Systematic functional analysis of the zinc cluster transcription factor family of the pathogenic yeast Candida albicans by artificial activation

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The regulation of gene expression is crucial for the ability of cells to adapt to changes in their environment, but it is also an important determinant in the development of organisms and in the evolution of species. The zinc cluster proteins are a family of transcriptional regulators that are unique to the fungal kingdom. In the pathogenic yeast Candida albicans they have key roles in the regulation of virulence-related traits. Furthermore, gain-of-function mutations in these transcription factors result in the generation of variants with novel phenotypes and are responsible for the acquisition of antifungal drug resistance in many clinical isolates. However, the functions of most members of this family in the biology and pathogenicity of C. albicans are still unknown. We have devised a method for artificially activating zinc cluster proteins, an approach that does not require knowledge about inducing conditions and can reveal their involvement in a specific phenotype even when deletion of the corresponding genes has no effect. We generated a comprehensive library of C. albicans strains expressing constitutively active forms of all 82 zinc cluster transcription factors of this fungus. The phenotypic analysis of this strain collection identified a novel regulator of the morphological transition to invasive hyphal growth and uncovered previously unknown mechanisms of resistance to the widely used antifungal drug fluconazole and against innate host defenses. Importantly, natural gain-of-function mutations in these transcription factors were detected in patient isolates with the same phenotypes, demonstrating the clinical relevance of our findings. By generating hybrid transcription factors with altered DNA-binding properties, we showed that the ability to efficiently induce the expression of multidrug efflux pumps in the presence of fluconazole strongly increases the intrinsic resistance of C. albicans to this drug. The aims of this project are now to uncover the molecular mechanisms by which specific transcription factors affect the properties and behavior of C. albicans and how important they are for the colonization and infection of different host niches. In addition, we investigate if acquiring the ability to react to a certain stimulus by inducing the expression of specific sets of previously unresponsive genes confers a selective advantage under adverse conditions and contributes to a better adaptation of C. albicans to environmental alterations in the human host.