cDNA microarray analysis of differential gene expression in Candida albicans biofilm exposed to farnesol

Abstract

Candida albicans biofilms are structured microbial communities with high levels of drug resistance. Farnesol, a quorum-sensing molecule that inhibits hyphal formation in C. albicans, has been found to prevent biofilm formation by C. albicans. There is limited information, however, about the molecular mechanism of farnesol against biofilm formation. We used cDNA microarray analysis to identify the changes in the gene expression profile of a C. albicans biofilm inhibited by farnesol. Confocal scanning laser microscopy was used to visualize and confirm normal and farnesol-inhibited biofilms. A total of 274 genes were identified as responsive, with 104 genes up-regulated and 170 genes down-regulated. Independent reverse transcription-PCR analysis was used to confirm the important changes detected by microarray analysis. In addition to hyphal formation-associated genes (e.g., TUP1, CRK1, and PDE2), a number of other genes with roles related to drug resistance (e.g., FCR1 and PDR16), cell wall maintenance (e.g., CHT2 and CHT3), and iron transport (e.g., FTR2) were responsive, as were several genes encoding heat shock proteins (e.g., HSP70, HSP90, HSP104, CaMSI3, and SSA2). Further study of these differentially regulated genes is warranted to evaluate how they may be involved in C. albicans biofilm formation. Consistent with the down-regulation of the cell surface hydrophobicity-associated gene (CSH1), the water-hydrocarbon two-phase assay showed a decrease in cell surface hydrophobicity in the farnesol-treated group compared to that in the control group. Our data provide new insight into the molecular mechanism of farnesol against C. albicans biofilm formation.

FIG. 1.

Confocal scanning laser microscopy showing the effect of farnesol on C. albicans biofilm (24 h) formation. ConA (green) and FUN-1 (red) staining were used to generate the images. Metabolically active cells are shown in red, and cell wall polysaccharides are shown in green. Cells treated with 40 μM farnesol (B) show poor biofilm architectures, with the biofilm predominantly composed of yeast cells and pseudohyphae. True hyphae were present in farnesol-treated cells at levels much less than those in the farnesol-free control (A).

FIG. 2.

Confirmation by RT-PCR of differential expression of selected important C. albicans genes found to be differentially expressed by cDNA microarray analysis. 18S rRNA was tested as a control.

FIG. 3.

Effect of farnesol on CSH of C. albicans biofilms. Different farnesol concentrations (0, 1, 10, and 100 μM) were added to C. albicans cells 1 h after attachment, and the cells were incubated under conditions favorable for biofilm growth. CSH was estimated by the water-hydrocarbon two-phase assay. Mean relative CSH values and standard deviations for three separate experiments are shown. Statistically significant differences between farnesol-treated biofilms and biofilms formed in the absence of farnesol were determined by Student's t test (*, P < 0.01).