Tyrosol is a quorum-sensing molecule in Candida albicans

Abstract

The human fungal pathogen Candida albicans shows a significant lag in growth when diluted into fresh minimal medium. This lag is abolished by the addition of conditioned medium from a high-density culture. The active component of conditioned medium is tyrosol, which is released into the medium continuously during growth. Under conditions permissive for germ-tube formation, tyrosol stimulates the formation of these filamentous protrusions. Because germ-tube formation is inhibited by farnesol, another quorum-sensing molecule, this process must be under complex positive and negative control by environmental conditions. The identification of tyrosol as an autoregulatory molecule has important implications on the dynamics of growth and morphogenesis in Candida.

Fig. 1.

Cell density controls the length of lag phase in C. albicans. An overnight culture of Candida strain SC5314 was diluted into SD (pH 4.3) at 5 × 10⁷ (A), 5 × 10⁵ (B), and 5 × 10³ (C) cells per ml in the absence (triangles) or presence (squares) of CM. The number of cells at each specific time point was acquired by measuring the OD absorption at 600 nm. The growth curve for each culture was prepared by plotting the logarithmic values of OD₆₀₀ vs. incubation time.

Fig. 2.

Tyrosol is responsible for the growth-promoting activity of CM. (A) A typical analytical scale reverse-phase HPLC elution profile of the activity. The active peak is labeled as QS (quorum signaling). The values on the x axis represent the elution time, and the values on the y axis represent the UV absorption at 215 nm. (B) NMR spectral data for the purified compound. HMBC, heteronuclear multiple bond correlation. (C) The chemically synthesized tyrosol shortens the lag phase of Candida after dilution. An overnight culture of Candida was diluted to 5 × 10³ cells per ml in SD (pH 4.3) in the absence (triangles) or presence (squares) of tyrosol (20 μM). The number of cells at each specific time point was acquired by plating and subsequently counting colony-forming units on yeast extract/peptone/dextrose agar plates.

Fig. 3.

The accumulation of tyrosol in the medium increases with increasing cell density. An overnight Candida culture was diluted to 1 × 10⁵ cells per ml in SD (pH 4.3), and its growth (squares) was measured by counting colony-forming units. The yield of tyrosol at specific time points (triangles) was obtained by measuring its concentration in the medium by analytical scale reverse-phase HPLC.

Fig. 4.

Tyrosol promotes the germ-tube formation in C. albicans.(A) Microscopy analysis of the morphogenesis of Candida in the presence or absence of tyrosol. Diluted Candida cultures (1 × 10⁴ cells per ml) were incubated in SD (pH 7) at 37°C in the presence or absence of tyrosol (20 μM) and photographed at a magnification of ×400. At 1 h in SD (pH 7) without tyrosol, ≈5% of cells formed germ tubes, whereas in the presence of tyrosol, ≈55% of cells formed germ tubes. At 2 h, only ≈15% of cells formed germ tubes when tyrosol was absent, whereas ≈80% of cells formed germ tubes when tyrosol was present. (Scale bar, 25 μm.) (B) Quantitation of the germ-tube formation in Candida when diluted to 10⁴ cells per ml (percentage).

Fig. 5.

Genes regulated by tyrosol. (A) Candida genome array analysis identified a cluster of genes regulated by cell density and tyrosol (≥2.0-fold changes). The level of these transcripts are reduced after dilution but not reduced when tyrosol is present. (B) Semiquantitative RT-PCR confirmed the genome array data. Cells from a saturated Candida culture were washed and reinoculated at either high (10⁸ cells per ml, lane 1) or low (10⁵ cells per ml, lanes 2-5) density in SD (pH 4.3), and total RNA was extracted for RT-PCR analysis. Lane 1, 10⁸ cells per ml; lane 2, 10⁵ cells per ml (30 min); lane 3, 10⁵ cells per ml (60 min); lane 4, 10⁵ cells per ml plus 20 μM tyrosol (30 min); lane 5, 10⁵ cells per ml plus 20 μM tyrosol (60 min). C. albicans rRNA was used as a loading control. (C) The level of CaEDT1 mRNA increases with cell density relative to rRNA controls. Cells from an overnight culture were washed and reinoculated in SD at different cell densities for 30 min, and mRNA was extracted for Northern analysis. Lane 1, 10⁵ cells per ml; lane 2, 10⁶ cells per ml; lane 3, 10⁷ cells per ml; lane 4, 10⁸ cells per ml; lane 5, a saturated overnight culture.