar avirulent phenotypes under the dox þ 2D conditions. These results corroborate the previous finding that ERG3 is required for virulence (24,25) and expands this conclusion to additional late stage ergosterol biosynthetic enzymes even though they are dispensable for growth under in vitro conditions.…”

Stress, Drugs, and Evolution: the Role of Cellular Signaling in Fungal Drug Resistance

“…2C), which blocks the accumulation of toxic sterol intermediates when Erg11 is inhibited by the azoles (3,32). While this mechanism of resistance has been identified in clinical isolates (87), the clinical relevance is less clear (135). Some resistance mechanisms may span both categories, with dual roles in minimizing the impact of the drug on the cell and enabling the cell to cope with stress.…”

Azole Resistance by Loss of Function of the Sterol Δ 5,6 -Desaturase Gene ( ERG3 ) in Candida albicans Does Not Necessarily Decrease Virulence

“…Besides these clinical isolates, a few laboratory-generated C. albicans erg3 mutants have been reported, either constructed through targeted deletion of both ERG3 alleles (25,38) or, in one other case, obtained from serial cultures of a wild-type strain in the presence of fluconazole (46). Typically, these erg3 mutants have lost the ability to produce hyphae.…”

“…However, a report from Martel et al described the first two filament-forming clinical erg3 mutants ever discovered (23). Perhaps as a consequence of being locked in the yeast form, all isolates tested so far (not including the recent filament-forming mutants), irrespective of their clinical or in vitro origin, showed attenuated virulence in animal models of disseminated candidiasis (5,25,29). Moreover, it has been suggested that despite the typical high-level in vitro resistance, ERG3 inactivity alone does not lead to fluconazole resistance in vivo (25).…”