The Role of Sfp1 in Candida albicans Cell Wall Maintenance

Abstract

The cell wall is the first interface for Candida albicans interaction with the surrounding environment and the host cells. Therefore, maintenance of cell wall integrity (CWI) is crucial for C. albicans survival and host-pathogen interaction. In response to environmental stresses, C. albicans undergoes cell wall remodeling controlled by multiple signaling pathways and transcription regulators. Here, we explored the role of the transcription factor Sfp1 in CWI. A deletion of the SFP1 gene not only caused changes in cell wall properties, cell wall composition and structure but also modulated expression of cell wall biosynthesis and remodeling genes. In addition, Cas5 is a known transcription regulator for C. albicans CWI and cell wall stress response. Interestingly, our results indicated that Sfp1 negatively controls the CAS5 gene expression by binding to its promoter element. Together, this study provides new insights into the regulation of C. albicans CWI and stress response.

Keywords: Candida albicans; Cas5; Sfp1; cell wall integrity; cell wall stress response.

Figure 1

Deletion of SFP1 leads to changes of C. albicans cell wall-related properties. The susceptibility of C. albicans to cell wall-disrupting agents calcofluor white (a) and congo red (b). The cells were ten-fold serially diluted and spotted onto SC agar plated with or without calcofluor white (600 μg/mL) and congo red (100 μg/mL). The plates were incubated at 30 °C for 5 d. Representative images of three independent experiments with similar results are shown. WT: wild type; (c) The zymolyase sensitivity assay. Cells were treated with 2 μg/mL of Zymolyase 100 T. Results are displayed as the mean ± standard deviation (SD) of two independent experiments; (d) Measurement of CSH. The CSH is expressed as percentage and displayed as the mean ± SD of three independent experiments. **, p < 0.01, ***, p < 0.001.

Figure 2

Deletion of SFP1 confers resistance to the antifungal caspofungin. Caspofungin susceptibility testing by spot assay. The ten-fold serially diluted cells were spotted onto SC agar plates with or without caspofungin (8 μg/mL). The plates were incubated at 30 °C for 5 days. Representative images of three independent experiments with similar results are shown.

Figure 3

SFP1 deletion also changes the cell wall and cell wall composition in C. albicans. (a) Cell wall thickness was examined by TEM and compared between the WT, sfp1∆/∆ and SFP1-reintegration strains. Representative images are shown. Scale bar: 100 nm; (b) For each C. albicans cells, 3 parts of cell wall were analyzed. Results are the mean ± SD from 20 individual cells. The statistical significance is assessed by Mann-Whitney U test. *, p < 0.05, ***, p < 0.001; (c) Total cell wall polysaccharide content was quantified. The wild type value was set as 100%. Results are displayed as the mean ± SD from three independent experiments. *, p < 0.05; (d) The cell wall chitin, glucan, and mannan content was quantified using HPAEC-PAD. Results are displayed as the mean ± SD of three independent experiments. *, p < 0.05, **, p < 0.01.

Figure 4

Sfp1 regulates expression of cell wall biosynthesis and remodeling genes. Gene expression was measured by real-time qPCR. The PMA1 transcripts were used as an internal control for the RNA input. Results are displayed as the mean ± SD of three independent experiments. For each gene, the relative fold changes were displayed as the expression levels of an individual strain normalized to the WT strain (as 1). *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Figure 5

Sfp1 regulates cell wall stress response through Cas5. Cell susceptibility to (a) calcofluor white; (b) congo red; and (c) caspofungin was determined by spot assay. The cells were ten-fold serially diluted and spotted onto SC agar plates with or without calcofluor white (600 μg/mL), congo red (100 μg/mL) and caspofungin (1 μg/mL). The plates were incubated at 30 °C for 5 d. Representative images of three independent experiments with similar results are shown; (d) The expression of CAS5 gene was detected using real-time qPCR. The PMA1 transcripts were used as an internal control for the RNA input. Results are displayed as the mean ± SD for three independent experiments. The relative fold changes were displayed as the expression levels of an individual strain normalized to the WT strain (as 1). *, p < 0.05, **, p < 0.01; (e) The ChIP assay for binding of Sfp1 to the CAS5 promoter. DNA fragments from the WT and HA-SFP1 strains were amplified by real-time qPCR. Results are displayed as the mean ± SD for three independent experiments. *, p < 0.05.