CTA4 transcription factor mediates induction of nitrosative stress response in Candida albicans

Abstract

This work has identified regulatory elements in the major fungal pathogen Candida albicans that enable response to nitrosative stress. Nitric oxide (NO) is generated by macrophages of the host immune system and commensal bacteria, and the ability to resist its toxicity is one adaptation that promotes survival of C. albicans inside the human body. Exposing C. albicans to NO induces upregulation of the flavohemoglobin Yhb1p. This protein confers protection by enzymatically converting NO to harmless nitrate, but it is unknown how C. albicans is able to detect NO in its environment and thus initiate this defense only as needed. We analyzed this problem by incrementally mutating the YHB1 regulatory region to identify a nitric oxide-responsive element (NORE) that is required for NO sensitivity. Five transcription factor candidates of the Zn(II)2-Cys6 family were then isolated from crude whole-cell extracts by using magnetic beads coated with this DNA element. Of the five, only deletion of the CTA4 gene prevented induction of YHB1 transcription during nitrosative stress and caused growth sensitivity to the NO donor dipropylenetriamine NONOate; Cta4p associates in vivo with NORE DNA from the YHB1 regulatory region. Deletion of CTA4 caused a small but significant decrease in virulence. A CTA4-dependent putative sulfite transporter encoded by SSU1 is also implicated in NO response, but C. albicans ssu1 mutants were not sensitive to NO, in contrast to findings in Saccharomyces cerevisiae. Cta4p is the first protein found to be necessary for initiating NO response in C. albicans.

FIG. 1.

NORE element position based on data from linker scanning substitution mutations of YHB1 regulatory region. A close-up of the mutations made closest to the NORE is shown. β-Galactosidase activity (on right) was determined as described in Materials and Methods and normalized to activity of wild-type cells under 10 mM sodium nitrite induction. Substituted nucleotides of mutations Msub19 through Msub31 are highlighted in gray. Each strain was tested in three independent trials with at least three independent transformants. Error bar, ±1 standard error of the mean.

FIG. 2.

Differential binding of proteins to wild-type and mutant NORE DNA sequences. Streptavidin-coated magnetic beads were attached to biotinylated DNA containing either the wild type or a mutant NORE and then incubated with 20 mg of whole-cell extract from C. albicans induced with 10 mM sodium nitrite. Lanes from left to right, 1 and 2, flowthrough after initial incubation with whole-cell extract and 3 nmol of competitor DNA; 3 and 4, wash 1 with buffer; 5 and 6, wash 2 with buffer and 1 μmol of poly(dI-dC) nonspecific competitor DNA; 7 and 8, wash 3 with buffer; 9 and 10, final high-salt eluate; 11, Benchmark ladder (Invitrogen). Three bands of proteins with greater affinity for wild-type NORE beads than mutant NORE beads are indicated by white arrows. Similar results were obtained in three other independent trials. MW, molecular weight (in thousands).

FIG. 3.

Growth inhibition of C. albicans strains by NONOates. (A) Growth of the cta4Δ/cta4Δ deletion mutant is hypersensitive to the nitric oxide donor DPTA NONOate. Transcription factor deletion mutants cta4Δ/cta4Δ (written in the format cta4Δ/Δ), stb5Δ/stb5Δ, war1Δ/war1Δ, zcf29Δ/zcf29Δ, and zcf36Δ/zcf36Δ were tested with parental strain SN152 and isogenic wild-type SLA1.1. Cultures were exposed to DPTA NONOate concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, or 1.0 mM and were incubated at 30°C for 3.5 h. OD₆₀₀ readings were then taken as a measure of cell density. Data have been normalized between trial repetitions by expressing cell density readings for a strain as a fraction of the 0 mM DPTA NONOate reading of that strain in each separate trial. Results are from four independent experiments. Error bar, ±1 standard deviation. Analysis of the data for 0.2 mM DPTA NONOate using one-way analysis of variance with Bonferroni's multiple comparison test shows that cta4Δ/Δ is significantly different than yhb1Δ/Δ (P < 0.001). (B) The CTA4 and YHB1 complementation strains recover resistance to the nitric oxide donor DPTA NONOate. Homozygous deletion strains with complementation plasmids (symbolized by cta4Δ/Δ + CTA4 and yhb1Δ/Δ + YHB1) were tested for DPTA NONOate sensitivity along with the SN152 background strain, deletion strains cta4Δ/cta4Δ and yhb1Δ/yhb1Δ, and the deletion strains transformed with the plasmid vector CIp20 used for complementation. The experiment was conducted as described for panel A. Results shown are from three independent experiments. Error bar, ±1 standard deviation. (C) SSU1 does not affect NO sensitivity in YPD medium. The ssu1Δ and yhb1Δ ssu1Δ, plus the SSU1-complemented controls and wild-type strains were grown for 4 h at 30°C in YPD medium with the indicated concentration of DETA NONOate. Cell densities were measured and expressed as a fraction of the control without DETA NONOate, as in panel A. Two independent ssu1Δ mutants and complemented strains were tested, and both behaved identically; for simplicity only one mutant is shown. The data are the average of two experiments. (D) SSU1 does not affect NO sensitivity in synthetic complete medium. The experiment was conducted as in panel C, using synthetic complete medium.

FIG. 4.

Lack of YHB1 RNA accumulation in response to nitrosative stress in the cta4Δ/cta4Δ mutant. Results of Northern blot assays on total RNA from deletion and complementation strains are shown. Cultures were grown to mid-exponential phase and then treated with nitric oxide donor or control solution for 15 min. Blots were probed with YHB1 DNA and loading control TEF1 DNA. (A) Deletion strains treated with 0 mM or 10 mM sodium nitrite. (B) Deletion strains and controls treated with 0 mM or 0.1 mM DPTA NONOate. Homozygous deletion strains are indicated a format where cta4Δ/cta4Δ is represented by cta4Δ/Δ, for example. (C) Complementation strains and controls were treated with 0 mM and 0.1 mM DPTA NONOate. The symbol cta4Δ/Δ + CTA4 indicates the homozygous CTA4 deletion mutant cta4Δ/cta4Δ with the CTA4 complementation plasmid transformed into the genome.

FIG. 5.

In vivo binding of Cta4p to the NORE in both the presence and absence of sodium nitrite. Results of ChIP assay using a yeast strain expressing a single copy of Cta4p with a Myc₉ tag at the C terminus are shown. Cells were grown in YPD medium, and immunoprecipitation (IP) was performed in the presence of mouse anti-Myc antibody 9E10 and protein G beads. Genomic DNA was amplified using PCR primers designed to flank the NORE of the YHB1 regulatory region and give a predicted PCR product of 241 bp. A yeast strain carrying wild-type Cta4p was used as a negative control. Results are representative of three independent experiments.

FIG. 6.

Virulence of cta4Δ/cta4Δ and yhb1Δ/yhb1Δ C. albicans mutant strains against the wild type in a murine tail vein injection model. For each C. albicans strain tested, 10 adult female ICR mice were injected intravenously with 10⁶ mid-exponential-phase yeast cells through the tail vein. (A) The cta4Δ/cta4Δ homozygous deletion strain is symbolized by cta4Δ/Δ. The complementation strain with one copy of CTA4 is indicated as cta4Δ/Δ + CTA4. This assay was repeated twice (n = 20 mice/strain total), and the data from both experiments are combined in the figure. (B) The YHB1 strains are denoted as in panel A. This assay was performed once (n = 10 mice/group).