cDNA Microarray Analysis of Differential Gene Expression in Candida albicans Biofilm Exposed to Farnesol

C. albicans collection strain SC5314 was kindly provided by William A. Fonzi (Department of Microbiology and Immunology, Georgetown University, Washington, D.C.). Cells were propagated in yeast peptone dextrose (YPD) medium (1% [wt/vol] yeast extract, 2% [wt/vol] peptone, 2% [wt/vol] dextrose). Batches of medium (20 ml) were inoculated from YPD agar plates containing freshly grown C. albicans and incubated overnight in an orbital shaker at 30°C. C. albicans grew in the budding-yeast phase under these conditions. Cells were harvested and washed in sterile phosphate-buffered saline (PBS; 10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride [pH 7.4]). The cells were then suspended in RPMI 1640 medium supplemented with l-glutamine and buffered with morpholinepropanesulfonic acid (MOPS), counted in a hemocytometer, and adjusted to the desired cell density (1.0 × 10⁶ cells/ml). Farnesol (mixed isomers; lot 082K2520; Sigma Chemical Co.) was obtained as a 4 M stock solution and then diluted in 100% (vol/vol) methanol to obtain a 40 mM working stock solution. The working concentration of farnesol was 40 μM when it was prepared in RPMI 1640 medium.

Standardized C. albicans cells (1.0 × 10⁶ cells/ml in RPMI 1640 medium) were prepared and added to 25-ml portions of RPMI 1640 medium in 75-cm² tissue culture flasks with vented caps. The flasks were incubated statically for 1 h to allow initial adherence of the cells, after which the medium was decanted and replaced with 50-ml portions of prewarmed (37°C) RPMI 1640 medium containing 40 μM farnesol. This farnesol concentration was chosen because it still maintained an inhibitory effect on biofilm formation, while it allowed the recovery of a sufficient cellular mass for RNA extraction. The flasks were then gently rocked (50 rpm) at 37°C for 24 h to promote biofilm formation (33). A farnesol-free control was also included.

The CSH of C. albicans was measured by the water-hydrocarbon two-phase assay, as described previously (18). Briefly, C. albicans cultures to which different concentrations (0, 1, 10, and 100 μM) of farnesol were added were cultured as described above to form biofilms, and the cells of the biofilm were removed from the flask surfaces with a sterile scraper to prepare a cell suspension (optical density at 600 nm [OD₆₀₀], 1.0 in YPD medium). A total of 1.2 ml of a suspension from each group was drawn into a clean glass tube and overlaid with 0.3 ml of octane. The mixture was vortexed for 3 min for phase separation. Soon after the two phases had separated, the OD₆₀₀ of the aqueous phase was determined. The OD₆₀₀ for the group without the octane overlay in YPD medium was used as the negative control. Three repeats were performed for each group. The relative hydrophobicity was obtained as [(OD₆₀₀ of the control − OD₆₀₀ after octane overlay)/OD₆₀₀ of the control] × 100.

Confocal scanning laser microscopy was performed as described in the literature (8) to demonstrate the inhibitory effect of farnesol on biofilm formation. The formation of C. albicans strain SC5314 biofilms was achieved by adding 4 ml of a standardized cell suspension onto six-well plates containing plastic disks. Following farnesol exposure and biofilm formation, the disks were removed and transferred to new six-well culture plates and incubated for 45 min at 37°C in 4 ml of PBS containing the fluorescent stains FUN-1 (10 μM; Molecular Probes, Eugene, Oreg.) and concanavalin A-Alexa Fluor 488 conjugate (ConA; 25 μg/ml; Molecular Probes). FUN-1 (excitation wavelength, 543 nm; emission wavelength, 560 nm; long-pass filter) is converted to an orange-red cylindrical intravacuolar structure by metabolically active cells, while ConA (excitation wavelength, 488 nm; emission wavelength, 505 nm; long-pass filter) binds to the glucose and mannose residues of cell wall polysaccharides and emits a green fluorescence. After incubation with the dyes, the disks were flipped and the stained biofilms were observed with a Leica TCS sp2 confocal scanning laser microscope equipped with argon and HeNe lasers.

Cells were washed in ice-cold sterile PBS in the flasks and then removed from the flask surfaces with a sterile scraper. Following collection of the cells by centrifugation, the cells were resuspended in 12 ml of AE buffer (50 mM sodium acetate [pH 5.2], 10 mM EDTA) at room temperature, after which 800 μl of 25% sodium dodecyl sulfate (SDS) and 12 ml of acid phenol were added. The cell lysate was then incubated for 10 min at 65°C with vortexing each minute, cooled on ice for 5 min, and subjected to centrifugation at 12,000 × g for 15 min. The supernatants were transferred to new tubes containing 15 ml of chloroform, mixed, and subjected to centrifugation at 200 × g for 10 min. RNA was precipitated from the resulting aqueous layer by transferring that portion to new tubes containing 1 volume of isopropanol and 0.1 volume of 2 M sodium acetate (pH 5.0) and mixed well. The mixture was centrifuged at 18,000 × g for 35 min at 4°C. The supernatants were removed. The pellet was resuspended in 10 ml of 70% ethanol and centrifuged at 18,000 × g for 20 min at 4°C to collect the RNA. The supernatants were removed again, and the RNA was resuspended in diethyl pyrocarbonate-treated water. The OD₂₆₀ and OD₂₈₀ were measured, and the integrity of the RNA was visualized by subjecting 2 to 5 μl of the sample to electrophoresis through a 1% agarose-MOPS gel. Poly(A) mRNA was extracted by using an Oligotex mRNA kit (Qiagen, Hilden, Germany) and was quantitated by using a RiboGreen RNA quantitation kit (Molecular Probes).

The microarray (3132 chip; United Gene Holdings, Ltd., Shanghai, People's Republic of China) used in our study consisted of 3,132 spots (3,102 sequences), including full-length and partial cDNA sequences representing the sequences of novel, known, and control genes of C. albicans. Most of the genes were obtained by sequencing the clones isolated from a library of C. albicans with full-length cDNA at a high ratio by using the switching mechanism at the 5′ end of the RNA transcript. Sequencing of the clones was carried out at United Gene Holdings. Other known genes involved in drug resistance and biofilm formation were selected from the National Center for Biotechnology Information Unigene set and were cloned into plasmid vectors. The control spots included human glycerol-3-phosphate dehydrogenase (8 spots), actin genes (8 spots), and spotting solution alone without DNA (16 spots). The 3132 chip was constructed by our previously described method (22). In brief, the cDNA inserts were amplified by PCR with universal primers specific for the plasmid vector sequences and then purified by isopropanol precipitation. All PCR products were examined by agarose gel electrophoresis to ensure the quality and the identity of the amplified clones, which were as expected. The amplified PCR products were dissolved in a buffer containing 3× SSC solution (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). These solutions were spotted onto sialylated slides (CEL Associates, Houston, Tex.) with a Cartesian PixSys 7500 motion-control robot (Cartesian Technologies, Irvine, Calif.) fitted with ChipMaker Micro-Spotting technology (TeleChem International, Sunnyvale, Calif.). The glass slides spotted with cDNA were then hydrated for 2 h in an atmosphere with 70% humidity, dried for 0.5 h at room temperature, and UV cross-linked (65 mJ/cm²). They were further processed at room temperature by soaking them in 0.2% SDS for 10 min, distilled water for 10 min, and 0.2% sodium borohydride for 10 min. The slides were dried again, at which time they were ready for use.

The labeling procedures were conducted as follows: the fluorescent cDNA probes were synthesized from purified mRNA with Cy3- or Cy5-dUTP (Amersham Pharmacia Biotech, Piscataway, N.J.) by oligo(dT)-primed polymerization with Superscript II reverse transcriptase (Invitrogen). The reaction buffer mixture contained deoxynucleoside triphosphates (200 μmol of dATP, dCTP, and dGTP per liter, 60 μmol of dTTP per liter, and 60 μmol of Cy3- or Cy5-dUTP per liter), 2 μl of Superscript II reverse transcriptase, and 1× reaction buffer. The reactions were carried out at 42°C for 2 h. Then the RNA was hydrolyzed by the addition of 4 μl of 2.5 mol of NaOH per liter and incubation at 65°C for 10 min, and then the RNA was neutralized with 4 μl of 2.5 mol of HCl per liter. Dye swap was used to avoid dye-associated effects on cDNA synthesis. That is, three independent hybridization experiments were performed, with RNA from the farnesol-treated group labeled with Cy5-dUTP two times and with Cy3-dUTP one time. The two color probes were then mixed and diluted to 500 μl with TE (Tris-EDTA), concentrated to 10 μl with a Microcon YM-30 filter (Millipore, Bedford, Mass.), and vacuum dried.

The probes were dissolved in 20 μl of hybridization solution (5× SSC [0.75 mol of NaCl per liter and 0.075 mol of sodium citrate per liter], 0.4% SDS, 50% formamide). The microarrays were prehybridized with hybridization solution containing 0.5 mg of denatured salmon sperm DNA per ml at 42°C for 6 h. The fluorescent probe mixtures were denatured at 95°C for 5 min and were then applied onto the prehybridized chip under a cover glass. The chip was hybridized in a homemade chamber at 42°C for 15 to 17 h. The hybridized chip was then washed at 60°C in solutions of 2× SSC-0.2% SDS, 0.1× SSC-0.2% SDS, and 0.1× SSC for 10 min in each solution and then dried at room temperature.

The chips were scanned with a ScanArray 3000 apparatus (GSI Lumonics, Bellerica, Mass.) at two wavelengths to detect the emissions from both Cy3 and Cy5. The acquired images were analyzed with ImaGene (version 3.0) software (BioDiscovery, Los Angeles, Calif.). The intensities of each spot at the two wavelengths represent the quantities of Cy3-dUTP and Cy5-dUTP, respectively, that hybridized to each spot. The ratios of Cy5 to Cy3 were calculated for each location on each microarray. To minimize artifacts that arise from low expression values, only genes with raw intensity values of >800 counts for both Cy3 and Cy5 were chosen for differential analysis. Genes were identified as differentially expressed if the absolute value of the natural logarithm of the ratios was >0.69 (22).

Farnesol acts as a naturally occurring quorum-sensing molecule that inhibits filamentation in C. albicans and that was also reported to inhibit C. albicans biofilm formation (16, 33). In the present study, we compared C. albicans biofilms cultured for 24 h in the presence of either the control solution or farnesol (40 μmol/liter). The 24-h time point was chosen since it was reported that the biofilm is mature at 24 h (33) and drug resistance appears at this time point (8, 32). Farnesol was used at a concentration of 40 μmol/liter and was added after the original incubation of 1 h to suppress biofilm formation while enough cells were retrieved for RNA extraction and hybridization (Fig. 1). After farnesol treatment, 274 genes (mainly identified at http://genolist.pasteur.fr/CandidaDB/ ) altered differentially with a threshold of a 2.0-fold change in the expression level (see the supplementary table available at http://www.chinagenenet.com/ca_microarray ). Differential expression for these genes was then confirmed independently by reverse transcription-PCR (RT-PCR). The important genes selected from the differentially expressed genes are shown in Table 1, and the RT-PCR results for some of these genes are shown in Fig. 2.

Consistent with previous findings that farnesol prevents hyphal development in a dose-dependent manner (33), we found that several hyphal formation-associated genes were differentially expressed. TUP1 encodes a global transcriptional corepressor. Deletion of the TUP1 gene causes hyperfilamentation under conditions favorable for growth of the yeast form (3, 4). Up-regulation of TUP1 may therefore contribute to the inhibition of hyphal formation. However, we did not find that the TUP1-repressed genes, which are associated with morphology, as reported previously (5), were differentially expressed.

CRK1 is a new member of the Cdc2 kinase subfamily (9). Deletion of CRK1 dramatically impaired hyphal formation under various hypha-inducing conditions, whereas the ectopic expression of its catalytic domain promoted hyphal colony formation, even under conditions favorable for growth of the yeast form (44). Down-regulation of the CRK1 gene may account for the yeast phenotype.

The cAMP-dependent pathway is one of the pathways that regulate yeast-to-hypha morphogenesis in C. albicans, which is controlled by changes in cyclic AMP (cAMP) levels determined by the processes of synthesis and hydrolysis. CaPDE2 encodes the high-affinity cAMP phosphodiesterase. Deletion of CaPDE2 causes elevated cAMP levels, elevated responsiveness to exogenous cAMP, and highly reduced levels of transcription of EFG1, which is an important hypha-associated gene. In vitro in hypha-inducing liquid medium, CaPDE2 deletion prohibits normal hyphal development (17). Thus, down-regulation of PDE2 may affect morphogenesis.

A typical laboratory model of fungal biofilm formation involves three steps: adhesion, biofilm growth, and maturation (2, 8). Using different species of Candida, previous researchers observed that fungal adherence to plastic surfaces is correlated with CSH (14, 18, 34, 38, 39). Recently, it was reported (21) that CSH plays a major role in biofilm formation in C. albicans. A positive correlation between biofilm formation abilities and CSH has been found. The CSH1 gene, which codes for the CSH-associated protein, is the first candidate gene that has been demonstrated to play a role in affecting the CSH phenotype in C. albicans. Knockout of this gene resulted in a decrease in measurable CSH and a decrease in the level of adhesion of C. albicans to fibronectin (40). We found that CSH1 was down-regulated about threefold in this experiment. Since initial adhesion is important in terms of the ability of farnesol to inhibit biofilm formation, there may be a relationship between CSH and the farnesol concentration. To confirm this, we determined the CSH of biofilms treated with different concentrations of farnesol. We found a negative correlation between the farnesol concentrations and CSH (Fig. 3). It is therefore possible that a decrease in CSH may contribute to the inhibition of biofilm formation.

C. albicans biofilms are highly resistant to the actions of clinically important antifungal agents, especially fluconazole (8, 13). CDR1, CDR2, and MDR1, which encode major multidrug efflux pumps in C. albicans and which play important roles in the drug resistance of planktonic cells, cannot be used to explain why mature biofilms show high-level drug resistance (26, 32). The antifungal resistance of biofilms involves multiple factors and phase-specific mechanisms. In the present study, FCR1 and PDR16, which are associated with drug resistance, were found to be differentially expressed.

FCR1 codes for a transcription factor of the C₆ zinc cluster family homologous to Saccharomyces cerevisiae Pdr1p. It behaves as a negative regulator of drug resistance in C. albicans. The fcr1/fcr1 mutant displays hyperresistance to fluconazole and other antifungal drugs (42). Up-regulation of FCR1 in the farnesol-treated group suggests that the level of fluconazole resistance decreased in inhibited biofilms.

PDR16 is another differentially expressed gene involved in drug resistance and belongs to the ABC family of transporters. De Deken and Raymond (12) recently reported that C. albicans azole-resistant clinical isolates overexpress the PDR16 gene. In S. cerevisiae, deletion of PDR16 led to strongly increased sensitivity to azole antifungals (43). Furthermore, experiments showed that this deletion affects the phospholipid and sterol compositions of the plasma membrane and that the changes in phospholipid and sterol compositions result in changes in the total yeast lipid composition. It was reported (19) that alteration of the phospholipid content in C. albicans is associated with drug resistance. Consistent with this, we observed INO1, which encodes inositol-1-phosphate synthase, a key enzyme in the synthesis of inositol for phosphotidylinositol synthesis (41), was down-regulated dramatically in the farnesol-treated group. Of note, INO1 was reported to be up-regulated in drug-resistant isolates (37). Down-regulation of PDR16 and INO1 thus suggests that altered phospholipid metabolism is associated with azole resistance.

Farnesol exposure resulted in the down-regulation of several genes encoding heat shock proteins (e.g., HSP70, HSP90, HSP104, CaMSI3, and SSA2). Presumably, the production of heat shock proteins contributes to the protection of cells from damage and repair of cell damage following stress, which may occur in biofilms. Besides, heat shock proteins play important roles in major growth-related processes, such as cell division, DNA synthesis, transcription, translation, protein folding and transport, and membrane translocation (23). Recently, a C. albicans biofilm microarray experiment revealed the overexpression of genes involved in protein synthesis (15), which indicated that, because of their role in helping with protein synthesis, up-regulation of heat shock proteins could also occur in normal biofilms, while down-regulation of these proteins could occur in impaired biofilms.

Chitinase is an essential component in the maintenance of cell wall plasticity during fungal growth and proliferation. C. albicans contains three chitinase genes, CHT1, CHT2, and CHT3. Previous reports (6, 24) showed that CHT2 and CHT3 are preferentially expressed in the yeast phase. The up-regulation of these two genes in our study is consistent with the yeast phenotype in the farnesol-treated group.

We found that the high-affinity iron permease gene FTR2 is up-regulated more than fourfold. Previous studies have indicated a relationship between iron accessibility and azole resistance in C. albicans. Moreover, FTR2 is down-regulated in fluconazole-resistant C. albicans isolates compared to its regulation in fluconazole-susceptible isolates (36). The up-regulation of FTR2 may therefore contribute to the decrease in the level of azole resistance.

To our knowledge, this study is the first to use cDNA microarray analysis to characterize changes in gene expression between normal biofilms and biofilms inhibited by farnesol, a quorum-sensing molecule in C. albicans. The ability of cDNA microarray analysis to examine thousands of genes simultaneously allows not only the identification of the differential expression of genes involved in hypha1 formation, which is the primary function of farnesol, but also that of several genes associated with CSH and drug resistance. This supports the interpretation of C. albicans biofilms. That is, the higher the level of CSH is, the easier it is for a biofilm to form; and when a biofilm has formed, high levels of drug resistance occur. In addition, genes functionally related to cell wall maintenance or iron transport as well as genes encoding heat shock proteins were found to be differentially expressed for the first time. The present study identifies new potential targets in the development of pharmacological approaches to circumventing biofilm formation and the antifungal resistance derived from biofilm formation.