Author:
DG Calise1,2*, JW Bok1, NP Keller1,3
Author address:
1Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA
2Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, WI, USA
3Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA
Full conference title:
10th Advances Against Aspergillosis and Mucormycosis
Date: 2 February 2022
Abstract:
Purpose:
Aspergillus fumigatus is an opportunistic pathogen causing many serious diseases such as invasive aspergillosis and allergic bronchopulmonary aspergillosis. A key developmental process in A. fumigatus is the balance between polar growth and lateral branching both of which are necessary for colony expansion within host substrates. Recently our lab found the transcription factor ZfpA to be essential in the hyperbranching response of A. fumigatus to the oxylipin 5,8-diHODE which is produced by the fungal enzyme PpoA. ZfpA deletion generates a hypobranching phenotype whereas its overexpression yields a hyperbranching phenotype. In order to begin to understand the regulatory role of ZfpA in the hyphal development of A. fumigatus, RNA-seq analysis was conducted to identify potential regulatory pathways of ZfpA.
Methods:
Total mRNA was extracted from 16 hour cultures of wild type (WT), ΔzfpA, and OE::zfpA strains in the Af293 background. The RNA samples were sequenced and analyzed, comparing each mutant to the WT strain. The ppoA gene was deleted in the OE::zfpA background by replacing the ppoA open reading frame with a hygromycin B resistance marker. Transformants were confirmed by PCR and Southern blotting.
Results:
In the Af293 ΔzfpA strain, 1273 genes were found to be significantly upregulated (padj. <0.01, log2FC≥1) and 348 significantly downregulated (padj. <0.01, log2FC≤–1). In the OE::zfpA strain 648 and 1076 genes were found to be significantly up- and downregulated respectively. Differentially expressed genes (DEGs) in the deletion mutant were enriched in 16 functional categories (FunCat), and DEGs in the overexpression mutant were enriched in 9 functional categories including secondary metabolism (FDR < 0.05). Manual analysis of the RNA-seq data revealed that genes involved in chitin metabolism were differentially regulated in both mutants suggestive of alterations in cell wall composition. In vitro testing showing ΔzfpA to be more sensitive to cell wall stressors caspofungin and calcofluor white while OE::zfpA was more resistant than WT supports the RNA-seq findings. Additionally, ppoA was the most highly upregulated gene in the OE::zfpA strain suggesting a potential feedback loop in PpoA and ZfpA activities. To start to assess this hypothesis, isogenic strains (ΔppoA mutant, OE::zfpA mutant, and ΔppoA OE::zfpA double mutants) have been created for further analysis of hyphal development.
Conclusions:
Current results suggest that ZfpA is an important developmental regulator of genes involved in diverse metabolic processes in A. fumigatus including chitin metabolism and cell wall synthesis. Candidate genes identified from the RNA-seq profiling will be characterized for roles in hyphal branching.
Abstract Number: 45
Conference Year: 2022
Link to conference website: https://aaam2022.org/
URL Conference abstract: