Inactivation of genes TEC1 and EFG1 in Candida albicans influences extracellular matrix composition and biofilm morphology

Figures & data

Figure 1. Biofilm characterization flowchart. ECM, extracellular matrix; eDNA, extracellular DNA; WSP, water-soluble polysaccharide; ASP, alkali-soluble polysaccharide.

Table 1. Biofilm and extracellular matrix (ECM) components of Candida albicans biofilms.^a

Biofilm components				ECM components
Strain	Log10 cfu mL^−1	Dry weight (mg)	Protein (μg)	ASP (μg)	WSP (μg)	eDNA (μg)	Matrix protein (μg)
C. albicans SN425 (WT)	6.89 ± 0.07a	24.5 ± 3.7a	47.8 ± 1.5a	74.5 ± 22.0a	47.7 ± 13.4a	20.1 ± 7.1a	13.8 ± 9.8a
C. albicans CJN2302 (Δ/Δ efg1)	7.16 ± 0.17b	18.4 ± 2.8b	50.3 ± 2.3b	14.7 ± 5.0b	52.2 ± 7.8a	27.4 ± 7.6a	20.4 ± 11.3a
C. albicans CJN2330 (Δ/Δ tec1)	6.83 ± 0.18a	18.5 ± 3.0b	46.5 ± 1.4a	44.0 ± 24.1c	46.1 ± 11.3a	34.2 ± 14.4a	17.4 ± 4.3a

^aData are shown as mean ± SD of colony count (log₁₀ cfu mL⁻¹), dry weight (mg), total protein (µg), alkali-soluble polysaccharide (ASP) (µg), water-soluble polysaccharide (WSP) (µg), extracellular DNA (eDNA) (µg), and matrix protein (µg) for C. albicans SN425 [wild-type (WT)], C. albicans CJN2302 (Δ/Δ efg1), and C. albicans CJN2330 (Δ/Δ tec1).

Comparisons by one-way ANOVA and Tukey’s post-hoc test: means followed by the same letter in a column are not significantly different from each other.

Table 2. Pearson’s correlation (r) of colony count (log₁₀ cfu mL⁻¹) and extracellular matrix (ECM) components for the wild-type (WT) strain (SN425).

WT strain (SN425)		Log10 cfu mL^−1	ASP	WSP	Total protein	Matrix protein	eDNA
Log10 cfu mL^−1	Pearson’s r	1	0.372	0.262	0.173	0.017	−0.167
	Sig. (2-tailed)		0.234	0.411	0.590	0.957	0.605
	N		12	12	12	12	12
ASP	Pearson’s r		1	0.184	0.666*	0.361	0.273
	Sig. (2-tailed)			0.567	0.018	0.249	0.391
	N			12	12	12	12
WSP	Pearson’s r			1	0.065	0.017	−0.326
	Sig. (2-tailed)				0.841	0.959	0.301
	N				12	12	12
Protein	Pearson’s r				1	0.670*	0.376
	Sig. (2-tailed)					0.017	0.229
	N					12	12
Matrix protein	Pearson’s r					1	−0.032
	Sig. (2-tailed)						0.921
	N						12
eDNA	Pearson’s r						1
	Sig. (2-tailed)
	N

ASP, alkali-soluble polysaccharide; WSP, water-soluble polysaccharide; eDNA, extracellular DNA; Sig., significance.

*Correlation is significant at the 0.05 level (two-tailed).

Table 3. Pearson’s correlation (r) of colony count (log₁₀ cfu mL⁻¹) and extracellular matrix (ECM) components for the Δ/Δ efg1 mutant strain (CJN2302).

Δ/Δ efg1 strain		Log10 cfu mL^−1	ASP	WSP	Total protein	Matrix protein	eDNA
Log10 cfu mL^−1	Pearson’s r	1	0.246	0.057	0.734*	−0.286	−0.315
	Sig. (2-tailed)		0.442	0.861	0.010	0.367	0.318
	N		12	12	12	12	12
ASP	Pearson’s r		1	0.477	−0.133	0.252	0.290
	Sig. (2-tailed)			0.117	0.696	0.429	0.361
	N			12	12	12	12
WSP	Pearson’s r			1	0.236	−0.160	0.080
	Sig. (2-tailed)				0.485	0.619	0.805
	N				12	12	12
Protein	Pearson’s r				1	−0.205	0.538
	Sig. (2-tailed)					0.545	0.071
	N					12	12
Matrix protein	Pearson’s r					1	−0.247
	Sig. (2-tailed)						0.463
	N						12
eDNA	Pearson’s r						1
	Sig. (2-tailed)
	N

ASP, alkali-soluble polysaccharide; WSP, water-soluble polysaccharide; eDNA, extracellular DNA; Sig., significance.

*Correlation is significant at the 0.05 level (two-tailed).

Table 4. Pearson’s correlation (r) of colony count (log₁₀ cfu mL⁻¹) and extracellular matrix (ECM) components for the Δ/Δ tec1 mutant strain (CJN2330).

Δ/Δ tec1 strain		Log10 cfu mL^−1	ASP	WSP	Total protein	Matrix protein	eDNA
Log10 cfu mL^−1	Pearson’s r	1	−0.080	0.171	−0.155	−0.171	−0.194
	Sig. (2-tailed)		0.804	0.596	0.630	0.595	0.567
	N		12	12	12	12	12
ASP	Pearson’s r		1	0.285	0.106	0.190	0.678*
	Sig. (2-tailed)			0.369	0.743	0.554	0.022
	N			12	12	12	12
WSP	Pearson’s r			1	−0.002	−0.535	0.219
	Sig. (2-tailed)				0.995	0.073	0.518
	N				12	12	12
Protein	Pearson’s r				1	0.000	0.098
	Sig. (2-tailed)					1.000	0.775
	N					12	12
Matrix protein	Pearson’s r					1	0.322
	Sig. (2-tailed)						0.334
	N						12
eDNA	Pearson’s r						1
	Sig. (2-tailed)
	N

ASP, alkali-soluble polysaccharide; WSP, water-soluble polysaccharide; eDNA, extracellular DNA; Sig., significance.

*Correlation is significant at the 0.05 level (two-tailed).

Figure 2. Visual macroscopic appearance and overall Candida albicans biofilm structure by variable-pressure scanning electron microscopy (VPSEM). The wild-type (WT) strain presents a typical thick biofilm architecture in visual appearance (a) with the presence of abundant extracellular matrix (ECM) (b, c), exemplified by the arrows (c). The Δ/Δ efg1 mutant shows sparse thin biofilm growth patterns (d) and an ECM (e, f) morphologically distinct from the WT (b, c) and Δ/Δ tec1 strains (h, i). The Δ/Δ tec1 mutant presents defects in visual appearance (g) compared to the WT strain (a); however, its ECM is morphologically similar to the WT (h, i, see arrows).

Figure 3. Candida albicans biofilm structures and corresponding quantitative data from confocal scanning laser microscopy assays. (a) Representative three-dimensional and orthogonal images of the structural organization of the 48 h biofilms of C. albicans wild-type (WT) (1) and mutant strains: Δ/Δ efg1 (2) and Δ/Δ tec1 (3) (green color denotes labeling with SYTO9 for live yeast cells). Mean and SD of biovolume (b) and average biofilm thickness (c) of C. albicans WT and mutant strains (Δ/Δ efg1 and Δ/Δ tec1) determined by COMSTAT2 analysis. The average biovolume and biofilm thickness were calculated from five independent samples from each strain. Values followed by the same letter are not significantly different (p > 0.05), as determined by an analysis of variance for all pairs using Tukey’s test. The profile of the distribution of C. albicans WT and mutant strains (Δ/Δ efg1 and Δ/Δ tec1) in the biofilms is represented in (d).