Chaminda Jayampath Seneviratne1,*, Guisheng Zeng2,*, Thuyen Truong1,*, Sarah Sze Wah Wong3, Lakshman Samaranayake3,4, Fong Yee Chan2, Yan-Ming Wang2, Haitao Wang2, Jiaxin Gao2, and Yue Wang2,5
1 Oral Sciences, Faculty of Dentistry, National University of Singapore
2 Institute of Molecular & Cell Biology, Agency for Science, Technology and Research, Proteos, Singapore
3 Faculty of Dentistry, The University of Hong Kong, Hong Kong
4 School of Dentistry, Queensland University, Australia
5 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
* equal contribution
Published online in Scientific Reports, on 23rd July, 2015.
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 legend: 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 24h, 48h, and 72h. ConA-Alexa 488 (green) was used to stain the glucose and mannose residues in the fungal cell wall and extended ECM. Bar, 15μm. (d) Visualization of haploid and diploid biofilms formed at 24h, 48h, and 72h with scanning electron microscopy. Bar, 15μm. (e) Confocal imaging of haploid and diploid biofilms formed at 24h, 48h, and 72h. 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 24h, 48h, and 72h. Three different sections of each biofilm confocal image were taken for height estimation using BioImage_L to calculate the average height
For more information on Yue WANG's laboratory, please click here.