IADR Abstract Archives
“New-haploid” Candida albicans unravelled IRA2 as a major biofilm regulator
Objectives: Candida albicans is the major fungal pathogen of humans which causes oral and systemic mycoses. Biofilm formation is a key virulence attribute of this fungus directly associated with treatment failure. Recent groundbreaking discovery of haploid state of C. albicans has opened up new opportunities to apply technologies which were unfeasible in invariably and heterozygous diploids. In present study, we aimed to comprehensively characterize C. albicans haploid biofilm model and identify novel biofilm regulators using haploid mutagenesis.
Methods: Biofilm formation of diploid strains (BWP17, SC5314), haploid strains (GZY803) and mutant library of C. albicans were examined using standard methodology. Biofilm biomass was evaluated in a time-course experiment by XTT-reduction assay and counting colony forming units. Ex-vivo biofilm formation of C. albicans strains were assessed using reconstituted human oral epithelial (RHOE) model. Ultrastructure and cellular viability of biofilms were assessed by scanning electron and confocal microscopes, respectively. Findings were further confirmed by diploid C. albicans biofilms. Appropriate statistical analysis was performed using SPSS software.
Results: Haploid C. albicans strains formed comparable biofilm to that of diploid biofilms by 48-72 h. Deletion of IRA2 or ORF19.3216 had inhibitory effect on biofilm formation while deletion of ARL1, IRG1, AGE2 seemed to promote the development of biofilm. IRA2 gene significantly attenuated biofilm formation in a temperature-independent manner. Consistently, the RHOE model also showed a significantly reduced ability of the haploid ira2∆ strain to form biofilm in comparison with the rescued strains. In addition, ira2∆/∆ diploid strains also showed a reduction in biofilm formation compared to the diploid BWP17 parental strain.
Conclusions: We have for the first time demonstrated the utility of the “new-haploid” C. albicans biofilm model for genetic studies. Interesting, we discovered IRA2, a novel morphogenesis-independent biofilm regulator, which will open up new therapeutic options for oral and systemic Candida infections in future.
Methods: Biofilm formation of diploid strains (BWP17, SC5314), haploid strains (GZY803) and mutant library of C. albicans were examined using standard methodology. Biofilm biomass was evaluated in a time-course experiment by XTT-reduction assay and counting colony forming units. Ex-vivo biofilm formation of C. albicans strains were assessed using reconstituted human oral epithelial (RHOE) model. Ultrastructure and cellular viability of biofilms were assessed by scanning electron and confocal microscopes, respectively. Findings were further confirmed by diploid C. albicans biofilms. Appropriate statistical analysis was performed using SPSS software.
Results: Haploid C. albicans strains formed comparable biofilm to that of diploid biofilms by 48-72 h. Deletion of IRA2 or ORF19.3216 had inhibitory effect on biofilm formation while deletion of ARL1, IRG1, AGE2 seemed to promote the development of biofilm. IRA2 gene significantly attenuated biofilm formation in a temperature-independent manner. Consistently, the RHOE model also showed a significantly reduced ability of the haploid ira2∆ strain to form biofilm in comparison with the rescued strains. In addition, ira2∆/∆ diploid strains also showed a reduction in biofilm formation compared to the diploid BWP17 parental strain.
Conclusions: We have for the first time demonstrated the utility of the “new-haploid” C. albicans biofilm model for genetic studies. Interesting, we discovered IRA2, a novel morphogenesis-independent biofilm regulator, which will open up new therapeutic options for oral and systemic Candida infections in future.