Interspecies pheromone signaling promotes biofilm formation and same-sex mating in Candida albicans

Abstract

The opportunistic pathogen Candida albicans undergoes a parasexual mating cycle in which cells must switch from the conventional "white" form to the alternative "opaque" form to become mating competent. Pheromones secreted by opaque cells induce the formation of polarized mating projections and result in cell-cell conjugation. In contrast, white cells are unable to undergo mating, but can still respond to pheromone by expression of adhesion genes that promote biofilm formation. In this study, we have analyzed the dual ability of pheromones to activate mating by opaque cells and biofilm formation by white cells. We first show that there is considerable plasticity in interactions between the α pheromone and its receptor, Ste2, by analysis of analogs of the α pheromone. Significantly, substituted forms of α pheromone can induce a response in opaque cells and this is sufficient to drive same-sex a-a cell fusion and homothallic mating. In addition, pheromone analogs were able to induce adhesion and biofilm formation in white cells of C. albicans. Because of the observed plasticity in pheromone signaling, we subsequently tested putative pheromones from multiple Candida species and identified nonnative ligands that can induce self-mating and biofilm responses in C. albicans. Our findings demonstrate that environmental signals can initiate C. albicans parasexual reproduction and biofilm formation, and highlight the role of the pheromone-signaling apparatus in mediating these functions.

Fig. 1.

Substituted pheromone peptides retain activity in C. albicans. (A) Sequences of di-alanine derivatives of the 13 amino acid C. albicans α pheromone. (B) Substituted pheromones induce a morphological change (wrinkled colonies) because of induction of mating. Colony images of opaque C. albicans a cells exposed to 25 μg of the indicated pheromone daily for 3 d. (Holes in colonies indicate where pheromone was added.) (C) Cells exposed to pheromones form filament-like mating projections. Images of cells taken from the colonies shown in B. (Scale bar, 16 μm.) (D) Quantification of mating gene expression induced by pheromone analogs using a pFIG1-GFP reporter strain (DSY700). Opaque a cells were incubated with 10 μg/mL of peptide in Spider medium for 24 h, then analyzed for GFP fluorescence by flow cytometry. Values are mean fluorescence of the entire cell population (arbitrary units). (E) Sequences of α pheromone analogs of different lengths. (F) Quantification of mating-gene expression induced by pheromone analogs A to G. Cells treated with C. albicans native α pheromone (Ca), cells treated with S. cerevisiae α pheromone (Sc). Error bars represent SEM from a minimum of three replicate experiments. *P < 0.05 vs. DMSO control.

Fig. 2.

Addition of α pheromone or analogs induces same-sex mating of C. albicans a cells. (A) Addition of pheromone to C. albicans opaque a cells results in cell–cell conjugation and progeny that have tetraploid DNA content. Flow cytometry plots showing DNA content of cells fixed and stained with Sytox Green. Three representative mating product strains are shown. (B) PCR confirming the mating type of a-a matings using primers directed at OBPa (Lower) and OBPα (Upper). Control a and α strains together with three pheromone-induced a-a mating products. (C) Frequency of mating induced by di-alanine–substituted pheromones. Opaque a cells from strains RBY1118 (-his) and RBY1179 (-arg) were coincubated with either 25 μg (light bars) or 0.5 μg (dark bars) of pheromone daily for 5 d on Spider medium. Mating products were detected by plating cells on selective medium (-his, -arg), as described (24). (D) Frequency of mating induced by pheromones of different lengths. Error bars represent SEM from a minimum of three experiments.

Fig. 3.

Pheromone analogs induce adhesion in C. albicans white cells. (A) Images of white cells adhering to plastic. C. albicans white P37005 a cells were grown in Lee's medium for 24 h in the presence of di-alanine pheromone analogs (10 μg). Nonadherent cells were removed by washing and pictures of the wells taken. (B) Quantification of adherent white cells shown in A. (C) Images of adherent white cells after exposure to 10 μg of pheromones A-G for 24 h. (D) Quantification of adherent white cells shown in C. Error bars represent SEM from a minimum of three experiments.

Fig. 4.

Pheromones from other Candida species induce same-sex mating and adherence in C. albicans. (A) Phylogenetic tree showing relationship between Candida species and their predicted α pheromones (not to scale). Two potential α pheromones are shown for C. dubliniensis and C. tropicalis. (B) Pheromones from multiple other Candida species induce a morphological change in C. albicans opaque a cells. Images of colonies incubated with 25 μg of pheromone daily for 3 d on Spider medium. (C) Quantification of FIG1 gene expression induced by 10 μg/mL pheromone for 24 h in Spider medium (arbitrary units). (D) Pheromone analogs induce C. albicans a-a conjugation. Quantification of mating products after 5 d culture on Spider medium in the presence of 25 or 0.5 μg pheromone per day. (E) Alternative pheromones induce adherence of C. albicans white cells. Images of adherent P37005 white cells grown in 12-well plastic dishes in Lee's medium and exposed to 10 μg of pheromone for 24 h. (F) Quantification of adherent white cells shown in E. Error bars represent SEM from a minimum of three experiments. *P < 0.05 vs. DMSO control.