CRISPR-Cas9 mutagenesis and single gene reintegration suggests functional diversity within the Candida albicans TLO gene family

Candida albicans has between 10-15 Telomere-associated ORF family(TLO)genes, whereas its closest relative, Candida dubliniensis, has two. The Tlo proteins are components of the Mediator complex which plays an important role in transcriptional regulation. CRISPR-Cas9 mutagenesis was used to generate a TLOnull mutant of C. albicans. Phenotypic analysis of the mutant showed significantly reduced fitness, with major defects in growth rate, morphogenesis, stress resistance and virulence in a Galleria mellonellamodel. Clade representative TLOα1, TLOβ2 and TLOγ11constructs were reintroduced into the null mutant background to determine if members of the TLO gene family exhibit functional differences. The genes were reintroduced under the control of the TET1 and ENO1promoters. TLOα1and TLOβ2expression restored stress tolerance and growth rate, in some cases to the level of the WT. TLOβ2expression also showed a dramatic effect on morphology resulting in constitutive true hyphal growth. Moderate expression of TLOγ11 had no detectable effect on many of the phenotypes tested, however overexpression increased biofilm formation in Spider medium, and also conferred increased resistance to cell wall stressors. These data suggest that individual TLO genes have distinct functions and that the diversity within the TLO family may contribute to the relative success of C. albicans as a coloniser and pathogen of humans.