Mechanism of fluconazole resistance in Candida albicans biofilms: phase-specific role of efflux pumps and membrane sterols

Abstract

Candida albicans biofilms are formed through three distinct developmental phases and are associated with high fluconazole (FLU) resistance. In the present study, we used a set of isogenic Candida strains lacking one or more of the drug efflux pumps Cdr1p, Cdr2p, and Mdr1p to determine their role in FLU resistance of biofilms. Additionally, variation in sterol profile as a possible mechanism of drug resistance was investigated. Our results indicate that parent and mutant strains formed similar biofilms. However, biofilms formed by double and triple mutants were more susceptible to FLU at 6 h (MIC = 64 and 16 microg/ml, respectively) than the wild-type strain (MIC > 256 microg/ml). At later time points (12 and 48 h), all the strains became resistant to this azole (MIC > or = 256 microg/ml), indicating lack of involvement of efflux pumps in resistance at late stages of biofilm formation. Northern blot analyses revealed that Candida biofilms expressed CDR and MDR1 genes in all the developmental phases, while planktonic cells expressed these genes only at the 12- and 48-h time points. Functionality of efflux pumps was assayed by rhodamine (Rh123) efflux assays, which revealed significant differences in Rh123 retention between biofilm and planktonic cells at the early phase (P = 0.0006) but not at later stages (12 and 48 h). Sterol analyses showed that ergosterol levels were significantly decreased (P < 0.001) at intermediate and mature phases, compared to those in early-phase biofilms. These studies suggest that multicomponent, phase-specific mechanisms are operative in antifungal resistance of fungal biofilms.

FIG. 1.

Percent growth inhibition of C. albicans biofilms exposed to high concentration of fluconazole. Percentages of inhibition for biofilms grown to the early (6 h), intermediate (12 h), or late (48 h) phase of development and exposed to 256 μg of fluconazole/ml were determined. Strains used were: CAF2-1 (wild type), DSY448 (Δcdr1), DSY465 (Δmdr1), DSY654 (Δcdr1 Δcdr2), and DSY1050 (Δcdr1 Δcdr2 Δmdr1). For each strain, drug susceptibility decreased from the early to late phase of biofilm development. Additionally, deletion of two and three efflux pumps led to progressively decreasing susceptibility to fluconazole. Metabolic activity was normalized to the control without fluconazole, which was taken as 100%. Data (means ± standard deviations) are representative of three separate experiments. ∗, the wild-type (CAF2-1) strain showed 0% inhibition at all the time points.

FIG. 2.

Expression of CDR and MDR1 genes in (P) planktonic (P) and biofilm (B) forms of C. albicans. Total RNA was isolated from biofilms and planktonic cells grown for 6, 12, and 48 h. Sixty micrograms of total RNA was analyzed by Northern blotting using a CDR- or MDR1-specific probe as described in Materials and Methods. Top, CDR transcript; middle, MDR1 transcript; bottom, 25S rRNA (loading control). Results are representative of three separate experiments.

FIG. 3.

Rh123 accumulation by early-, intermediate-, and mature-phase biofilms and planktonic cells of C. albicans. Data were analyzed by two-way analysis of variance, and a value of P <0.05 was considered significant. ∗, P < 0.05 versus 6-h biofilms; ∗∗, P < 0.0001 versus 6-h planktonic cells. AFU, arbitrary fluorescence units.

FIG. 4.

Variations in sterol profiles of C. albicans biofilms at different developmental phases. Sterol patterns for biofilms grown to the early (A), intermediate (B), or mature (C) phase were determined by GLC. (D) Percentages of sterols identified in C. albicans biofilms and planktonic cells (chromatograph not shown), determined from the corresponding peak areas and retention times relative to ergosterol. Peaks 1 to 7 (A to C) represent sterols described in panel D. SD, standard deviation.