Fig. 7: Defined elements within the AOX2 promoter are required for response to specific stressors and support C. albicans virulence.
a Strains were treated with: AA (10 μM), KCN (1 mM), menadione (MD, 0.8 mM) in YPGal, sodium sulfite (0.1 mM for SC5314 and aox2 + AOX2(A) or 0.2 mM for transcription factor (TF) mutant reference strain) in YPGal (pH 4.0), sodium nitroprusside (SNP, 1 mM), or DPTA NONOate (DPTA/NO, 1 mM) in YPGal (pH 7.2). Exposure time was 60 min for MD and 20 min for all other agents. Relative AOX2 level after induction is presented as percentage of ACT1 level (ACT1%) from the same sample and plotted in log2 scale. AOX2 expression varied little with pH (ACT1% range: 2−6.5 to 2−8.0). b Left: Comparison of AOX2 levels post induction ([AOX2]in) between strains with an intact or mutant (nucleotides −1278 to −1000 deleted) promoter. Right: Comparison of AOX2 induction between strains with individual or triple TF-deletion versus the WT strain. Treatments were performed as in a. [AOX2]in ratios (log2) are presented in heatmap (see scale bar). Effects on [AOX2]in are presented in the same manner throughout this figure. c Top: Schematic of AOX2 promoter depicting Mac1-binding consensus sequence (bold font). AOX2(B) encodes two copies of ‘KAGGAATTTGCTCA’ (K: G or T), while the A allele encodes one. Bottom: Mutants with rtg3-deletion or altered CuREs within the AOX2 promoter were cultured in the presence of AA (10 μM; 20 min) or in the absence of copper (-Cu). [AOX2]in was compared between the mutant and WT strain. d AOX2(A) promoters with mutation of GTAC motifs, CuREs (ATTTGCTCA to ATTTtaTaA), or with deletion of nucleotides −1278 to −1000 were used to drive AOX2(A) in aox2-deletion strain. Relative AOX2 induction by AA (10 μM; 20 min), KCN (1 mM; 20 min), or copper starvation in mutant strains were compared to WT. See scale bar in b. e Mutation of AOX2 promoter impairs C. albicans virulence. Survival of mice (n = 8) after challenge by each strain was compared to parental aox2-deletion strain. p value: Mantel-Cox test (n = 8). b–d present mean of technical triplicates from an experiment representative of biological duplicates. See source data file.
