Control of Candida albicans metabolism and biofilm formation by Pseudomonas aeruginosa phenazines

Abstract

Candida albicans has developmental programs that govern transitions between yeast and filamentous morphologies and between unattached and biofilm lifestyles. Here, we report that filamentation, intercellular adherence, and biofilm development were inhibited during interactions between Candida albicans and Pseudomonas aeruginosa through the action of P. aeruginosa-produced phenazines. While phenazines are toxic to C. albicans at millimolar concentrations, we found that lower concentrations of any of three different phenazines (pyocyanin, phenazine methosulfate, and phenazine-1-carboxylate) allowed growth but affected the development of C. albicans wrinkled colony biofilms and inhibited the fungal yeast-to-filament transition. Phenazines impaired C. albicans growth on nonfermentable carbon sources and led to increased production of fermentation products (ethanol, glycerol, and acetate) in glucose-containing medium, leading us to propose that phenazines specifically inhibited respiration. Methylene blue, another inhibitor of respiration, also prevented the formation of structured colony biofilms. The inhibition of filamentation and colony wrinkling was not solely due to lowered extracellular pH induced by fermentation. Compared to smooth, unstructured colonies, wrinkled colony biofilms had higher oxygen concentrations within the colony, and wrinkled regions of these colonies had higher levels of respiration. Together, our data suggest that the structure of the fungal biofilm promotes access to oxygen and enhances respiratory metabolism and that the perturbation of respiration by bacterial molecules such as phenazines or compounds with similar activities disrupts these pathways. These findings may suggest new ways to limit fungal biofilms in the context of disease. IMPORTANCE Many of the infections caused by Candida albicans, a major human opportunistic fungal pathogen, involve both morphological transitions and the formation of surface-associated biofilms. Through the study of C. albicans interactions with the bacterium Pseudomonas aeruginosa, which often coinfects with C. albicans, we have found that P. aeruginosa-produced phenazines modulate C. albicans metabolism and, through these metabolic effects, impact cellular morphology, cell-cell interactions, and biofilm formation. We suggest that the structure of C. albicans biofilms promotes access to oxygen and enhances respiratory metabolism and that the perturbation of respiration by phenazines inhibits biofilm development. Our findings not only provide insight into interactions between these species but also provide valuable insights into novel pathways that could lead to the development of new therapies to treat C. albicans infections.

FIG 1

Effects of phenazines on C. albicans colony development and cellular morphology. (A) C. albicans SC5314 colony morphology when near P. aeruginosa WT or the Δphz strain. (B) Top, C. albicans colonies grown on Glu-AA-GlcNAc at 37°C (inducing conditions) or 30°C in the absence of GlcNAc (noninducing conditions) for 48 h in the absence or presence of 5 µM PMS or 20 µM PYO; bottom, microscopic view of cells from colonies, captured using differential interference contrast (DIC) microscopy. (C) Viable cell counts from C. albicans colonies exposed to phenazines. CFUs from colonies grown on Glu-AA at 30°C without or with 5 µM PMS or 20 µM PYO for 48 h (*, P < 0.05; n = 4).

FIG 2

Phenazines modulate C. albicans extracellular alkalinization and morphogenesis. (A) Fungal colonies grown on Glu+AA or Glu alone under filament-inducing and noninducing conditions without or with 5 µM of PMS and 20 µM PYO for 48 h; pH was monitored using bromocresol purple. The images were obtained by using a stereoscope (7.5×). (B and C) Growth (B) and pH (C) in liquid cultures grown with Glu-AA in the presence and absence of 5 µM PMS at 37°C.

FIG 3

Phenazines promote glucose fermentation. (A) Top, extracellular changes in pH were visualized by addition of bromocresol green; bottom, C. albicans colonies grown for 24 h at 37°C on polycarbonate filters suspended on Glu-AA liquid medium in the presence or absence of 20 µM PYO and 5 µM PMS without bromocresol green. (B) Quantification of acetic acid (black bars) and ethanol (white bars) in supernatants of colonies grown on filters (n = 3; *, P < 0.05).

FIG 4

Phenazines alter C. albicans growth in fermentable and nonfermentable carbon sources. Growth curves of liquid cultures on media containing only glucose (A) or amino acids (B) in the presence (triangles) or absence (open circles) of 5 µM PMS at 37°C. The optical density (OD₆₀₀) of each culture was monitored every hour.

FIG 5

Phenazine-modulated inhibition of C. albicans wrinkled colony morphology is not solely due to decreased extracellular pH. (A and B) C. albicans colonies grown on Glu-AA-GlcNAc with the pH adjusted to 7.0 or 5.0 prior to inoculation. A total of 5 µM of PMS, PYO, or methylene blue (MB) was added to the medium when specified, and the pH indicator bromocresol purple, which yields a red-purple color under alkaline conditions and a yellow-orange color under acidic conditions, was included. In panel A, the medium was buffered with 40 mM MOPS (pH 7) or 100 mM citrate (pH 5) as indicated. Colonies were incubated for 48 h at 37°C.

FIG 6

Effects of phenazines on respiratory metabolism and oxygen consumption. (A) C. albicans colonies grown in Glu-AA medium alone or with 5 µM PMS or 20 µM PYO under noninducing conditions. The respiratory activity of colonies was assessed using TTC, and images were captured using a stereoscope at 10× (top). Internal oxygen measurements in similarly grown colonies were made from the top to the bottom of control colonies (black), with PMS (light gray) or PYO (dark gray). (B) Oxygen gradients in colonies grown under noninducing (smooth) and inducing conditions (wrinkled) were measured at room temperature. The respiratory activity in wrinkled colonies was greater in wrinkles (gray arrow) than in valleys (black arrow) (inset).