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. 2022 Jul 7;23(14):7555.
doi: 10.3390/ijms23147555.

Transcriptional Profiling of the Candida albicans Response to the DNA Damage Agent Methyl Methanesulfonate

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Transcriptional Profiling of the Candida albicans Response to the DNA Damage Agent Methyl Methanesulfonate

Yuting Feng et al. Int J Mol Sci. .

Abstract

The infection of a mammalian host by the pathogenic fungus Candida albicans involves fungal resistance to reactive oxygen species (ROS)-induced DNA damage stress generated by the defending macrophages or neutrophils. Thus, the DNA damage response in C. albicans may contribute to its pathogenicity. Uncovering the transcriptional changes triggered by the DNA damage-inducing agent MMS in many model organisms has enhanced the understanding of their DNA damage response processes. However, the transcriptional regulation triggered by MMS remains unclear in C. albicans. Here, we explored the global transcription profile in response to MMS in C. albicans and identified 306 defined genes whose transcription was significantly affected by MMS. Only a few MMS-responsive genes, such as MGT1, DDR48, MAG1, and RAD7, showed potential roles in DNA repair. GO term analysis revealed that a large number of induced genes were involved in antioxidation responses, and some downregulated genes were involved in nucleosome packing and IMP biosynthesis. Nevertheless, phenotypic assays revealed that MMS-induced antioxidation gene CAP1 and glutathione metabolism genes GST2 and GST3 showed no direct roles in MMS resistance. Furthermore, the altered transcription of several MMS-responsive genes exhibited RAD53-related regulation. Intriguingly, the transcription profile in response to MMS in C. albicans shared a limited similarity with the pattern in S. cerevisiae, including COX17, PRI2, and MGT1. Overall, C. albicans cells exhibit global transcriptional changes to the DNA damage agent MMS; these findings improve our understanding of this pathogen's DNA damage response pathways.

Keywords: Candida albicans; DNA damage response; RNA-seq; Rad53; methyl methanesulfonate.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of MMS on the growth of C. albicans cells. (A,B) Morphology of C. albicans cells with the MMS treatment. The log phase WT (SN148) cells were treated with 0.015% or 0.03% MMS for 90 min, before being stained with CFW (×400). The single and connected cells in panel (A) were counted, and the ratio of connected cells was listed. (C) Survival analysis of C. albicans cells with the MMS treatment. The log phase WT (SN148) cells were treated with 0.015% or 0.03% MMS for 90 min before being spread on YPD plates. The CFUs surviving from MMS treatment were counted and compared with the non-treated group (n = 3). The connected cells were indicated by arrows. * represents p < 0.05 and ** represents p < 0.01.
Figure 2
Figure 2
DNA damage response genes affected by MMS in C. albicans. (A) Overview of pooling transcriptional altered genes induced by MMS. Two RNA-seq assays were run independently with similar MMS treatment. The genes were pooled out from two independent RNA-seq assays by log2 fold cut-off of 1. The 306 common genes repeated in two RNA-seq assays were considered as defined MMS-responsive genes. The remaining genes with log2 fold change higher than 1 and p value less than 0.05 were considered as putative MMS-responsive genes in C. albicans. (B) Potential DNA damage response genes affected by MMS treatment. The genes with predicted roles in DNA damage response were selected. The log2 fold change for each gene was shown. The gene with dark background came from the defined MMS-responsive gene group, and the gene with light background came from the putative MMS-responsive gene group. The phenotype in response to MMS of mutant strains of mentioned genes was shown. Sc: S. cerevisiae; Ca: C. albicans.
Figure 3
Figure 3
Oxidative stress genes affected by MMS in C. albicans. (A) Summary of typical oxidation response genes affected by MMS. The log2 fold change for each gene from the defined MMS-responsive group was listed behind the gene name. (B) The Cap1 related genes are induced by MMS. The mentioned genes were analyzed by the STRING platform. (C) The phenotypic assay of the CAP1 deletion strain to H2O2 and genotoxic stresses. (D&E) The total GST activity (D) and the total SOD activity (E) affected by MMS treatment. The WT strain (SN148, n = 3) was treated with 0.015% or 0.03% MMS for 90 min before being harvested for activity assay. The relative activity was compared to the enzyme activity in the untreated group, where its activity was normalized as 1. * represents p < 0.05 and ** represents p < 0.01.
Figure 4
Figure 4
Summary of IMP biosynthetic process, nucleosome assembly, glutathione metabolism, and cell wall structure genes affected by MMS in C. albicans. (A) IMP biosynthetic process related genes. (B) Nucleosome assembly related genes. (C) Glutathione metabolism genes. (D) Phenotypic assay by increasing the transcription of GST2 and GST3. (E) Cell wall structure genes. (F) Anti-HA Western blot of WT with Rad53-HA that was incubated with MMS, Congo Red, or CFW for 90 min. In panels (A,B), the genes with a dark background came from the defined MMS-responsive gene group, and the genes with a light background came from the putative MMS-responsive gene group. In panels (C,E), all the genes came from the defined MMS-responsive gene group. The value of log2 fold for each gene was listed behind the gene name.
Figure 5
Figure 5
Confirmation of gene transcription by qRT-PCR. The WT strain (SN148) and the RAD53 deletion strain were treated with 0.015% MMS for 90 min before being harvested for RNA extraction. The qRT-PCR assay for each strain contained at least 3 biological replicates. The difference between each group was compared using paired t test with GraphPad Prism 8 software. * represents p < 0.05 and ** represents p < 0.01.
Figure 6
Figure 6
Comparison of the transcriptional profile to MMS in S. cerevisiae and C. albicans. The MMS-responsive transcriptional data was collected from published papers with fold changes over 2 fold. These data sets were checked and combined. The genes reported twice or more were considered as core MMS-responsive genes in S. cerevisiae and were used to check the similarity with the transcriptional profile to MMS in C. albicans. Both the defined MMS-responsive gene set and the putative MMS-responsive gene group were compared with the core MMS-responsive genes in S. cerevisiae. The similarity between different data sets was compared (A), and the common genes were listed in panel (B). The gene name with a dark background came from the defined MMS-responsive gene group, and the gene with a light background came from the putative MMS-responsive gene group. Sc: S. cerevisiae; Ca: C. albicans. (C) The genes with opposite transcription responses to MMS in S. cerevisiae and C. albicans were shown. (DF) qRT-PCR assay of COX17, PRI2, and MGT1 in S. cerevisiae and C. albicans. The WT strains of S. cerevisiae (BY4741) and C. albicans (SN148) were treated with 0.015% MMS for 90 min before being harvested for RNA extraction. The qRT-PCR assay for each strain contained at least 3 biological replicates. The difference between each group was compared using paired t test with GraphPad Prism 8 software. * represents p < 0.05 and ** represents p < 0.01.
Figure 7
Figure 7
Overview of the transcriptional profile to MMS exposure in C. albicans. MMS functions on DNA and introduces lesions, such as SSBs or DSBs. The DNA damage checkpoints are activated to, maybe partially, regulate MMS-responsive genes. Genes involved in DNA damage repair, antioxidation process, and glutathione metabolism are induced by MMS, while nucleosome assembly ‘de novo’ IMP biosynthetic process and cell-wall-related genes are repressed.

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