Purpose: Aspergillus fumigatus causes 90% of all Aspergillus-related infections. It has been demonstrated that virulence of this fungus is multifactorial and several virulence factors have been identified. In particular, the ability of the fungus to respond to various host-imposed stresses has repeatedly been proven to be essential for pathogenicity. Therefore, regulation of genes by transcription factors plays a critical role in establishing infection. However, we have a limited understanding of the molecular basis of A. fumigatus pathogenicity.
Methods: In this study, we carried out a systematic phenotyping of a transcription factor knockout (TFKO) collection to define the cohort of transcription factors required for pathogenicity. A high throughput analytical pipeline was developed in a liquid microculture 96-well plate format to analyze relative fitness of 401 A. fumigatus TFKO mutants. Data analysis was automated to convert optical density readings collected directly from the spectrophotometer into fitness indices.
Results: Fitness was assessed for the following host-imposed stress conditions: iron starvation, temperature stress and pH tolerance. For iron starvation one previously uncharacterized transcription factor was found, besides the previously described HapX. Alkaline stress resulted in the identification of four previously uncharacterized transcription factors, as well as the pacC mutant, to have a reduced fitness. Ten TFKO mutants exhibit reduced fitness under 48 °Celsius.
Conclusion: Our study yields a robust high throughput phenotyping protocol for functional genomic analyses of A. fumigatus stress tolerance, and for the first time its application to identifying the full cohort of A. fumigatus transcription factors that govern responses to host-imposed stresses.
Full conference title:
- AAA 8th (2018)