The Candida Albicans Telomeric Associated ORF (TLO) Gene Family Plays a Role in Antifungal Drug Tolerance.
Objectives: Candida albicans is an opportunistic cause of oral infection. Development of resistance and tolerance to antifungal drugs is a growing concern. This study investigated the role of the TLO gene family in the response to the azole drugs, which inhibit fungal sterol biosynthesis. The TLO family is comprised of 14 genes which encode Med2, a subunit of the multiprotein Mediator complex, which is involved in the global control of transcription by bridging DNA bound transcription factors with RNA Polymerase II. The present study is focused on the effect of Telomeric Associated ORFs (TLOs) on the drug tolerance of the fungal pathogen Candida albicans.
Methods: A TLO deficient mutant of C. albicans in which all 14 genes were deleted was generated using CRISPR-Cas9 technology. Representative TLO genes were reintroduced to complement the deletions. RNA-Seq analysis was used to compare the strains when grown in YPD at 37°C for 4 hours. Expression of TLO genes was examined by qPCR. As part of a comprehensive comparison of the phenotypes of the WT and mutant stains their fluconazole MICs were investigated using E-tests and microdilution assays, and the membrane sterol content identified using mass spectrometry.
Results: Drug sensitivity assays revealed increased tolerance of the TLO null mutant to fluconazole compared to the parent strain, which was reversed upon reintroduction of representative TLO genes. mRNA transcripts of the TLO genes were reduced in the parent strain upon exposure to fluconazole. RNA sequencing data suggested changes in cell wall organisation and reduction in expression of sterol biosynthesis genes in the TLO null mutant. Mass spectrometry revealed reduced ergosterol content in the TLO mutant cell membrane.
Conclusions: These data confirm the involvement of Tlo proteins in the development of C. albicans tolerance of azole drugs, highlighting their potential as targets for the development of novel antifungal drugs.