Depletion of thegene family reveals a requirement for alphagenes for wild-type virulence.

Abstract

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