IADR Abstract Archives
The TLO Gene Family Influences Fitness and Virulence of the Human Fungal Pathogen Candida Albicans
Objectives: Candida albicans is a fungal commensal of the human oral cavity, however, if the immune system is compromised, it can cause oral candidiasis. One major difference between C. albicans and other Candida spp. is the expansion of the Telomere-associated ORF (TLO) gene family. C. albicans strains can have 10-15 different TLO genes, while its closest (and less virulent) relative, C. dubliniensis, has only two. We believe that expansion of this gene family allows C. albicans to be such a successful opportunistic pathogen.
Methods: CRISPR-Cas9 mutagenesis was used to knock out all 14 TLO genes from C. albicans AHY940). Three representative TLO genes (TLO1, 2 and 11) which had been engineered to be under the control of a low-level or strong promoter were selected for reintroduction to the null background. Phenotypic assays and transcript profiling were performed to compare the fitness of the mutant strains to the WT.
Results: Phenotypic analysis of the TLO null mutant showed that in the absence of TLOs, C. albicans is highly defective, with a heavily reduced growth rate in nutrient rich media and a constitutively pseudohyphal morphology. In stress inducing conditions, including oxidative stress and cell wall stress inducing, the null mutant was more sensitive than WT. Reintroduction of specific TLOs to the null background restored many phenotypes to WT level, with some differences in each TLO’s effect. The transcript profile of the TLO mutant was significantly different to that of the WT.
Conclusions: The wide range of phenotypes affected by deletion of TLOs indicates the role of these genes in a large number of cellular processes. The ability of singular TLOs to affect many phenotypes, with some clade specificity, suggests that the expansion of the TLO family in C. albicans contribute to its relative success as a colonizer and pathogen of humans. Determination of precise roles for each gene may allow their use as targets for the development of novel antifungal treatments.
Methods: CRISPR-Cas9 mutagenesis was used to knock out all 14 TLO genes from C. albicans AHY940). Three representative TLO genes (TLO1, 2 and 11) which had been engineered to be under the control of a low-level or strong promoter were selected for reintroduction to the null background. Phenotypic assays and transcript profiling were performed to compare the fitness of the mutant strains to the WT.
Results: Phenotypic analysis of the TLO null mutant showed that in the absence of TLOs, C. albicans is highly defective, with a heavily reduced growth rate in nutrient rich media and a constitutively pseudohyphal morphology. In stress inducing conditions, including oxidative stress and cell wall stress inducing, the null mutant was more sensitive than WT. Reintroduction of specific TLOs to the null background restored many phenotypes to WT level, with some differences in each TLO’s effect. The transcript profile of the TLO mutant was significantly different to that of the WT.
Conclusions: The wide range of phenotypes affected by deletion of TLOs indicates the role of these genes in a large number of cellular processes. The ability of singular TLOs to affect many phenotypes, with some clade specificity, suggests that the expansion of the TLO family in C. albicans contribute to its relative success as a colonizer and pathogen of humans. Determination of precise roles for each gene may allow their use as targets for the development of novel antifungal treatments.