Candida albicans Sun41p, a putative glycosidase, is involved in morphogenesis, cell wall biogenesis, and biofilm formation

Abstract

The SUN gene family has been defined in Saccharomyces cerevisiae and comprises a fungus-specific family of proteins which show high similarity in their C-terminal domains. Genes of this family are involved in different cellular processes, like DNA replication, aging, mitochondrial biogenesis, and cytokinesis. In Candida albicans the SUN family comprises two genes, SUN41 and SIM1. We demonstrate that C. albicans mutants lacking SUN41 show similar defects as found for S. cerevisiae, including defects in cytokinesis. In addition, the SUN41 mutant showed a higher sensitivity towards the cell wall-disturbing agent Congo red, whereas no difference was observed in the presence of calcofluor white. Compared to the wild type, SUN41 deletion strains exhibited a defect in biofilm formation, a reduced adherence on a Caco-2 cell monolayer, and were unable to form hyphae on solid medium under the conditions tested. Interestingly, Sun41p was found to be secreted in the medium of cells growing as blastospores as well as those forming hyphae. Our results support a function of SUN41p as a glycosidase involved in cytokinesis, cell wall biogenesis, adhesion to host tissue, and biofilm formation, indicating an important role in the host-pathogen interaction.

FIG. 1.

Microscopic imaging of different C. albicans strains growing exponentially in liquid YPD. After 6 h, cells were fixed with glutaraldehyde and imaged using light microscopy (A and B) or prepared for further imaging with either scanning electron microscopy (C to E) or transmission electron microscopy (F to H). Bars, 2 μm.

FIG. 2.

Distribution of cell sizes of different C. albicans strains. The sizes of the cell surfaces of C. albicans strains on microscopic images were measured and plotted on a box-whisker plot. The distribution differed significantly between the wild type and Δsun41 mutant (P = 7.2 × 10⁻¹⁴). The boxes indicate the interquartile ranges (25th to 75th percentiles), the horizontal thick lines symbolize the medians, the black squares represent the means, the whiskers extend to 1.5 times the interquartile range, and the circles illustrate the outliers.

FIG. 3.

Influence of Congo red on different C. albicans strains. Serial dilutions of C. albicans cells of the indicated strains were spotted onto agar plates containing YPD (A) or YNB (B) supplemented with 300 μg/ml Congo red. Plates were incubated for 5 days at 30°C. (C) C. albicans wild-type cells were incubated in the indicated media at 30°C for 7 h. (D) C. albicans wild-type and sun41Δ cells were incubated in YPD supplemented with 10% serum and 300 μg/ml Congo red at 37°C for 7 h. Bars, 10 μm.

FIG. 4.

Phenotype of different C. albicans strains growing on solidified media. (A) Approximately 100 cells of the indicated C. albicans strains were spotted onto agar plates containing α-MEM or YPD supplemented with 10% bovine calf serum. Plates were incubated for 5 days at 37°C. (B) The rim of the colonies grown on YPD supplemented with 10% bovine calf serum was imaged using light microscopy (upper three panels). Additionally, cells were scraped off the colonies and examined microscopically (lower three panels). (C) The ability to adhere on YPD agar was tested by washing layers formed by the indicated strains on plates with or without serum. Bars, 10 μm.

FIG. 5.

(A) Quantitative assay to determine adherence of different C. albicans strains on a Caco-2 monolayer. C. albicans strains were applied to a monolayer of Caco-2 cells. The percentage of adherent C. albicans cells after 30, 60, 120, and 240 min of infection is shown. Results are average values with standard deviations of four independent experiments. (B) Comparison of biofilm formation of different C. albicans strains. C. albicans cells of the indicated strains were incubated in 96-well plates for 2 days. The amount of biofilm-forming cells was measured in an XTT activity assay. Results represent means with standard deviations (error bars) from two independent experiments.

FIG. 6.

Differential regulation in sun41Δ: total RNA of C. albicans wild-type and sun41Δ mutant cells growing as hyphae in liquid α-MEM were collected and reverse transcribed to cDNA, which was used as a template in a real-time PCR. The transcript amount of the indicated genes in the sun41Δ mutant was compared to that of the wild type. Dashed lines represent two times and one-half the amount of transcript in the sun41Δ mutant. The dotted line in the middle indicates an equivalent amount (1.0×). Error bars indicate the standard errors of the means from three independent experiments.

FIG. 7.

Comparison of proteins found in supernatants of different C. albicans strains growing in YNB. Different C. albicans strains were grown exponentially in the liquid synthetic medium YNB. Cells were removed and media collected. Proteins were precipitated and digested with trypsin. Shown are the mass spectra of the resulting peptide mixtures from 700 to 4,000 Da from the MALDI-TOF MS. The sequences of the indicated peptides were identified by MS/MS and associated to proteins using the MASCOT software. Peaks: 1 and 5, Sun41p; 2, Sim1p; 3, MP65p; 4, Tos1p.