Depletion of the Candida albicans TLO gene family reveals a requirement for alpha TLO genes for wild-type virulence

<i>Candida albicans</i> uniquely possesses an expanded family of genes (the <i>TLO</i> gene family) that encodes 10-15 paralogues of the Med2 component of the transcriptional regulator Mediator. Previous studies have shown that <i>TLO</i> null mutants are unable to form hyphae and are hypersensitive to environmental stress. However, the reason for the <i>TLO</i> gene expansion remains unclear, and the current study aimed to determine if reduction in the <i>TLO</i> family copy number affected virulence. In order to investigate this, we used CRISPR-Cas9 mutagenesis to generate two <i>TLO</i>-depleted mutants: one mutant retaining only <i>TLO</i>β<i>2</i> (CaTLO2) and the second mutant containing only <i>TLO</i>γ<i>5</i> (CaTLO5). Both <i>TLO</i>-depleted mutants exhibited increased filamentous growth, increased susceptibility to specific stresses and reduced virulence in a murine model of oropharyngeal candidiasis (OPC). <i>In vitro</i>, the CaTLO5 mutant also exhibited impaired hyphal escape from macrophages and reduced hyphal invasion of oral keratinocytes. We then investigated if complementation with <i>TLO</i>α<i>1</i>, a gene previously shown to restore wild-type growth in a Δ<i>tlo</i> null mutant, could restore virulence. <i>In vitro</i> infection models showed that <i>TLO</i>α<i>1</i> could restore true hypha formation, epithelial invasion and hyphal escape from macrophages in the CaTLO5 background. The murine OPC model showed that <i>TLO</i>α<i>1</i> could restore wild-type virulence in both CaTLO2 and CaTLO5 strains, suggesting an essential role for α-<i>TLO</i> in oral mucosal infection. Together, these findings highlight the functional specialization between the α, β and γ <i>TLO</i> gene groups and establish α-<i>TLO</i> as a major regulator of virulence in <i>C. albicans</i>.