Adaptation of Candida albicans to Reactive Sulfur Species

Abstract

Candida albicans is an opportunistic fungal pathogen that is highly resistant to different oxidative stresses. How reactive sulfur species (RSS) such as sulfite regulate gene expression and the role of the transcription factor Zcf2 and the sulfite exporter Ssu1 in such responses are not known. Here, we show that C. albicans specifically adapts to sulfite stress and that Zcf2 is required for that response as well as induction of genes predicted to remove sulfite from cells and to increase the intracellular amount of a subset of nitrogen metabolites. Analysis of mutants in the sulfate assimilation pathway show that sulfite conversion to sulfide accounts for part of sulfite toxicity and that Zcf2-dependent expression of the SSU1 sulfite exporter is induced by both sulfite and sulfide. Mutations in the SSU1 promoter that selectively inhibit induction by the reactive nitrogen species (RNS) nitrite, a previously reported activator of SSU1, support a model for C. albicans in which Cta4-dependent RNS induction and Zcf2-dependent RSS induction are mediated by parallel pathways, different from S. cerevisiae in which the transcription factor Fzf1 mediates responses to both RNS and RSS. Lastly, we found that endogenous sulfite production leads to an increase in resistance to exogenously added sulfite. These results demonstrate that C. albicans has a unique response to sulfite that differs from the general oxidative stress response, and that adaptation to internal and external sulfite is largely mediated by one transcription factor and one effector gene.

Keywords: Candida albicans; ZCF2; sulfide; sulfite.

Figure 1

Sulfite-pretreated wild-type cells have an increase in sulfite resistance compared to non-pretreated cells. Cultures in log phase were pretreated for 15 min with 0.25 mM sulfite at 30° before being exposed to sulfite concentrations of 0, 2, 4, and 8 mM for 16 hr. Cell density readings were measured at a wavelength of 600 nm for (A) nontreated wild-type cells, (B) pretreated wild-type cells, (C) nontreated Δzcf2 cells, and (D) pretreated Δzcf2 cells. Results are averaged from three independent experiments (n = 9).

Figure 2

Sulfate assimilation pathway in C. albicans.

Figure 3

C. albicans lacking ECM17 are resistant to sulfite. Cell cultures in log phase were challenged for 16 hr with 0 and 12 mM sulfite (filled and open markers, respectively). Results are averages of three biological replicates (n = 3).

Figure 4

Sulfide-pretreated wild-type cells have an increase in sulfite resistance compared to non-pretreated cells. Cultures in log phase were pretreated for 15 min with 0.012 mM sulfide at 30° before being exposed to sulfite concentrations of 0, 2, 4, and 8 mM for 16 hr. Cell-density readings were measured at a wavelength of 600 nm for (A) nontreated wild-type cells, (B) pretreated wild-type cells, (C) nontreated Δzcf2 cells, and (D) pretreated Δzcf2 cells. Results are averaged from three independent experiments (n = 9).

Figure 5

SSU1-gLUC induction, shown as Relative Luminescence Unit (RLU)/OD₆₀₀, by sulfite and sulfide is Zcf2-dependent. Cultures in log phase were grown in the presence or absence (white) of (A) 1.0 mM sulfite (gray) or 0.5 mM sulfide (black) for 30 min, or (B) 1.25 mM cysteine (black) for 1 hr, at 30°. SSU1-gLUC induction by sulfite and sulfide was observed in the wild-type strain, as well as in cells unable to endogenously produce sulfite (Δmet16), sulfide (Δecm17), or either sulfite and sulfide (Δecm17 Δmet16). No SSU1-gLUC induction was observed in the Δzcf2 mutant. Induction by cysteine was seen in all three strains tested. Results are averages of three biological replicates. *** and **** indicate that the detected differences were significant: P < 0.001 and P < 0.0001, respectively.

Figure 6

Mutating motifs 4 and 6 reduced SSU1-gLUC induction, shown as Relative Luminescence Unit (RLU)/OD₆₀₀, by nitrite and sulfite, respectively. Cultures in log phase were grown in the presence (black) or absence (white) of (A and B) 0.5 mM nitrite for 45 min, or (C) 1.0 mM sulfite for 30 min, at 30°. Results are averages of three biological replicates. * and ** indicate that the detected differences were significant: * P < 0.05 and ** P < 0.01, respectively.

Figure 7

Increased sulfate assimilation pathway flux induces C. albicans sulfite resistance. Wild-type and ∆met16 mutant strains were challenged for 16 hr in YEPD pH 4 with 0 and 6 mM sulfite (filled and open markers, respectively) after being grown to log phase for 3 hr in YEPD pH 4, and then in SD medium (supplemented with ammonium chloride and glucose) with or without methionine for 1 hr. Results are averages of four experiments, each with three biological replicates for each condition.

Figure 8

Increased sulfate assimilation pathway flux increases endogenous sulfite levels. SSU1-gLUC reporter luminescence, shown as Relative Luminescence Unit (RLU)/OD₆₀₀, was measured in C. albicans wild-type (white), ∆zcf2 (gray), and ∆met16 (black) strains that were grown for 1 hr at 30° in SD medium containing or lacking ammonium chloride, glucose, and methionine. Nutrients essential for growth, ammonia (NH₃) or glucose, are required for SSU1-gLUC induction, consistent with growth-dependent activity of the sulfur assimilation pathway (Figure S11). Results are averages of three biological replicates. ** indicates that the detected differences were significant: P < 0.01.