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Deletion of the Candida albicans TLO gene family using CRISPR-Cas9 mutagenesis allows characterisation of functional differences in α-, β- and γ- TLO gene function

Fletcher, Jessica, O’Connor-Moneley, James, Frawley, Dean, Flanagan, Peter R., Alaalm, Leenah, Menendez-Manjon, Pilar, Estevez, Samuel Vega, Hendricks, Shane, Woodruff, Andrew L., Buscaino, Alessia, and others. (2023) Deletion of the Candida albicans TLO gene family using CRISPR-Cas9 mutagenesis allows characterisation of functional differences in α-, β- and γ- TLO gene function. PLoS Genetics, 19 (12). Article Number e1011082. ISSN 1553-7404. (doi:10.1371/journal.pgen.1011082) (KAR id:104333)

Abstract

The Candida albicans genome contains between ten and fifteen distinct TLO genes that all encode a Med2 subunit of Mediator. In order to investigate the biological role of Med2/Tlo in C. albicans we deleted all fourteen TLO genes using CRISPR-Cas9 mutagenesis. ChIP-seq analysis showed that RNAP II localized to 55% fewer genes in the tloΔ mutant strain compared to the parent, while RNA-seq analysis showed that the tloΔ mutant exhibited differential expression of genes required for carbohydrate metabolism, stress responses, white-opaque switching and filamentous growth. Consequently, the tloΔ mutant grows poorly in glucose- and galactose-containing media, is unable to grow as true hyphae, is more sensitive to oxidative stress and is less virulent in the wax worm infection model. Reintegration of genes representative of the α-, β- and γ-TLO clades resulted in the complementation of the mutant phenotypes, but to different degrees. TLOα1 could restore phenotypes and gene expression patterns similar to wild-type and was the strongest activator of glycolytic and Tye7-regulated gene expression. In contrast, the two γ-TLO genes examined (i.e., TLOγ5 and TLOγ11) had a far lower impact on complementing phenotypic and transcriptomic changes. Uniquely, expression of TLOβ2 in the tloΔ mutant stimulated filamentous growth in YEPD medium and this phenotype was enhanced when Tloβ2 expression was increased to levels far in excess of Med3. In contrast, expression of reintegrated TLO genes in a tloΔ/med3Δ double mutant background failed to restore any of the phenotypes tested, suggesting that complementation of these Tlo-regulated processes requires a functional Mediator tail module. Together, these data confirm the importance of Med2/Tlo in a wide range of C. albicans cellular activities and demonstrate functional diversity within the gene family which may contribute to the success of this yeast as a coloniser and pathogen of humans.

Item Type: Article
DOI/Identification number: 10.1371/journal.pgen.1011082
Additional information: For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.
Subjects: Q Science
Q Science > QH Natural history > QH426 Genetics
Divisions: Divisions > Division of Natural Sciences > Biosciences
Funders: Biotechnology and Biological Sciences Research Council (https://ror.org/00cwqg982)
Depositing User: Alessia Buscaino
Date Deposited: 18 Dec 2023 14:45 UTC
Last Modified: 03 May 2024 03:16 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/104333 (The current URI for this page, for reference purposes)

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