Regulatory role of Mss11 in Candida glabrata virulence: adhesion and biofilm formation

Abstract

Introduction: Candida glabrata has emerged as a fungal pathogen with high infection and mortality rates, and its primary virulence factors are related to adhesion and biofilm formation. These virulence factors in C.glabrata are primarily mediated by epithelial adhesins (Epas), most of which are encoded in subtelomeric regions and regulated by subtelomeric silencing mechanisms. The transcription factor Mss11, known for its regulatory role in adhesion, biofilm formation, and filamentous growth in Saccharomyces cerevisiae and Candida albicans, has also been implicated in the expression of EPA6, suggesting its potential influence on C.glabrata virulence. The present study aims to determine the regulatory role of Mss11 in the virulence of C. glabrata.

Methods: In this work, a Δmss11 null mutant and its complemented strain were constructed from a C.glabrata standard strain. The impact of the transcription factor Mss11 on the virulence of C.glabrata was investigated through a series of phenotypic experiments, including the microbial adhesion to hydrocarbons (MATH) test, adherence assay, biofilm assay, scanning electron microscopy and Galleria mellonella virulence assay. Furthermore, transcriptome sequencing, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and chromatin immunoprecipitation sequencing (ChIP-seq) were employed to investigate the molecular mechanisms behind the regulation of Mss11.

Results: In C.glabrata, the loss of MSS11 led to a significant reduction in several virulence factors including cell surface hydrophobicity, epithelial cell adhesion, and biofilm formation. These observations were consistent with the decreased virulence of the Δmss11 mutant observed in the Galleria mellonella infection model. Further exploration demonstrated that Mss11 modulates C. glabrata virulence by regulating EPA1 and EPA6 expression. It binds to the upstream regions of EPA1 and EPA6, as well as the promoter regions of the subtelomeric silencing-related genes SIR4, RIF1, and RAP1, indicating the dual regulatory role of Mss11.

Conclusion: Mss11 plays a crucial role in C. glabrata adhesion and biofilm formation, and thus has a broad influence on virulence. This regulation is achieved by regulating the expression of EPA1 and EPA6 through both promoter-specific regulation and subtelomeric silencing.

Keywords: Candida glabrata; EPA; Mss11; adhesion; biofilm; subtelomeric silencing; virulence.

Figure 1

CSH and epithelial cell adhesion of C. glabrata ATCC 2001, Δmss11 mutant, and the complemented strains. (A) CSH of ATCC 2001, Δmss11 mutant, and the complemented strains. The higher the n-octane affinity, the greater is the hydrophobicity. The Δmss11 mutant exhibited significantly lower CSH than ATCC 2001 and the complemented strains—suggesting a major influence of MSS11 disruption on CSH in C. glabrata. All data are presented as mean ± SD from three independent experiments. (B) Adhesion of C. glabrata strains to epithelial cells. C. glabrata strains were co-incubated with 293T or Caco-2 cells, and the adhesion rate was calculated using the final colony-forming units observed on the plate (relative to ATCC 2001 as 1.0). The adhesion rates of the Δmss11 mutant to 293T and Caco-2 cells were significantly lower than ATCC 2001 and the complemented strain, suggesting that the absence of MSS11 in C. glabrata significantly impaired its adhesion to epithelial cells. ***P < 0.001, “ns” denotes no significance.

Figure 2

C. glabrata biofilm formation analysis. (A) In vitro biofilm formation assay for quantifying ATCC 2001, Δmss11 mutant, and the complemented strain biofilms. Absorbance at 570 nm of the mature biofilms was determined after crystal violet staining. The Δmss11 mutant exhibited significantly lower biofilm biomass than ATCC 2001 and the complemented strains at 4, 6, 8, 12, and 24 (h) (B) XTT assay for biofilm viability. Absorbance at 450 nm indicated that the Δmss11 mutant had significantly lower biofilm metabolic activity than ATCC 2001 and the complemented strains. Results in (A) and (B) collectively highlight that the absence of MSS11 not only reduces biofilm biomass but also impairs the biofilm viability significantly in C. glabrata. (C) SEM of contrasting biofilm structures of ATCC 2001, Δmss11 mutant, and the complemented strains. Observed variations in cell–cell adhesion (tight to loose arrangement) and extracellular matrix contents (abundant to nearly absent) were consistent with the differences noted in the virulence phenotypes. **P < 0.01, ***P < 0.001, “ns” denotes no significance.

Figure 3

Impact of Mss11 on C. glabrata virulence in G. mellonella infection model. Healthy larvae were divided into groups and injected with the C. glabrata strain suspension or sterile PBS (negative control). Mortality rate of G. mellonella larvae was monitored daily based on their response to touch. The virulence of the Δmss11 mutant was notably diminished, with a 60% survival rate at post-infection day 5; however, it was only 30% for ATCC 2001 and the complemented strains. ***P < 0.001.

Figure 4

Analysis of EPA1 and EPA6 expression in C. glabrata. (A) RT-qPCR validating EPA1 and EPA6 expression levels in various C. glabrata strains. A substantial reduction was noted in EPA1 and EPA6 expression in the Δmss11 mutant compared with that in ATCC 2001 and the complemented strains, confirming that Mss11 had a regulatory impact on EPA1 and EPA6 expression. (B) Transcriptome sequencing for DEGs between ATCC 2001 and Δmss11 mutant strains. In total, 640 and 820 genes were upregulated and downregulated after MSS11 deletion, respectively. Notably, EPA1 (CAGL0E06644g) and EPA6 (CAGL0C00110g) were among the downregulated genes. (C) GO enrichment analysis on DEGs between ATCC 2001 and Δmss11 mutant strains for Mss11-associated pathways. The identified top 10 enriched pathways were primarily linked to fungal cell wall biosynthesis and extracellular matrix formation. ***P < 0.001, “ns” denotes no significance.

Figure 5

Mss11 binding patterns in C. glabrata. (A) Comprehensive overview of the binding patterns of Mss11 across the C. glabrata genome. (B) Pie chart of the distribution of Mss11 DNA binding regions. Most Mss11 binding sites were situated within gene promoter regions, consistent with the expected transcription factor function of Mss11. (C) RT-qPCR validating that Mss11 disruption increased expression of subtelomeric silencing-associated genes SIR4, RIF1, and RAP1 in C. glabrata. ***P < 0.001. (D) Differential enrichment peaks in the comparison of ChIP-seq data between FLAG-tagged and untagged Mss11 visualized using IGV. The presented figure is a screenshot from IGV. Red bars above the peaks represent the regions of the differentially enriched peaks and their genomic positions, while black bars below indicate the target genes. The results reveal that Mss11 not only binds to EPA1 and EPA6 upstream regions but also to SIR4, RIF1, and RAP1 promoters.