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-3beta-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.

FIG. 1.

Fluconazole susceptibility of a C. albicans erg3 homozygote at 24 and 48 h. Fluconazole susceptibility of CAF2-1 (ERG3/ERG3 ura3/URA3) and CAE3DU3 (erg3/erg3 ura3/URA3) was examined by broth microdilution tests following the NCCLS M27-A2 protocol (25). The optical density at 600 nm (OD₆₀₀) was measured at 24 and 48 h of incubation. The assay was performed in triplicate, and representative data are shown.

FIG. 2.

Northern blot analysis of ERG3 and ERG11. CAE3DU (erg3/erg3) showed a lack of ERG3 transcript and increased ERG11 expression compared to that of CAF2-1 (ERG3/ERG3) and CAD1U (erg3/ERG3). The visible rRNA bands serving as controls were approximately equivalent. The numbers to the right indicate the approximate sizes of mRNA in kilobases.

FIG. 3.

Survival of mice infected with CAF2-1 (ERG3/ERG3 ura3/URA3) and CAE3DU3 (erg3/erg3 ura3/URA3). Immunocompetent mice (n = 10) were infected intravenously with 0.904 × 10⁶ cells of CAF2-1 (open squares), 4.52 × 10⁵ cells of CAF2-1 (solid squares), 0.922 × 10⁶ cells of CAE3DU3 (open triangles), or 4.61 × 10⁵ cells of CAE3DU3 (solid circles). Representative data of two independent experiments are shown.

FIG. 4.

Histopahologic analysis of kidney tissues obtained from mice infected with CAF2-1 (ERG3/ERG3 ura3/URA3) and CAE3DU3 (erg3/erg3 ura3/URA3). Groups of three immunocompetent mice were infected intravenously with 4.91 × 10⁵ cells of CAF2-1 or 4.85 × 10⁶ cells of CAE3DU3. Kidneys were excised 4 days after injection, and tissue sections were stained with Grocott-Gomori methenamine silver stain. Note the aborted hyphal formation of CAE3DU3, shown in arrows, compared to abundant and intact hyphae of CAF2-1.

FIG. 5.

Fluconazole treatment in a murine model of candidiasis. Immunocompetent mice (n = 10) were injected intravenously with 4.91 × 10⁵ cells of CAF2-1 or 4.85 × 10⁶ cells of CAE3DU3. Of note, the inoculum size of CAE3DU3 was 10-fold higher than that of CAF2-1 because of the difference in virulence between these strains. Fluconazole was administered by gavage at 40 mg/kg once a day for 4 days starting at 3 h after injection. Sterile saline was used as a control. Kidneys were excised 4 days after injection, and kidney CFU were determined. The scatter plot shows kidney CFU of CAF2-1 treated with saline (solid circles), CAF2-1 treated with fluconazole (open circles), CAE3DU3 treated with saline (solid triangles), and CAE3DU3 treated with fluconazole (open triangles). The geometric means and the standard deviations are shown in each group.