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

Abstract

Candida albicans is a member of the human commensal microbiota but can also cause opportunistic infections, including life-threatening invasive candidiasis, particularly in immunocompromised patients. One of the important features of C. albicans commensalism and virulence is its ability to adapt to diverse environmental stress conditions within the host. Rap1 is a DNA-binding protein identified in yeasts, protozoa, and mammalian cells, and it plays multiple functions, including telomere regulation. Intriguingly, our previous study showed that Rap1 is also involved in cell wall integrity, biofilm formation, and virulence in C. albicans. In this work, using RNA-seq analysis and other approaches, the role of C. albicans Rap1 in oxidative stress response was further revealed. The RAP1-deletion mutant exhibited greater resistance to the superoxide generator menadione, a lower level of intracellular reactive oxygen species (ROS) upon menadione treatment, and higher expression levels of superoxide dismutase genes, all in response to oxidative stress. Moreover, the association between Rap1-mediated oxidative stress response and the mitogen-activated protein kinase (MAPK) Hog1, the transcription factor Cap1 and the TOR signalling was also determined. Together, these findings expand our understanding of the complex signalling and transcriptional mechanisms regulating stress responses in C. albicans.

Keywords: Candida albicans; Rap1; oxidative stress.

Figure 1. Gene expression comparison using RNA-seq

(A) Differentially expressed genes (DEGs) in the rap1Δ/Δ mutant compared with the wild-type strain. Numbers of the DEGs with an adjusted P<0.05 and fold-change more than and equal to 1.5-, 2- and 3-fold were shown. (B) Gene ontology (GO) analysis of the DEGs. The most significantly (adjusted P<0.05) enriched GO term in biological process, molecular function, and cellular component branches are presented. Gene numbers in each GO term are indicated. The entire result of this GO analysis is listed in Supplementary Table S3. BP, Biological process; CC, Cellular component; MF, Molecular Function.

Figure 2. Menadione susceptibility assay and superoxide dismutase (SOD) gene expression

(A) Cellular susceptibility to menadione. Cells were ten-fold serially diluted and spotted onto SC agar plates with or without 55 μM menadione. Cells were incubated at 30°C for 3 days. Representative images of three independent experiments with identical results are shown. (B) Measurement of the superoxide content measurement using DHE staining. Cells were stained with DHE (20 μM) and the mean fluorescence intensity (MFI) of 20,000 cells was determined by flow cytometry. The results are expressed as the mean ± standard deviation (SD) of three independent experiments. **, P<0.01; ***, P<0.001. (C) Assessment of SOD gene expression. Cells were treated with 300 μM menadione and incubated at 30°C for 1 h, and the expression of SOD genes was analyzed using real-time qPCR. The ACT1 transcript was used as an endogenous control. The results are displayed as the mean ± SD from three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 3. The Hog1 signaling and the transcription factor Cap1 are associated with Rap1-mediated oxidative stress response

(A) Activation of the Hog1 MAPK. Cells were treated with 300 μM menadione for 0, 30, and 60 min. Then, Hog1 phosphorylation was detected by Western blotting and analyzed by ImageJ software. The total Hog1 band of each sample was served as the loading control to normalize the Hog1-p levels and the fold-change values were indicated. The data are representative of three independent experiments with identical results. (B) The expression of the CAP1 gene. Cells were treated with 300 μM menadione for 1 h, and the gene expression level of CAP1 was analyzed by real-time qPCR. The ACT1 transcript was used as an endogenous control. The results are displayed as the mean ± SD from three independent experiments. ***, P<0.001.

Figure 4. The association of the TOR signaling with Rap1-mediated oxidative stress response

(A) Heat map of differentially expressed genes (DEGs) probably related to the TOR pathway. The DEGs were sorted based on their functions and analyzed by hierarchical clustering calculation with their expression fold changes from RNA-seq analysis to generate Z-score. The Z-score of each data was then used to generate the heat map. a, b, c: the wild-type strain; d, e, f: the rap1Δ/Δ mutant strain. (B) Cellular susceptibility to rapamycin. Cells were ten-fold serially diluted and spotted onto YPD agar plates with or without rapamycin. Cells were cultivated at 30°C for 3 days. Representative images of three independent experiments with identical results are shown. (C) Phosphorylation of the ribosomal protein Rps6 was assayed by Western blotting. Rps6 and Hog1 protein was used as the loading controls, and the phosphorylation ratio (Rps6-p/Rps6) was analyzed by using ImageJ software. The data are representative of three independent experiments with identical results. (D) Cellular susceptibility to menadione with or without blocking the TOR signalling. Cells were pre-treated with or without rapamycin (200 ng/mL) for 1 h and spotted onto SC plates with or without menadione. Viability was recorded after cell growth at 30°C for 3 days. Representative images of three independent experiments with identical results are shown.