Transcriptional profiling reveals the role of Candida albicans Rap1 in oxidative stress response

Candida albicans is a commensal fungus that can cause opportunistic infections, particularly in immunocompromised patients. Worst of all, C. albicans can even cause bloodstream infections, and the mortality rate of these infections can reach more than 50% [1]. An important feature related to C. albicans pathogenicity and virulence is its adaptability to harsh environmental conditions, including changes in pH and temperature, limitation of nutrients, and attack from the immune system [2–5]. Through different stress responsive mechanisms, C. albicans is able to overcome adversities, allowing the pathogen to grow and proliferate within the host [6,7]. These stress responses are coordinately regulated by various components of signalling and regulatory networks [7].

The maintenance of oxidation-reduction balance, known as redox homeostasis, is closely associated with generation and elimination of reactive oxygen species (ROS) that are by-products of aerobic metabolism [8,9]. Excessive ROS can induce oxidative stress and damage DNA, lipids, and proteins, leading to cell death. C. albicans encounters ROS-induced oxidative stress during the respiratory burst in macrophages and neutrophils, as well as from ROS-generating antifungal drugs such as amphotericin B, miconazole, and caspofungin [10–12]. To cope with oxidative stress in C. albicans, the involvement of antioxidants (superoxide dismutase, catalase, glutathione, and thioredoxin system), the Hog1 mitogen-activated protein kinase (MAPK) signalling, and the transcription regulator Cap1 are well recognized [9,10,13].

Repressor activator protein 1 (Rap1) is a multifunctional DNA-binding protein identified in different eukaryotic organisms [14]. In Saccharomyces cerevisiae, Rap1 plays an important role in subtelomeric gene silencing [15], telomere length control [16], and telomere end protection [17–19]. In addition, S. cerevisiae Rap1 also possesses other non-telomeric functions, including transcriptional regulation and metabolic control [20,21]. Similarly, C. albicans Rap1 participates in the control of telomere length and structure [22–24]. However, interestingly, we have previously found that C. albicans Rap1 is also associated with cell wall integrity and biofilm formation, and modulates phagocytosis and cytokine release by host immune cells [25]. Importantly, Rap1 is also related to C. albicans virulence as shown in a Galleria mellanella infection model.

In the present study, we aimed to explore more functions of Rap1 in C. albicans. Using RNA sequencing (RNA-seq) data with Gene Ontology (GO) analysis, different categories of the differentially expressed genes (DEGs) between the RAP1-deletion (rap1Δ/Δ) and wild-type strains were revealed, including the category of cell wall, membrane, and redox process. Here, we characterized the role of Rap1, particularly with respect to its function in oxidative stress response, which is associated with redox process. The rap1Δ/Δ mutant was much more resistant to the superoxide generator menadione, altered intracellular ROS levels, and had higher expression levels of superoxide dismutase genes. In addition, the Hog1 MAPK signaling and Cap1 were somehow associated with the Rap1-mediated response to menadione. Finally, using Gene Set Enrichment Analysis (GSEA) and other approaches, the link between the Target of Rapamycin (TOR) signalling pathway and Rap1-mediated response to menadione was also uncovered. Together, our findings not only reveal new functions of Rap1 but also highlight the complex signaling and regulation of oxidative stress response in C. albicans.

Rap1 is a DNA-binding protein identified in different eukaryotes including yeasts and mammals. Other than telomeric functions, nontelomeric activities have been revealed in S. cerevisiae and mammalian cells. Therefore, we hypothesized that C. albicans Rap1 may also possess nontelomeric functions. Indeed, our previous study linked C. albicans Rap1 to cell wall integrity, biofilm formation and interactions with host immune cells [25]. In the present study, we used genomic profiling and other approaches to further explore new functions of C. albicans Rap1 and found its role in oxidative stress response and a new relationship with the TOR signalling.

C. albicans strains used throughout this study include SC5314 (RAP1/RAP1), RAP1-deletion (rap1Δ/Δ), and RAP1-reintegrated (rap1Δ::RAP1/rap1Δ::RAP1) strain [25]. All strains were routinely maintained at −80°C and plated on YPD agar (1% of yeast extract, 2% of peptone [Condalab, Madrid, Spain], and 2% of glucose). A single colony was inoculated in YPD broth and grown overnight at 30°C with shaking (180 rpm). The overnight grown cells were subcultured in synthetic complete (SC) medium (0.67% of yeast nitrogen base [YNB] with ammonium sulfate, 2% of glucose and 0.079% complete supplement mixture [MP Biomedicals, Santa Ana, CA, USA]) with an initial optical density at 600 nm (OD₆₀₀) of 0.5 and grown at 30°C to the early exponential phase. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless indicated otherwise.

C. albicans cells were collected and resuspended in sterile phosphate-buffered saline (PBS). Ten microliters of 10-fold serial dilution of cells were spotted onto agar plates supplemented with menadione or rapamycin (Cayman Chemical, Ann Arbor, MI, USA) as indicated. Cell viability was recorded after incubation at 30°C for 3 days.

Total RNA was extracted using TRIzol Reagent (Invitrogen, Waltham, MA, USA), and ribosomal RNA (rRNA) was removed using the RiboMinus Eukaryote Kit (Thermo Fisher Scientific, Waltham, MA, USA). The yield and quality of purified RNA were assessed using an ND-1000 spectrophotometer (Nanodrop Technology, Wilmington, DE, USA) and a Bioanalyzer 2100 with the RNA 6000 Nano kit (Agilent Technology, Santa Clara, CA, USA). The RNA library was prepared using the SureSelect XT HS2 mRNA Library Preparation kit (Agilent), followed by DNA cleanup and size selection using AMPure XP beads (Beckman Coulter, Brea, CA, USA). Sequencing data (FASTQ reads) were generated using the Illumina Solexa platform (Illumina, San Diego, CA, USA) based on Illumina's basecalling program bcl2fastq v2.20. Sequencing reads were aligned to the C. albicans SC5314 genome assembly 22 (http://www.candidagenome.org/) with the HISAT2 alignment program to generate read counts [26]. RNA-seq data are available in the NCBI Sequence Read Archive (SRA) with the accession number PRJNA1114919. Three independent biological replicates of C. albicans cultures grown in SC medium to the early exponential phase were used for transcriptome analysis.

Transcriptome analysis was performed using raw read counts, and normalization along with differential expression analysis was carried out using the Bioconductor DESeq2 package (version 3.18) within the RStudio environment (version 4.3) under default parameters. DESeq2 models gene counts with a generalized linear model (GLM) that uses a negative binomial distribution to account for biological variability. The Wald test was used for hypothesis testing between two conditions [27]. Dispersion estimation was performed using parametric fitting (fitType), modeling dispersion as a function of the mean, with empirical Bayes shrinkage applied for stabilization. Additionally, multiple testing was corrected using the Benjamini-Hochberg method to control the false discovery rate (FDR) and to generate adjusted p-values shown in Supplementary Table S1. Gene Ontology enrichment analysis was subsequently conducted using the FungiFun2 tool (version 2.2.8 BETA) to identify enriched biological processes, cellular components, and molecular functions associated with the DEGs [28].

For GSEA, the pre-rank analysis was conducted using the Gseapy package (version 1.0.6), a Python wrapper for GSEA. Pre-ranked correlation table was generated from the differential expression results from DESeq2, ranked by log2 fold change. The gene set collection for running GSEA was obtained from Andre Nantel (http://www.candidagenome.org/download/community/GSEA_Nantel_2012/). Default parameters were applied, except for the maximum and minimum gene set sizes, which were adjusted to 250 and 15, respectively. To enhance the robustness of the analysis, the number of permutations was increased to 30,000. The pre-ranked results were sorted by FDR. For the results with an FDR of 0, the value was set to less than 1 divided by the number of permutations. The results were output as a table for further analysis.

For creating heatmaps, the normalization results from the differential expression analysis were converted into z-scores using the ‘zscore’ function from the ‘scipy.stats' module (SciPy version 1.11.2) along the gene axis. The heatmap was then generated using the ’clustermap’ function from the Seaborn library (Seaborn version 0.12.2). All analyses were performed using Python 3.11.5 in the Visual Studio Code environment.

RNA isolation and reverse transcription for cDNA synthesis were performed as previously described [29]. Real-time qPCR was conducted using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The primers used for real-time qPCR are listed in Supplementary Table S2. Gene expression levels were normalized to the ACT1 transcript levels [30]. All experiments were performed in triplicate, with three independent experiments for each strain, and the average cycle threshold (C_T) values were determined. The 2^−ΔΔCT method was used to calculate the relative fold change in the expression of each gene [31].

Cells were treated with or without menadione (300 μM) for 1 hour and washed twice with sterile PBS. To measure superoxide anion accumulation, cells were stained with dihydroethidium (DHE, 20 μM, Thermo Fisher Scientific) for 10 min. After staining, cells were washed twice with PBS, and the mean fluorescence intensity was measured with PE detector using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA).

Cells were harvested and resuspended in Radio-Immunoprecipitation Assay (RIPA) lysis buffer containing various protease inhibitors [25]. Cells were broken by vortexing with acid-washed glass beads. Supernatants were collected by centrifugation, and total proteins were extracted [25]. The total protein concentration was determined using a Bio-Rad Bradford assay (Bio-Rad, Hercules, CA, USA).

For western blotting to detect phosphorylated Hog1 (Hog1-p) and Rps6 (Rps6-p), proteins were separated by 10% of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis as previously described [32]. Hog1-p and total Hog1 were detected using anti-phospho-p38 MAPK (catalog no. 9215; Cell Signalling Technology, Danvers, MA, USA) and anti-Hog1 antibodies (catalog no. sc-9079; Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively. Moreover, anti-phospho-(Ser/Thr) Akt substrate antibody (catalogue no. 9611; Cell Signalling Technology) was used to detect Rps6-p, and anti-ribosomal protein S6/RPS6 antibody (catalogue no. AF5436; R&D Systems, Minneapolis, MN, USA) was used to detect total Rps6. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (no. GTX213110-01, GeneTex, Hsinchu, Taiwan), and HRP-conjugated anti-sheep IgG (catalogue no. HAF016, R&D Systems) were used as the secondary antibodies. HRP was detected by the Western Lightning Plus chemiluminescent substrates (PerkinElmer, Waltham, MA, USA), and the blot images were taken using an ImageQuant LAS 4000 biomolecular imager (GE Healthcare, Chicago, IL, USA).

As mentioned above, Rap1 is involved in cell wall integrity, biofilm formation, and virulence in C. albicans [25]. To explore other functions of Rap1 in this important pathogen, RNA-seq was used to profile gene expression in the rap1Δ/Δ and wild-type strains. A total of 926 genes were significantly modulated (≥ 1.5-fold change and adjusted P<0.05, see Supplementary Table S2). Among them, 478 genes were up-regulated, and 448 genes were down-regulated in the rap1Δ/Δ compared with the wild-type strain (Figure 1A).

GO analysis was then performed to identify enriched biological categories for the DEGs using the FungiFun2 functional enrichment tool [28]. A total of 23 and 11 GO terms were independently identified in the up-regulated and down-regulated genes of the rap1Δ/Δ mutant compared with the wild-type strain. These categories involve various biological processes, molecular functions, and cellular components (Figure 1B and Supplementary Table S3). Among the up-regulated genes, the GO terms include “telomere maintenance via recombination,” “interstrand cross-link repair,” “damaged DNA binding,” and “fungal-type cell wall”. The identification of these categories is consistent with previous findings that Rap1 plays a role in telomere regulation and cell wall integrity [24,25,33]. Moreover, the GO terms of the down-regulated genes include metabolic processes, such as “purine nucleobase biosynthetic process”, and “de novo IMP biosynthetic process”. Interestingly, many up-regulated and down-regulated genes were related to “oxidoreductase activity” and “oxidation-reduction process”. However, the relationship between Rap1 and redox-related events has yet to be studied.

Redox processes are primarily linked to aerobic metabolism for energy generation and are associated with the production of ROS [8,9]. ROS not only modulate redox reactions, but also influence intracellular redox homeostasis [34]. Moreover, ROS can cause oxidative damage, and the response to ROS-mediated oxidative stress is critical for C. albicans pathogenicity and virulence [10]. For example, massive ROS generation by host immune cells of the host can cause irreversible damage and death of C. albicans cells [10]. As shown in Figure 1B and Supplementary Table S3, GO enrichment analysis suggested an association between Rap1 and redox process. Therefore, we aimed to investigate potential differences in oxidative stress response among the wild-type, rap1Δ/Δ, and RAP1-reintegrated strains.

To test this hypothesis, we examined cell susceptibility to menadione using a spot assay. Figure 2A shows that the rap1Δ/Δ mutant exhibited significantly higher resistance to the superoxide generator menadione compared with the wild-type and RAP1-reintegrated cells. Superoxide anions represent the main resource of ROS in cells and are produced as by-products of the mitochondrial electron transport chain (ETC). In addition, superoxide levels were measured using DHE staining and analyzed by flow cytometry. The result indicated that intracellular superoxide levels increased in wild-type and RAP1-reintegrated cells treated with menadione compared with untreated cells. However, in the rap1Δ/Δ mutant, intracellular superoxide levels decreased slightly after menadione treatment compared with untreated mutant cells. Furthermore, menadione-treated rap1Δ/Δ mutant cells exhibited lower intracellular superoxide levels than wild-type and RAP1-reintegrated strains (Figure 2B).

ROS levels and cellular redox state are regulated by various antioxidant enzymes [35,36], which are closely associated with cellular responses to oxidative stress [10]. In C. albicans, superoxide dismutases (Sods) play a critical role by converting superoxide into hydrogen peroxide (H₂O₂) [10,37]. To further explore the function of Rap1 in oxidative stress response, the expression of SOD genes was monitored using real-time qPCR. The results revealed that the expression of SOD genes was upregulated in the rap1Δ/Δ mutant upon menadione treatment compared with the control strains (Figure 2C). Together, these results suggest a link between Rap1 and oxidative stress response in C. albicans.

In C. albicans, the Hog1 MAPK pathway is known to function in the regulation of oxidative stress response [10,38,39]. Moreover, independent of the Hog1 pathway, Cap1 is a major regulator in the transcriptional responses to oxidative stress [10,39–41]. Based on the results that correlate Rap1 to oxidative stress response (Figure 2A–C), we were therefore interested in examining the relationship among Hog1, Cap1, and Rap1. As shown by semi-quantitative analysis of western blots, the rap1Δ/Δ mutant had an overall higher level of phosphorylated Hog1 (Hog1-p) than the wild-type and RAP1-reintegreated strains, particularly in cells treated with menadione for 30 min (Figure 3A). Interestingly, the expression of the CAP1 gene was upregulated in the rap1Δ/Δ mutant compared with the other two control strains (Figure 3B). These results suggest that the Hog1 pathway and Cap1 may also be involved in the Rap1-mediated oxidative stress response.

In response to nutrient availability and stress conditions, the yeast TOR signalling pathway regulates various cellular processes, including protein synthesis, ribosome biogenesis, and autophagy. Thereby, the TOR pathway is critical in coordination of cell growth, proliferation, metabolism, and cell cycle progression [42,43].

To derive more insights into Rap1 functions from our RNA-seq data, GSEA was further performed, using an extensive gene data set generated by Nantel and colleagues [44]. Using a false discovery rate (FDR) ≤ 0.25 as a cut-off, 227 gene sets were enriched (Supplementary Table S4). Interestingly, the enriched groups include ‘ribosome biogenesis & assembly,’ ‘translation,’ and ‘ribosome’, which are all related to protein biosynthesis. More intriguingly, several groups of our DEGs were also found to be related to translation, nitrogen starvation response, amino acid metabolism, autophagy, and PHO regulon (Supplementary Table S2 and Figure 4A). Together, we suspected that the TOR signalling may also be involved in Rap1 functions.

To examine the possible link between the TOR pathway and Rap1, spot assay of viability of cells treated with or without rapamycin was conducted. Rapamycin is an inhibitor of the Tor1 kinase, which is the key component of the TOR pathway. The rap1Δ/Δ mutant had much higher resistance to rapamycin than the wild-type and RAP1-reintegrated strains (Figure 4B). In addition, the ribosomal protein S6 (Rps6) is a downstream target of the TOR signalling and is phosphorylated through the TOR activity in C. albicans [45]. To further connect the TOR pathway and Rap1, levels of phosphorylated Rps6 (Rps6-p) were measured by western blotting. In Figure 4C, as a control, cells treated with rapamycin had no detectable Rps6-p. However, without rapamycin treatment, the levels of Rps6-p were increased in the wild-type and RAP1-reintegrated cells, but not in the rap1Δ/Δ mutant. Finally, the relationship between the TOR pathway and Rap1-mediated oxidative stress response was tested. Cells were pre-treated with or without rapamycin for 1 h, followed by spotted onto SC agar plates with or without menadione. As shown in Figure 4D, the wild-type and RAP1-reintegrated strains were more resistant to menadione in cells pretreated with rapamycin than those without rapamycin pretreatment. However, the rap1Δ/Δ mutant was not affected by rapamycin and exhibited a much more resistance to menadione. These results suggest the role of Rap1 in oxidative stress response modulated by the TOR signalling.

Rap1 is a multifunctional DNA binding protein identified in various eukaryotic organisms, including mammals, S. cerevisiae, and C. albicans [20]. In S. cerevisiae and C. albicans, Rap1 shares similar functions in telomere maintenance [20]. However, divergent structures and functions exist between Rap1 in S. cerevisiae and C. albicans. First, C. albicans Rap1 exhibits an unusual truncated protein structure lacking the C-terminal domain, which is implicated in telomere regulation of S. cerevisiae Rap1 [24]. Secondly, S. cerevisiae RAP1 is an essential gene, whereas RAP1 is not essential for cell viability of C. albicans [24]. Intriguingly, our recent study demonstrated that C. albicans Rap1 is involved in cell wall integrity, biofilm formation, and virulence [25], functions not displayed in S. cerevisiae Rap1. Therefore, expanding our knowledge of new functions of Rap1 in C. albicans is of great interest.

To explore this further, RNA-seq analysis was performed. Using GO analysis on RNA-seq data measuring differential expression between the rap1Δ/Δ and wild-type strain, many GO terms including redox process were enriched (Figure 1B and Supplementary Table S3). By measuring cell susceptibility to menadione, intracellular ROS accumulation, and the expression levels of the superoxide dismutase SOD genes (Figure 2A–C), we further demonstrated that Rap1 is involved in the oxidative stress response, which is associated with redox process.

Both menadione and H₂O₂ are commonly used oxidants in studies of cellular oxidative stress response [46]. As shown in Figure 2A, the rap1Δ/Δ mutant exhibited much greater resistance to menadione compared with the wild-type and RAP1-reintegrated strains. Interestingly, however, we observed no significant differences in susceptibility to H₂O₂ among all the tested strains (data not shown). It is well known that menadione and H₂O₂ function differently and elicit distinct cellular responses in both yeast and mammalian cells [46–48]. Menadione primarily triggers an integrated stress response, whereas H₂O₂ broadly influences biological processes [46]. Although the observation that RAP1 deletion leads to different cellular responses to menadione and H₂O₂ is intriguing, further studies are needed to elucidate the detail mechanisms. Additionally, while all SOD genes were upregulated in the rap1Δ/Δ mutant, the upregulation of SOD3 was more pronounced (Figure 2C). The SOD3 gene encodes a cytoplasmic manganese-containing superoxide dismutase in C. albicans, whose activation is regulated by the copper-responsive transcription factor Mac1 during copper limitation in the stationary phase [49–51]. Notably, overexpression of SOD3 alleviate menadione toxicity and rescues cell growth [49], suggesting that the reduced ROS accumulation and enhanced resistance to menadione observed in rap1Δ/Δ mutant may be partially attributed to the elevated expressed SOD3 (Figure 2A–C). Investigating how Rap1 regulates SOD3 expression and the potential connection between Rap1, copper and Mac1 will be valuable for further research.

Rap1 has been implicated in oxidative stress response in mammalian cells through the NF-κB signalling pathway and sirtuins [20]. Moreover, Rap1 levels are affected by oxidative stress, and Rap1-deficient mice exhibit elevated oxidative stress [52,53]. Also, in response to mild ethanol stress, increased expression of Rap1 and sirtuin proteins (Sir1, Sir2, and Sir3) is accompanied by the increase in total ROS and superoxide in industrial wine yeast strains [54]. Sirtuins are a class of nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylases, involved in signal transduction and various biological processes [55]. Interestingly, the association between telomeres and sirtuins has been a focus of some research [56,57], and several subtelomeric genes were identified as differentially expressed in the rap1Δ/Δ mutant in this study (Supplementary Tables S1 and S5). Therefore, whether telomeres and Sir proteins affect Rap1-mediated oxidative stress response is worth further investigation.

C. albicans encounters oxidative stress from host phagocytes and antifungals [10,11]. The well-known mechanism to respond to oxidative stress and activate antioxidant gene expression is primarily mediated by the Hog1 MAPK signalling and the transcription factor Cap1 [58,59]. In response to oxidative stress, C. albicans Hog1 is rapidly activated and accumulates in the nucleus, though it does not play a major role in regulating oxidative stress-related gene expression [60,61]. Additionally, Cap1 is normally oxidized in response to H₂O₂, leading to its nuclear accumulation and activation of target genes [59,62]. Although our results showed that RAP1 deletion affects Hog1 phosphorylation and CAP1 expression level, the detailed mechanisms still remain to be elucidated.

Recently, TOR signalling has been linked to oxidative stress in C. albicans. Lack of the N-terminal HEAT repeats in the Tor1 kinase led to significantly impaired cellular response to oxidative stress [63]. Furthermore, overexpression of Gtr1, the activating GTPase of the TOR complex 1, rescues the Sod3 deficiency in the manganese transporter PHO84-deletion mutant [63]. Interestingly, our previous studies found that the transcription factor Sfp1, which functions downstream of the Tor1 signalling, also regulates oxidative stress response in C. albicans [64,65]. In the present study, we further established a correlation between Rap1-mediated oxidative stress response and TOR signalling (Figure 4B–D). Future studies will be needed to examine the relationship between Rap1, TOR signalling, Sod3, Pho84, and Sfp1.

In conclusion, our findings not only reveal new functions of Rap1 but also underscore the complex regulatory machinery involved in the oxidative stress response of C. albicans.

Based on our findings in the present study, at least two main directions are worth considering for future research. First, whether and how telomeres and Sir proteins are associated with Rap1-mediated oxidative stress response in C. albicans. Secondly, how Rap1 interplays with different signalling pathways and other transcription regulators in controlling oxidative stress response in C. albicans.

The data presented in this study are available within the article and the Supplementary material. RNA-seq data are available in the NCBI Sequence Read Archive (SRA) with the accession number PRJNA1114919.

The authors declare that there are no competing interests associated with the manuscript.

This work was supported by the National Science and Technology Council (NSTC), Taiwan (grant numbers 113-2311-B-007-001, 112-2311-B-007-001 and 109-2311-B-007-001-MY3 to C.-Y. Lan). This funder had no role in the design of this study, data analyses, and interpretation of the results.

Wen-Han Wang: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review & editing. Hsuan-Yu Chen: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review & editing. Sheng-Yuan Chen: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review & editing. Chung-Yu Lan: Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review & editing.

DEGs

differentially expressed genes

DHE

dihydroethidium

FDR

false discovery rate

GSEA

gene set enrichment analysis

MAPK

mitogen-activated protein kinase

PBS

phosphate-buffered saline

qPCR

quantitative PCR

RAP1

repressor activator protein 1

ROS

reactive oxygen species

SD

standard deviation

SOD

superoxide dismutase