Production of tyrosol by Candida albicans biofilms and its role in quorum sensing and biofilm development

Abstract

Tyrosol and farnesol are quorum-sensing molecules produced by Candida albicans which accelerate and block, respectively, the morphological transition from yeasts to hyphae. In this study, we have investigated the secretion of tyrosol by C. albicans and explored its likely role in biofilm development. Both planktonic (suspended) cells and biofilms of four C. albicans strains, including three mutants with defined defects in the Efg 1 and Cph 1 morphogenetic signaling pathways, synthesized extracellular tyrosol during growth at 37 degrees C. There was a correlation between tyrosol production and biomass for both cell types. However, biofilm cells secreted at least 50% more tyrosol than did planktonic cells when tyrosol production was related to cell dry weight. The addition of exogenous farnesol to a wild-type strain inhibited biofilm formation by up to 33% after 48 h. Exogenous tyrosol appeared to have no effect, but scanning electron microscopy revealed that tyrosol stimulated hypha production during the early stages (1 to 6 h) of biofilm development. Experiments involving the simultaneous addition of tyrosol and farnesol at different concentrations suggested that the action of farnesol was dominant, and 48-h biofilms formed in the presence of both compounds consisted almost entirely of yeast cells. When biofilm supernatants were tested for their abilities to inhibit or enhance germ tube formation by planktonic cells, the results indicated that tyrosol activity exceeds that of farnesol after 14 h, but not after 24 h, and that farnesol activity increases significantly during the later stages (48 to 72 h) of biofilm development. Overall, our results support the conclusion that tyrosol acts as a quorum-sensing molecule for biofilms as well as for planktonic cells and that its action is most significant during the early and intermediate stages of biofilm formation.

FIG. 1.

Typical HPLC chromatograms for the determination of tyrosol. (A) A supernatant extract containing 7 μM tyrosol. (B) Uninoculated, extracted growth medium showing no interfering peaks at or around the tyrosol retention time (7.7 min).

FIG. 2.

Relationship between tyrosol production (□) and cell dry weight (▪) for planktonic cells of C. albicans strains SC5314 (A), JKC19 (B), HLC52 (C), and HLC54 (D). Results are means ± SEMs of three independent experiments.

FIG. 3.

Relationship between tyrosol production (□) and cell dry weight (▪) for biofilms of C. albicans SC5314 (A), JKC19 (B), HLC52 (C), and HLC54 (D). Results are means ± SEMs of three independent experiments.

FIG. 4.

Tyrosol production expressed as a function of cell dry weight for planktonic cells (○) and biofilms (•) of C. albicans SC5314 (A), JKC19 (B), HLC52 (C), and HLC54 (D). Results are means ± SEMs of three independent experiments carried out in triplicate.

FIG. 5.

Effect of farnesol concentration on biofilm formation by C. albicans GDH 2346. Farnesol, at concentrations of 50 μM, 100 μM, and 1 mM, was added at different stages of biofilm development. Biofilm formation (XTT reduction) is expressed as a percentage of that of control biofilms incubated in the absence of farnesol. Results are means ± SEMs from at least two independent experiments carried out in triplicate. Mean (±SEM) control values (A₄₉₂) ranged from 1.999 ± 0.399 to 2.311 ± 0.142. Farnesol was added at adhesion (□) adhesion and time zero (▤) time zero (), 2 h of biofilm formation (▥), 4 h of biofilm formation (░⃞), and 24 h of biofilm formation (). ▪, control biofilm (no farnesol added). P was <0.05 (*) and <0.001 (**) for treated biofilms compared with untreated controls (Student's t test).

FIG. 5.

Effect of farnesol concentration on biofilm formation by C. albicans GDH 2346. Farnesol, at concentrations of 50 μM, 100 μM, and 1 mM, was added at different stages of biofilm development. Biofilm formation (XTT reduction) is expressed as a percentage of that of control biofilms incubated in the absence of farnesol. Results are means ± SEMs from at least two independent experiments carried out in triplicate. Mean (±SEM) control values (A₄₉₂) ranged from 1.999 ± 0.399 to 2.311 ± 0.142. Farnesol was added at adhesion (□) adhesion and time zero (▤) time zero (), 2 h of biofilm formation (▥), 4 h of biofilm formation (░⃞), and 24 h of biofilm formation (). ▪, control biofilm (no farnesol added). P was <0.05 (*) and <0.001 (**) for treated biofilms compared with untreated controls (Student's t test).

FIG. 5.

Effect of farnesol concentration on biofilm formation by C. albicans GDH 2346. Farnesol, at concentrations of 50 μM, 100 μM, and 1 mM, was added at different stages of biofilm development. Biofilm formation (XTT reduction) is expressed as a percentage of that of control biofilms incubated in the absence of farnesol. Results are means ± SEMs from at least two independent experiments carried out in triplicate. Mean (±SEM) control values (A₄₉₂) ranged from 1.999 ± 0.399 to 2.311 ± 0.142. Farnesol was added at adhesion (□) adhesion and time zero (▤) time zero (), 2 h of biofilm formation (▥), 4 h of biofilm formation (░⃞), and 24 h of biofilm formation (). ▪, control biofilm (no farnesol added). P was <0.05 (*) and <0.001 (**) for treated biofilms compared with untreated controls (Student's t test).

FIG. 6.

Effect of simultaneous addition of farnesol and tyrosol on biofilm formation by C. albicans GDH 2346. Farnesol and tyrosol were present during the adhesion period and throughout the 48-h incubation. Biofilm formation (XTT reduction) is expressed as a percentage of that of control biofilms incubated in the absence of farnesol or tyrosol. Results are means ± SEMs from two independent experiments performed in duplicate. The mean (±SEM) control value for biofilm formation in the absence of farnesol (A₄₉₂) was 2.477 ± 0.099. P was <0.05 (*) and <0.001 (**) for treated biofilms compared with untreated controls (Student's t test).

FIG. 7.

Scanning electron micrographs of C. albicans SC5314 biofilms grown on polystyrene disks in the presence of farnesol and tyrosol. Biofilms were grown for 48 h in the presence of 1 mM farnesol and/or 500 μM tyrosol. (A) Control biofilm. (B) Farnesol-treated biofilm. (C) Farnesol-plus-tyrosol-treated biofilm. (D) Tyrosol-treated biofilm. Bar, 10 μm.

FIG. 8.

Scanning electron micrographs showing the effect of tyrosol on the early stages of biofilm formation by C. albicans SC5314. Biofilms were grown on polyvinyl chloride catheter disks in the presence or absence of 50 μM tyrosol. (A) Control biofilms incubated for 1, 2, 3, and 6 h. (B) Tyrosol-treated biofilms incubated for 1, 2, 3, and 6 h. Bar, 10 μm.

FIG. 9.

Effects of planktonic and biofilm culture supernatants on germ tube formation by C. albicans SC5314. The inhibitory effect of culture supernatants on germ tube formation (⋄) is shown, together with cell dry weight over 72 h (▪) for planktonic cells (A) and biofilms (B). This percentage of inhibition of germ tube formation is also shown as a function of cell dry weight (C) for planktonic cultures (□) and biofilms (░⃞). Germ tube formation was determined as a percentage of that for control cells incubated in the absence of culture supernatants. Results are means ± SEMs of two independent experiments carried out in triplicate. Mean (±SEM) values for the controls ranged from 158 ± 7 to 180 ± 12 cells/200 cells counted.