The function of the TLO gene family of Candida albicans

Abstract

Candida albicans is the most common cause of Candida infection worldwide. It is more pathogenic than all the other members of the genus, and it is thought that expansion of several key gene families accounts for this enhanced virulence. One such gene family is the TLO gene family of telomere associated ORFs. Candida albicans strains can have between 10-15 TLO genes, divided into α, β and γ clades, while close relative C. dubliniensis has only two, and all other species have a single TLO homologue. In this work, expansion of the C. dubliniensis TLO repertoire with representative genes from C. albicans enhanced the fitness and virulence of this species, while deletion of the entire gene family from C. albicans generated a severely unfit, morphologically defective, and less virulent strain, which appeared to be undergoing a constitutive starvation and stress response in the absence of external stimuli. This ΔΔtlo mutant was more tolerant of azole drug stress, likely due to alterations in the ergosterol biosynthesis pathway. Experiments to reintroduce representative TLO genes in to the ΔΔtlo mutant background showed that individual TLO genes appear to have overlapping and specific functions, with phenotypic and transcriptomic approaches highlighting similarities and differences in function. Reintroduction of the TLOγ11 resulted in expression of mRNA, but there was no protein detected in the cells by Western blotting, indicating the possibility of sequence specific targeted protein degradation at play, and no significant phenotypic or transcriptomic effects of TLOγ11 expression were detected. Expression of TLOα1 and TLOβ2 restored growth rate and morphology, while uniquely affecting biofilm formation and hyphal morphology, respectively. There were also differences in the ability of these strains to tolerate oxidative stress. RNA-seq analysis showed that the reintroduction of TLOα1 and TLOβ2 could restore the expression of genes in pathways important for the viability of the cell, including those related to cellular morphology and membrane composition. ChIP-seq highlighted that interaction of Tloα1 and Tloβ2 with genes could promote expression or repression, and that these Tlos were found to interact with and activate transcription factors important for fungal virulence. ChIP-seq did detect Tloγ11 protein and showed that this protein localised at telomeres and repeat sequences indicating a possible role in maintaining chromosome stability. Tloβ2 also localised at these sites but was more notably involved in interacting with ORFs, while Tloα1 was not found to significantly interact with repeat regions or telomeres. These data support the hypothesis that expansion and diversification of the TLO gene family in C. albicans may have contributed to the enhanced ability of this species to colonise and infect humans.