A novel role for the transcription factor Cwt1p as a negative regulator of nitrosative stress in Candida albicans

Abstract

The ability of Candida albicans to survive in the presence of nitrosative stress during the initial contact with the host immune system is crucial for its ability to colonize mammalian hosts. Thus, this fungus must activate robust mechanisms to neutralize and repair nitrosative-induced damage. Until now, very little was known regarding the regulatory circuits associated with reactive nitrogen species detoxification in fungi. To gain insight into the transcriptional regulatory networks controlling nitrosative stress response (NRS) in C. albicans a compilation of transcriptional regulator-defective mutants were screened. This led to the identification of Cwt1p as a negative regulator of NSR. By combining genome-wide location and expression analyses, we have characterized the Cwt1p regulon and demonstrated that Cwt1p is directly required for proper repression of the flavohemoglobin Yhb1p, a key NO-detoxification enzyme. Furthermore, Cwt1p operates both by activating and repressing genes of specific functions solicited upon NSR. Additionally, we used Gene Set Enrichment Analysis to reinvestigate the C. albicans NSR-transcriptome and demonstrate a significant similarity with the transcriptional profiles of C. albicans interacting with phagocytic host-cells. In summary, we have characterized a novel negative regulator of NSR and bring new insights into the transcriptional regulatory network governing fungal NSR.

Figure 1. Growth inhibition assay by the nitric oxide donor DPTA NONOate.

(A) Growth inhibition assay of selected mutant strains exposed to 0.3 and 1 mM DPTA NONOate during 3.5 hours. Growth inhibition rate was normalized relative to the 0 mM DPTA NONOate condition for each strain. Results are from three experiments. (B) Growth defect of cwt1 and cta4 strains was confirmed by an independent NO inhibition assay using different DPTA NONOate concentrations: 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 1 mM. Growth of the wt (SN152) and cwt1/cwt1/CWT1 strains is also shown. Values are average of three replicates. Error bars are standard deviations of triplicates. The symbol (*) and (**) indicate a significant difference compared to the WT strain using t-test (P<0.0005 and P<0.0001, respectively).

Figure 2. Confirmation of ChIP-chip by ChIP-Qpcr.

In vivo occupancy of Cwt1p at ten different intergenic regions. Immunoprecipitated DNA was subjected to qPCR to validate Cwt1p binding at 100–200 bp regions surrounding the probes showing a significant binding ratio. SDs were based on data from two independent experiments.

Figure 3. Gene ontology analysis of Cwt1p-bound promoters.

GO biological process, molecular function and cellular component annotations of Cwt1p bound promoters. P-values were calculated using hypergeometric distribution exploiting the Generic GO Term Finder (http://go.princeton.edu/cgi-bin/GOTermFinder).

Figure 4. Cwt1p bound the promoter region of the nitric oxide dioxygenase YHB1 and controls negatively its transcription.

(A) Cwt1p is detected at the YHB1 promoter using tiled ChIP-qPCR, and the enrichment signal overlaps precisely its DNA-binding motif. Cwt1p occupancy was assessed in the presence and the absence of nitrosative stress. The nitric oxide-responsive element (NORE) recognized by the TF Cta4p is also shown. (B) Average expression of the nitric oxide dioxygenase YHB1 in response to 0.1 mM DPTA NONOate is shown in the wt and cwt1 strains in two independent biological replicates. Fold changes were estimated by using the coding sequence of the C. albicans ACT1 ORF as a reference. Fold enrichments of the tested coding sequences were estimated using the comparative ΔΔCt method.

Figure 5. Cwt1p is a direct regulator of nitrosative stress responsive-genes.

Relationship between Cwt1p-bound genes and genes showing altered expression in the cwt1 mutant challenged by 0.1 mM DPTA NONOate during 15 min. The symbol (*) and (**) indicate a significant difference compared to the WT strain using t-test (P<0.0005 and P<0.0001, respectively).

Figure 6. Cwt1p occupancy of gluconeogenesis, pyruvate metabolic and TCA cycle genes.

Cwt1p binding ratio and transcription level of genes are shown in blue and green, respectively. Genes bound and requiring Cwt1p for their activation were highlighted in red.