K143R Amino Acid Substitution in 14-α-Demethylase (Erg11p) Changes Plasma Membrane and Cell Wall Structure of Candida albicans

Abstract

The opportunistic pathogen Candida albicans is responsible for life-threating infections in immunocompromised individuals. Azoles and polyenes are two of the most commonly used antifungals and target the ergosterol biosynthesis pathway or ergosterol itself. A limited number of clinically employed antifungals correspond to the development of resistance mechanisms. One resistance mechanism observed in clinical isolates of azole-resistant C. albicans is the introduction of point mutations in the ERG11 gene, which encodes a key enzyme (lanosterol 14-α-demethylase) on the ergosterol biosynthesis pathway. Here, we demonstrate that a point mutation K143R in ERG11 (C. albicans ERG11^K143R/K143R) contributes not only to azole resistance, but causes increased gene expression. Overexpression of ERG11 results in increased ergosterol content and a significant reduction in plasma membrane fluidity. Simultaneously, the same point mutation caused cell wall remodeling. This could be facilitated by the unmasking of chitin and β-glucan on the fungal cell surface, which can lead to recognition of the highly immunogenic β-glucan, triggering a stronger immunological reaction. For the first time, we report that a frequently occurring azole-resistance strategy makes C. albicans less susceptible to azole treatment while, at the same time, affects its cell wall architecture, potentially leading to exposure of the pathogen to a more effective host immune response.

Keywords: Candida albicans; cell wall; ergosterol; plasma membrane.

Figure 1

(A) The growth curve of the C. albicans SC5314 wild type (WT), erg11Δ/Δ (KS058) and the ERG11^K143R/K143R (10C1B1I1) strains in a YPD medium (28 °C, 120 rpm; n = 3, ±SD). (B) The percent viability of the C. albicans WT, KS058, and 10C1B1I1 strains cultured for 24 h, 28 °C, in a YPD medium supplemented with FLC (left) or AMB (right) (n = 3, ±SD).

Figure 2

The relative ERG11 gene expression in the C. albicans SC5314 wild type (WT) and ERG11^K143R/K143R (10C1B1I1) strains determined for different growth times (8, 14, and 24 h) and culture conditions: control (YPD medium alone), and with the addition of FLC (4 μg/mL) or AMB (0.25 μg/mL); ±SD; n = 3. Statistical analysis was performed by comparing the expression of the C. albicans WT and 10C1B1I1, or between the different culture conditions (*, p < 0.05; **, p < 0.01).

Figure 3

The representative, normalized fluorescence intensities (IFs) spectra for a Laurdan fluorescent probe incorporated into the plasma membrane of the C. albicans SC5314 (WT), erg11Δ/Δ (KS058), and ERG11^K143R/K143R (10C1B1I1) strains in control conditions at 8 h (A) and 24 h (B).

Figure 4

Triple staining of the C. albicans cell wall components for the SC5314 wild type (WT), erg11Δ/Δ (KS058), and ERG11^K143R/K143R (10C1B1I1) strains (A). Total chitin was detected using calcofluor white (CFW) (purple). Exposed β-glucan was stained with Fc-hDectin-1 (FC-Dec1) and Alexa Fluor 448-conjugated anti-human IgG Fc antibodies (green). Mannans were detected using streptavidin and concanavalin A (ConA) conjugated with Alexa Fluor-568 (red). Scale bars of all presented images are equal to 2 μm. White squares on the merged picture represent region of interest (ROI). Fluorescence intensities for particular CW components for ROI (regions of bud scars; white lines) for investigated C. albicans strains (B). The distance [nm] between CW components is presented from outside to inside of the cell at the area of the bud scars.

Figure 5

The exposure of chitin (panel (A)), β-glucan (panel (B)), and mannans (panel (C)) in the C. albicans SC5314 wild type (WT), erg11Δ/Δ (KS058) and ERG11^K143R/K143R (10C1B1I1) strains grown for 24 h in YPD (28 °C, 120 rpm). In each case, the C. albicans cells were stained with wheat germ agglutinin conjugated with FITC (WGA-FITC; in case of exposed chitin, panel (A)), Fc-hDectin-1 and Alexa Fluor 448-conjugated anti-human IgG Fc antibodies (in the case of unmasked β-glucan, panel (B)) or streptavidin and concanavalin A (ConA) conjugated with Alexa Fluor 568 (in case of mannans, panel (C)) and analyzed by FACS. Median fluorescence intensities (MFIs) were quantified for three independent experiments. Statistical analyses were performed using the WT strain, or between KS058 and 10C1B1I1 (above lines) (*, p < 0.05 **, p < 0.01).