Candida albicans-conditioned medium protects yeast cells from oxidative stress: a possible link between quorum sensing and oxidative stress resistance

Abstract

Candida albicans, the most frequent fungal pathogen of humans, encounters high levels of oxidants following ingestion by professional phagocytes and through contact with hydrogen peroxide-producing bacteria. In this study, we provide evidence that C. albicans is able to coordinately regulate the oxidative stress response at the global cell population level by releasing protective molecules into the surrounding medium. We demonstrate that conditioned medium, which is defined as a filter-sterilized supernatant from a C. albicans stationary-phase culture, is able to protect yeast cells from both hydrogen peroxide and superoxide anion-generating agents. Exponential-phase yeast cells preexposed to conditioned medium were able to survive levels of oxidative stress that would normally kill actively growing yeast cells. Heat treatment, digestion with proteinase K, pH adjustment, or the addition of the oxidant scavenger alpha-tocopherol did not alter the ability of conditioned medium to induce a protective response. Farnesol, a heat-stable quorum-sensing molecule (QSM) that is insensitive to proteolytic enzymes and is unaffected by pH extremes, is partly responsible for this protective response. In contrast, the QSM tyrosol did not alter the sensitivity of C. albicans cells to oxidants. Relative reverse transcription-PCR analysis indicates that Candida-conditioned growth medium induces the expression of CAT1, SOD1, SOD2, and SOD4, suggesting that protection may be mediated through the transcriptional regulation of antioxidant-encoding genes. Together, these data suggest a link between the quorum-sensing molecule farnesol and the oxidative stress response in C. albicans.

FIG. 1.

Susceptibility of C. albicans cells to hydrogen peroxide. Cells were grown (30°C in SD medium) to the specified optical densities (OD₆₀₀), harvested, and washed in PBS. Standardized cell suspensions (1 × 10⁷ cells) were challenged with 1.25 mM hydrogen peroxide for 60 min at 30°C, and viable counts were determined following dilution and plating on SAB plates. Percentages of survival are expressed as the means ± standard deviations of triplicate samples. A survival rate of >100% reflects the inherent variability associated with the plating process between control and test cultures. **, P < 0.001 for sample survival compared to sample survival at an OD₆₀₀ of 0.1 (Student's t test).

FIG. 2.

Survival of early-log-phase C. albicans cells pretreated with fresh medium or 1-day-old culture supernatants and subsequently challenged with hydrogen peroxide or superoxide anion-generating agents. C. albicans cells were grown in SD medium at 30°C until an OD₆₀₀ of 0.15 was reached. Cells were harvested and resuspended in either fresh medium (SD or RPMI) (A to C), filter-sterilized spent medium (A to C), or spent medium that had been diluted with a volume of fresh medium (1:2, 1:5, 1:10, and 1:20) (B). Following 90 min of incubation at 30°C, cells were harvested and washed in PBS, and standardized cell suspensions (1 × 10⁷ cells) were challenged with hydrogen peroxide (1.25 mM for 80 min), menadione (0.6 mM for 60 min), or plumbagin (0.05 mM for 60 min). Yeast cell survival was assessed by dilution and plating on SAB plates. Percentages of survival are expressed as the means ± standard deviations of triplicate samples. A survival rate of >100% reflects the inherent variability associated with the plating process between control and test cultures. **, P < 0.001 for conditioned medium- compared to fresh medium-treated samples (Student's t test). F and S, fresh and spent medium, respectively; N, undiluted sample.

FIG. 3.

Addition of the antioxidant alpha-tocopherol to conditioned medium does not neutralize the protective factor. C. albicans was grown to early log phase (OD₆₀₀ of 0.15) at 30°C in SD medium, harvested, and resuspended in fresh SD medium or spent SD medium with or without alpha-tocopherol (12.5, 25.0, or 50.0 μM). Following 90 min of incubation at 30°C, cells were harvested and washed in PBS, and standardized cell suspensions (1 × 10⁷ cells) were challenged with hydrogen peroxide (1.25 mM for 80 min). Viable counts were determined by dilution and plating on SAB plates. Percentages of survival are expressed as the means ± standard deviations of triplicate samples. A survival rate of >100% reflects the inherent variability associated with the plating process between control and test cultures. P = 0.100 for conditioned medium supplemented with 50 μM alpha-tocopherol compared to conditioned medium without any antioxidant (Student's t test).

FIG. 4.

Protection is not due to the metabolic waste product ethanol. C. albicans was grown to early log phase (OD₆₀₀ of 0.15) at 30°C in SD medium, harvested, and resuspended in fresh SD medium or SD medium with ethanol (0.25 to 1% [vol/vol]). Following 90 min of incubation at 30°C, cells were harvested and washed in PBS, and standardized cell suspensions (1 × 10⁷ cells) were challenged with hydrogen peroxide (1.25 mM for 80 min). Viable counts were determined by dilution and plating on SAB plates. Percentages of survival are expressed as the means ± standard deviations of triplicate samples. P = 0.810 for fresh medium supplemented with 1% (vol/vol) ethanol compared to fresh medium without ethanol (Student's t test).

FIG. 5.

Farnesol, in comparison to conditioned medium, partially protects yeast cells from hydrogen peroxide. C. albicans was grown to early log phase (OD₆₀₀ of 0.15) at 30°C in SD medium, harvested, and resuspended in either fresh SD medium (A and B), SD medium with farnesol (17.5 or 35.0 μM) (A), SD medium with tyrosol (5.0 to 25.0 μM) (B), or spent SD medium (A). Following 90 min of incubation at 30°C, cells were harvested and washed in PBS, and standardized cell suspensions (1 × 10⁷ cells) were challenged with hydrogen peroxide (1.25 mM for 60 min). Viable counts were determined by dilution and plating on SAB plates. Percentages of survival are expressed as the means ± standard deviations of triplicate samples. * and **, P < 0.1 and P ≤ 0.001, respectively, for farnesol- or conditioned medium-treated cells compared to fresh medium-treated cells (Student's t test).

FIG. 6.

Relative RT-PCR analysis of antioxidant gene expression. C. albicans was grown to early log phase (OD₆₀₀ of 0.15) at 30°C in SD medium, harvested, and resuspended in either fresh or conditioned SD medium. Following 90 min of incubation at 30°C, cells were harvested for RNA analysis. Lane 1, negative template control; lane 2, cells exposed to fresh medium minus RT control; lane 3, cells exposed to conditioned medium minus RT control; lane 4, RT-PCR products from analysis of RNAs isolated from cells exposed to fresh medium; and lane 5, RT-PCR products from analysis of RNAs isolated from cells exposed to conditioned medium. EFB1, elongation factor 1β gene; SOD1, copper/zinc-superoxide dismutase gene; SOD2, manganese-superoxide dismutase gene; SOD4, copper/zinc-superoxide dismutase gene; and CAT1, catalase gene. M, molecular marker.