Analysis of the spatial distribution of the key components of the HOG signaling pathway, TcsC and Ypd1, and its impact on their function in the pathogenic mold Aspergillus fumigatus.
Final Report Abstract
Aspergillus fumigatus is an important fungal pathogen and systemic Aspergillus infections are associated with a high mortality, which underlines the urgent need for new therapeutic options. The High Osmolarity Glycerol (HOG) pathway is a central stress response pathway of many fungi. Its activation by fludioxonil or related compounds is lethal for A. fumigatus. These compounds are therefore interesting candidates for the development of novel therapeutic options. Both, the HOG- mediated protective responses and the impact of fludioxonil rely on the sensor kinase TcsC. Activated TcsC modulates the activity of the Ypd1 protein to activate the downstream response regulators SskA and Skn7. In a first paper published 2021 in Scientific Reports, we demonstrate that Skn7 is particular important for the lethal impact of fludioxonil. In a second paper, published 2021 in Frontiers in Fungal Biology, we show that a loss of the ypd1 gene is lethal for A. fumigatus. Only a strain that already lacks sskA und skn7 can tolerate a loss of ypd1. In the wild type, such a loss results in an uncontrolled and fatal activation of SskA and Skn7. In a recent study, we have compared fludioxonil-induced transcriptional changes in three different strains: in the A. fumigatus wild type and the deletion mutants ΔtcsC and Δskn7. As expected, we observed the strongest response in the fludioxonil-sensitive wild type strain and the weakest response in the fludioxonil-resistant ΔtcsC mutant. In combination with novel biochemical and previously published immunofluorescence data, these RNA-Seq data demonstrate that fludioxonil employs Skn7 to weaken the fungal cell wall, which in combination with an increase in the internal osmotic pressure causes the characteristic and lethal ballooning of fungal cells. Investigations on the localization of TcsC were at the heart of this research project. It was already known from the start that TcsC resides in the nucleus, an unexpected localization for a sensor kinase. We could show that the N-terminal 208 amino acids guide TcsC to the nucleus and are also essential for the adaptive response to hyperosmotic stress. These data initially suggested a functional link between the nuclear localization and the physiological function of TcsC. In the Frontiers in Fungal Biology paper, we show that Skn7 is a nuclear and SskA a cytoplasmic protein, whereas the small Ypd1 protein shuttles between both compartments. Fludioxonil has no significant impact on these patterns, but it triggers a translocation of TcsC to the cytoplasm. We have identified two N-terminal nuclear localization signals and their deletion or even an exchange of a single basic amino acid prevented the nuclear targeting. Unexpectedly, these cytoplasmic variants of TcsC were still able to mount an adaptive response to hyperosmotic stress. This and other data are part of a recently submitted manuscript. In summary, the results of the current project have significantly improved our understanding of the HOG signaling pathway in A. fumigatus. However, new questions arose and an initial question remained: Why is TcsC targeted to the nucleus of A. fumigatus?
Publications
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54. Wissenschaftliche Tagung der Deutschsprachigen Mykologischen Gesellschaft, 16.09 – 18.09.2020 (oral presentation online): Novel insights in the antifungal activity of fludioxonil in Aspergillus fumigatus.
Schruefer, S., Böhmer, I., Dichtl, K., Pschibul, A., Kniemeyer, O., Wolf, T., Sae-Ong, T., Panagiotou, G., Brakhage, A. A. & Ebel, F.
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73. Jahrestagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie, 12.09. – 14.09.2021 in Berlin und online (oral presentation): The high osmolarity glycerol (HOG) pathway in Aspergillus fumigatus: a key player in stress resistance and a potential drug target.
Schruefer, S., Böhmer, I., Dichtl, K., Kleinemeier, C. & Ebel, F.
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Tagung der DVG-Fachgruppe „Bakteriologie und Mykologie“, 14.06. – 16.06.2021 (oral presentation online): The HOG pathway of Aspergillus fumigatus: an important element of stress resistance and a potential drug target.
Schruefer, S., Böhmer, I., Dichtl, K., Pschibul, A., Kniemeyer, O., Wolf, T., Sae-Ong, T., Panagiotou, G., Brakhag, A. A. & Ebel, F.
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The High Osmolarity Glycerol (HOG) Pathway 54. Wissenschaftliche Tagung der Deutschsprachigen Mykologischen Gesellschaft, 27.09.2021 (oral presentation online): The High Osmolarity Glycerol (HOG) Pathway - New Insights into a Crucial Fungal Signaling Pathway.
Ebel, F.
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The response regulator Skn7 of Aspergillus fumigatus is essential for the antifungal effect of fludioxonil. Scientific Reports, 11(1).
Schruefer, Sebastian; Böhmer, Isabella; Dichtl, Karl; Spadinger, Anja; Kleinemeier, Christoph & Ebel, Frank
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Ypd1 Is an Essential Protein of the Major Fungal Pathogen Aspergillus fumigatus and a Key Element in the Phosphorelay That Is Targeted by the Antifungal Drug Fludioxonil. Frontiers in Fungal Biology, 2.
Schruefer, Sebastian; Spadinger, Anja; Kleinemeier, Christoph; Schmid, Laura & Ebel, Frank
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56. Wissenschaftliche Tagung der Deutschsprachigen Mykologischen Gesellschaft zusammen mit der Österreichischen Gesellschaft für Medizinische Mykologie, 12.09. – 14.09.2022 in Wien (oral presentation): The Ying and Yang of Ypd1, a protein of the HOG pathway that is essential for the viability of Aspergillus fumigatus and the antifungal activity of fludioxonil.
Schruefer, S., Spadinger, A., Kleinemeier, C., Schmid, L. & Ebel F.
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74. Jahrestagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie, 05.09.-07.09.2022 in Berlin (oral presentation): Characterization of Ypd1, an essential element of the HOG pathway of Aspergillus fumigatus that is required for the antifungal mechanism of fludioxonil.
Schruefer, S., Spadinger, A., Kleinemeier, C., Schmid, L. & Ebel F.
