New "haploid biofilm model" unravels IRA2 as a novel regulator of Candida albicans biofilm formation

Abstract

Clinical isolates of the fungal human pathogen Candida albicans are invariably diploid and heterozygous, impeding genetic study. Recent isolation of C. albicans haploids opens opportunities to apply technologies unfeasible in diploids. However, doubts remain on whether the haploids, derived from chromosome loss, can represent the diploids. Here, we use C. albicans haploids to investigate biofilm, a key virulence attribute. We conducted the first comprehensive characterization of biofilm formation of the haploids in comparison with the diploids. We demonstrate that the haploids form biofilms with essentially the same characteristics as the diploids. Screening a haploid mutant library has uncovered novel GTPase-related genes as biofilm regulators, including IRA2 that encodes an activator of the Ras GTPase. IRA2-deletion mutants develop poorly constructed biofilm in both haploid and diploid C. albicans. Our results demonstrate that the haploids are a valid model for C. albicans biofilm research and a powerful tool for uncovering novel regulators.

Figure 1. Comparison of biofilms formed by C. albicans haploid and diploid in vitro.

(a,b) Quantification of the biomass of biofilms formed by diploid strains (SC5314 and BWP17) and haploid strains (GZY792 and GZY803) at different time points with XTT reduction assay (a) and CFU counting method (b). Cells were cultured in GMM medium (supplemented with required amino acids when necessary) at 30 °C overnight and re-inoculated to allow the development of biofilm at 37 °C. The assays were performed in triplicates, and the means were used to generate the curve with standard error. (c) Visualization of extracellular materials (ECM, indicated by arrows) on haploid and diploid biofilms formed at 24 h, 48 h, and 72 h. ConA-Alexa 488 (green) was used to stain the glucose and mannose residues in the fungal cell wall and extended ECM. Bar, 15 μm. (d) Visualization of haploid and diploid biofilms formed at 24 h, 48 h, and 72 h with scanning electron microscopy. Bar, 15 μm. (e) Confocal imaging of haploid and diploid biofilms formed at 24 h, 48 h, and 72 h. For each time point, the upper panels show the top view (with the depth of biofilm color-coded) and the bottom panels show the side view of the biofilm. (f) Comparison of the average heights of haploid and diploid biofilms formed at 24 h, 48 h, and 72 h. Three different sections of each biofilm confocal image were taken for height estimation using BioImage_L to calculate the average height.

Figure 2. Ploidy examination of haploid biofilms and dispersed cells.

Haploid strains GZY792 (a,b) and GZY803 (c,d) were cultured at 30 °C overnight and re-inoculated to allow the development of biofilm at 37 °C. Samples were taken from both biofilm (a,c) and dispersed cells (b,d) at the time points as indicated for ploidy examination by flow cytometry analysis. SC5314 was used as the standard for diploidy.

Figure 3. Comparison of biofilms formed by C. albicans haploid and diploid on ex vivo reconstituted human oral epithelial cells.

Reconstituted human oral epithelia were infected with the diploid SC5314 and haploid GZY803, respectively, and incubated at 37 °C with 5% CO₂. Samples were taken at the time points as indicated and processed for acid-Schiff staining to visualize C. albicans biofilm formation and tissue invasion.

Figure 4. Comparison of biofilms formed in vitro by different C. albicans haploid mutants with their parental strain GZY803.

(a,b) Haploid mutants ira2Δ, orf19.3216Δ, arl1Δ, lrg1Δ, age2Δ and the parental strain GZY803 were cultured at 30 °C overnight and re-inoculated to allow the development of biofilm at either 30 °C (a) or 37 °C (b) for 72 h. The biomass of biofilm formed by each strain was quantified with XTT reduction assay. Each sample was tested in triplicates and the means were used to generate the bar with standard error. (*): p-value < 0.05.

Figure 5. Examination of biofilms formed by haploid ira2Δ mutants.

(a,b) Quantification of the biomass of biofilms formed in vitro by haploid ira2Δ mutants (GZY893 and GZY918) and the rescued strain (GZY941) with XTT reduction assay (a) and CFU counting (b). Cells were cultured at 30 °C overnight and re-inoculated to allow the development of biofilm at 37 °C for 72 h. The parental strain GZY803 was used as the control. The assays were performed in triplicates and the means were used to generate the bar with standard error. (c) Visualization of biofilms formed by GZY803, GZY893, GZY918 and GZY941 at 24 h, 48 h, and 72 h with scanning electron microscopy. Bar, 15 μm. (d) Confocal imaging of biofilms formed by GZY803, GZY893, GZY918 and GZY941 at 24 h, 48 h, and 72 h. For each time point, the upper panels show the top view (with the depth of biofilm color-coded) and the bottom panels show the side view of the biofilm. Bar, 15 μm. (e) Comparison of the average heights of biofilms formed by GZY803, GZY893, GZY918 and GZY941 at 24 h, 48 h, and 72 h. Three different sections of each biofilm confocal image were taken for height estimation using BioImage_L to calculate the average height (with standard error). (f) Comparison of ex vivo biofilms formed by haploid ira2Δ mutant and its rescued strain. Reconstituted human oral epithelia was infected with the GZY893 and GZY941, respectively, and incubated at 37 °C for 72 h. Samples were taken and processed for acid-Schiff staining to visualize C. albicans biofilm formation and tissue invasion.

Figure 6. Quantification of biofilms formed by haploid ira2Δ and diploid ira2Δ/Δ mutants.

The biomass of biofilms formed in vitro by haploid ira2Δ (GZY893) and its rescued strain (ira2Δ+IRA2, GZY941), as well as diploid ira2Δ/Δ (GZY921) and its rescued strain (ira2Δ/Δ+IRA2, GZY1022) were quantified with XTT reduction assay (a) and CFU counting (b). GZY803 with bcr1Δ (GZY1095), and BWP17 with bcr1Δ/Δ (GZY1094), were used as the positive and negative controls for haploid and diploid, respectively. Cells were cultured at 30 °C overnight and re-inoculated to allow the development of biofilm at 37 °C for 72 h.