Five C. albicans transcription factor mutants exhibited hypersensitivity to H2O2
To systematically identify C. albicans regulators of the ROS response, we screened a comprehensive TF mutant library consisting of 211 TF mutant strains in the presence and absence of H2O2 using spot assays on YPD plates. Prior to performing the genetic screen, we assessed the sensitivity of the isogenic wildtype (WT) strain (SN250) to concentrations of H2O2 ranging from 0.5-10.0 mM and found a dose-dependent relationship between increasing H2O2 concentration and increasing oxidative sensitivity (data not shown). When the H2O2 concentration reached 5 mM, the growth of the WT strain was hindered by approximately 50% (Fig. 1), and this concentration was chosen for the screen. We found that, in addition to the known hypersensitive TF mutant strains, cap1Δ/Δ (TF140) and skn7Δ/Δ (TF083), three additional TF mutant strains, dpb4Δ/Δ (TF094), dal81Δ/Δ (TF155) and stp2Δ/Δ (TF162), showed significant sensitivity to H2O2 relative to the WT strain (Fig. 1). No C. albicans TF mutant strains were observed as clearly resistant to H2O2 in our screen (data not shown).
RNA-sequencing of the five TF mutant strains hypersensitive to H2O2
To determine the transcriptome of the five hypersensitive TF mutant strains, we performed RNA-seq on the five mutant strains and the isogenic WT strain in the presence and absence of H2O2. A total of 6,376 transcriptionally active regions were detected from the sequencing, 285 of which were novel transcriptionally active regions (nTARs, Dataset S1 Sheet A). The number of differentially expressed genes among relevant RNA-seq comparisons with fold changes of greater than or equal to twofold (Dataset S1 Sheet B and Fig. S1). For the WT strain SN250, 62 genes were significantly upregulated and 80 genes were significantly downregulated greater than or equal to twofold in the presence of H2O2 (Dataset S1 Sheet B and Fig. S1). Compared to that of SN250, the responses of the TF mutant strains to H2O2 were far more striking. 550, 240, 961, 148 and 105 genes were upregulated greater than or equal to twofold in the presence of H2O2 for the skn7Δ/Δ (TF083), dpb4Δ/Δ (TF094), cap1Δ/Δ (TF140), dal81Δ/Δ (TF155) and stp2Δ/Δ (TF162) mutant strains, respectively (Dataset S1 Sheet B, Fig. S1, and Fig. 2). Additionally, in the presence of H2O2, 828, 778, 888, 278 and 214 genes were downregulated greater than or equal to twofold in these five TF mutant strains, respectively (Dataset S1 Sheet B, Fig. S1 and Fig. 2). Moreover, 15 genes were upregulated greater than or equal to twofold and 48 genes were downregulated greater than or equal to twofold in common among all five TF mutant strains in the presence of H2O2 (Fig. 2 and Fig. 3).
KEGG pathway enrichment and GO term analyses of the differentially expressed genes among the five TF mutant strains hypersensitive to H2O2
To gain an understanding of the biological pathways involved in response to H2O2 for the five hypersensitive TF mutant strains, we performed KEGG pathway enrichment analyses for each strain in the presence and absence of H2O2 (Fig. 4). The enriched pathway in common among these five mutant strains was amino sugar and nucleotide sugar metabolism. Interestingly, the patterns of enrichment for these five mutant strains appeared to be divided into two groups. In the first group, consisting of the skn7Δ/Δ (TF083), dpb4Δ/Δ (TF094) and cap1Δ/Δ (TF140) mutant strains, we observed enrichment mainly in fatty acid metabolism, carbohydrate metabolism, proteasome metabolism, glutathione metabolism, and ribosome synthesis. In the second group, consisting of the dal81Δ/Δ (TF155) and stp2Δ/Δ (TF162) mutant strains, enrichment was mainly in meiosis and the cell cycle. Overall, glycolysis/gluconeogenesis, amino sugar and nucleotide sugar metabolism, and ribosome synthesis were the three most enriched KEGG pathways across all five TF mutant strains (Fig. 5).
We further performed GO term and KEGG enrichment analyses on the 15 upregulated genes and the 48 downregulated genes in common among all five of the TF mutant strains from Fig. 3. The GO term results included molecular function, biological process and cell component as enriched. The KEGG pathways results included metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, human diseases, and drug development as enriched. Tables 1 and 2 present the top five significantly enriched GO terms and KEGG pathways, respectively, of the commonly regulated genes among all five TF mutant strains in the presence of H2O2.
Confirmation of RNA-sequencing data by RT-qPCR
Based on the RNA-seq results presented in Fig. 3, we chose several genes of interest to validate by RT-qPCR. The selected genes were primarily divided into five functional categories: oxidation reduction, membrane, cell wall, transporters, and other (Table 3).
With the exceptions of CFL2 and CFL4 in the dpb4Δ/Δ (TF094) mutant strain, as well as SOD4 in the dal81Δ/Δ (TF155) and stp2Δ/Δ (TF162) mutant strains, which trended in the right directly, but did not meet the significance threshold, the expression patterns of all genes as determined by RT-qPCR were consistent with the RNA-seq results. The transcription factor Cap1 is reported to regulate the catalase-encoding gene CAT1 during oxidative stress [28]; however, we observed by RNA-seq and RT-qPCR that the expression of CAT1 in the cap1Δ/Δ mutant strain was, unexpectedly, not significantly changed in the presence of H2O2, relative to the WT strain. In the presence of H2O2, some genes in certain functional categories displayed clear expression level directions by RNA-seq and RT-qPCR, while others showed varied expression levels. For example, genes encoding membrane regulators/components (e.g. ECM21 and ATO1) were significantly downregulated in all five TF mutant strains in the presence of H2O2 relative to the WT strain by RNA-seq and RT-qPCR. Conversely, genes encoding two transporters involved in antimicrobial resistance, QDR1 and ROA1, showed varied expression patterns relative to the WT strain by RNA-seq and RT-qPCR.