The Iron-Dependent Regulation of the Candida albicans Oxidative Stress Response by the CCAAT-Binding Factor

Abstract

Candida albicans is the most frequently encountered fungal pathogen in humans, capable of causing mucocutaneous and systemic infections in immunocompromised individuals. C. albicans virulence is influenced by multiple factors. Importantly, iron acquisition and avoidance of the immune oxidative burst are two critical barriers for survival in the host. Prior studies using whole genome microarray expression data indicated that the CCAAT-binding factor is involved in the regulation of iron uptake/utilization and the oxidative stress response. This study examines directly the role of the CCAAT-binding factor in regulating the expression of oxidative stress genes in response to iron availability. The CCAAT-binding factor is a heterooligomeric transcription factor previously shown to regulate genes involved in respiration and iron uptake/utilization in C. albicans. Since these pathways directly influence the level of free radicals, it seemed plausible the CCAAT-binding factor regulates genes necessary for the oxidative stress response. In this study, we show the CCAAT-binding factor is involved in regulating some oxidative stress genes in response to iron availability, including CAT1, SOD4, GRX5, and TRX1. We also show that CAT1 expression and catalase activity correlate with the survival of C. albicans to oxidative stress, providing a connection between iron obtainability and the oxidative stress response. We further explore the role of the various CCAAT-binding factor subunits in the formation of distinct protein complexes that modulate the transcription of CAT1 in response to iron. We find that Hap31 and Hap32 can compensate for each other in the formation of an active transcriptional complex; however, they play distinct roles in the oxidative stress response during iron limitation. Moreover, Hap43 was found to be solely responsible for the repression observed under iron deprivation.

Fig 1. The CCAAT-binding factor regulates CAT1 in response to iron availability.

(A) Northern blot analysis of CAT1 mRNA expression in the wild-type (DMC146) and hap5Δ/Δ mutant (DMC117) following growth in iron-replete (+iron) and iron-limiting (-iron) medium. The 26s rRNA was the loading control. mRNA levels were normalized to the 26s rRNA control using the WT as the reference value. (B) C. albicans wild-type (DMC146) and hap5Δ/Δ mutant (DMC117) were grown in iron-replete (+iron) or iron-limited (-iron) medium and subsequently exposed to hydrogen peroxide at the indicated concentrations for 2 h at 30°C. Ten-fold serial dilutions were spotted to YPD medium and incubated at 30°C for 3 days. (C) Catalase enzymatic activity in cell extracts derived from the wild type (DMC146) and hap5Δ/Δ mutant (DMC117) following growth in iron-replete (+iron) and iron-limiting (-iron) medium. The enzymatic assays are the average of three independent experiments with the error bars indicating the standard error. (D) C. albicans wild-type (DMC356) and hap5Δ/Δ mutant (DMC357) were grown in iron-replete (+iron) or iron-limited (-iron) medium and subsequently assayed for expression of Renilla luciferase driven by the CAT1 promoter. The luciferase assays are the average of three independent experiments with the error bars indicating the standard error.

Fig 2. SOD1, SOD2, SOD3, and SOD4 are regulated by the CCAAT-binding factor.

Northern blot analysis was performed to examine the expression of SOD1, SOD2, SOD3, and SOD4 mRNA as indicated in the wild-type (DMC146) and hap5Δ/Δ mutant (DMC117) following growth in iron-replete (+iron) and iron-limiting (-iron) medium. The 26s rRNA was the loading control.

Fig 3. GRX2, GRX5, and TRX1 expression is regulated by the CCAAT-binding factor.

Northern blot analysis was performed to examine the expression of GRX2, GRX3, GRX5, and TRX1 mRNA as indicated in the wild-type (DMC146) and hap5Δ/Δ mutant (DMC117) following growth in iron-replete (+iron) and iron-limiting (-iron) medium. The 26s rRNA was the loading control.

Fig 4. Hap31 and Hap32 display a differential response to hydrogen peroxide stress.

(A) C. albicans wild-type (DMC146), hap2Δ/Δ (DMC249), and hap5Δ/Δ mutant (DMC117), were grown in iron-replete (+iron) or iron-limited (-iron) medium and subsequently exposed to hydrogen peroxide at the indicated concentrations for 2 h at 30°C. Ten-fold serial dilutions were spotted to YPD medium and incubated at 30°C for 2 days. (B) C. albicans wild-type (DMC146), hap31Δ/Δ (DMC280), hap32Δ/Δ (DMC285), and the hap31Δ/Δ hap32Δ/Δ (DMC290) mutants were grown in iron-replete (+iron) or iron-limited (-iron) medium and subsequently exposed to hydrogen peroxide at the indicated concentrations for 2 h at 30°C for 3 days.

Fig 5. Hap31 and Hap32 are interchangeable in the function of the CCAAT-binding factor.

(A) Northern blot analysis of CAT1 mRNA expression in the wild-type (DMC146) and the indicated hap mutants following growth in iron-replete (+iron) and iron-limiting (-iron) medium. The 26s rRNA was the loading control. mRNA levels were normalized to the 26s rRNA control using the WT as the reference value. (B) Catalase activity in cell extracts derived from the wild type (DMC146), hap31Δ/Δ (DMC280), hap32Δ/Δ (DMC285), and hap31Δ/Δ hap32Δ/Δ (DMC290) mutants following growth in iron-replete (+iron) and iron-limiting (-iron) medium. The enzymatic assays are the average of three independent experiments with the error bars indicating the standard error.

Fig 6. The role of the individual Hap4-like subunits in regulating the OSR.

(A) C. albicans wild-type (DMC146), hap41Δ/Δ (DMC190), hap42Δ/Δ (DMC350), hap43Δ/Δ (DMC351) and hap5Δ/Δ mutant (DMC117) were grown in iron-replete (+iron) or iron-limited (-iron) medium and subsequently exposed to hydrogen peroxide at the indicated concentrations for 2 h at 30°C. Ten-fold serial dilutions were spotted to YPD medium and incubated at 30°C for 3 days. (B) Catalase activity assays were performed on extracts from the wild type (DMC146), hap41Δ/Δ (DMC190), hap42Δ/Δ (DMC350), hap43Δ/Δ (DMC351) and hap5Δ/Δ mutant (DMC117) mutants following growth in iron-replete (+iron) and iron-limiting (-iron) medium. The enzymatic assays are the average of three independent experiments with the error bars indicating the standard error.

Fig 7. Hap43 is the sole Hap4 subunit involved in the CCAAT-binding factor-mediated oxidative stress response.

(A) C. albicans wild-type (DMC146) and indicated hap4 combination mutants were grown in iron-replete (YPD) or iron-limited (YPD+BPS) liquid medium and ten-fold serial dilutions were spotted to YPD or YPD+BPS, respectively, and incubated at 30°C for 3 days. (B) C. albicans wild-type (DMC146), hap5Δ/Δ (DMC117) and indicated hap4Δ/Δ combination mutants were grown in iron-replete (+iron) or iron-limited (-iron) medium and subsequently exposed to hydrogen peroxide at the indicated concentrations for 2 h at 30°C. Ten-fold serial dilutions were spotted to YPD medium and incubated at 30°C for 3 days.

Fig 8. Hap43 is necessary for the CCAAT-binding factor-mediated regulation of CAT1 in response to iron.

Northern blot analysis of CAT1 mRNA expression in the wild-type (DMC146), hap5Δ/Δ mutant (DMC117) and the indicated hap4Δ/Δ single or combination mutants following growth in (A) iron-replete (+iron) and (B) iron-limiting (-iron) medium. The rRNA was the loading control. mRNA levels were normalized to the rRNA control using the WT as the reference value.