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

Abstract

Aspergillus fumigatus is a major fungal pathogen causing life threatening infections in immunocompromised humans and certain animals. The HOG pathway is for two reasons interesting in this context: firstly, it is a stress signaling pathway that contributes to the ability of this pathogen to adapt to various stress conditions and secondly, it is the target of antifungal agents, such as fludioxonil or pyrrolnitrin. In this study, we demonstrate that Ypd1 is an essential protein in A. fumigatus. As the central component of the multistep phosphorelay it represents the functional link between the sensor histidine kinases and the downstream response regulators SskA and Skn7. A GFP-Ypd1 fusion was found to reside in both, the cytoplasm and the nucleus and this pattern was only slightly affected by fludioxonil. A strain in which the ypd1 gene is expressed from a tet-on promoter construct is unable to grow under non-inducing conditions and shows the characteristic features of A. fumigatus wild type hyphae treated with fludioxonil. Expression of wild type Ypd1 prevents this lethal phenotype, but expression of an Ypd1 mutant protein lacking the conserved histidine at position 89 was unable to do so, which confirms that A. fumigatus Ypd1 is a phosphotransfer protein. Generation of ypd1tet−on variants of several mutant strains revealed that the lethal phenotype associated with low amounts of Ypd1 depends on SskA, but not on TcsC or Skn7. The ΔsskA ypd1tet−on, but not the ΔsskAΔskn7 ypd1tet−on mutant, was sensitive to fludioxonil, which underlines the importance of Skn7 in this context. We finally succeeded to delete ypd1, but only if sskA and skn7 were both inactivated, not in a ΔsskA single mutant. Hence, a deletion of ypd1 and an inactivation of Ypd1 by fludioxonil result in similar phenotypes and the two response regulators SskA and Skn7 are involved in both processes albeit with a different relative importance.

The N-terminus of the Aspergillus fumigatus group III hybrid histidine kinase TcsC is essential for its physiological activity and targets the protein to the nucleus

“…In A. fumigatus , Ypd1 is present in the whole nucleo-cytoplasm and shuttles between both compartments. Ypd1 translocates much faster than TcsC ( 12 ), which is likely due to its much smaller size.…”

“…Recent data showed that TcsC executes its activity via the histidine phosphotransfer (Hpt) protein Ypd1 and the downstream response regulators Skn7 and SskA ( 11 , 12 ); all four proteins form a so-called multistep phosphorelay ( 13 ). Our current knowledge of the mechanisms that activate TcsC and other group III HHK is still in its infancy.…”

<i>In silico</i> screening of phytochemicals against Ypd1 protein of a destructive storage fungi, <i>Aspergillus</i>

“…Ypd1 protein was selected for the in silico study. The Ypd1 protein is important for the physiology of selected fungi as it contributes to the ability of the pathogen to adapt to various stressed conditions (Schruefer, 2021). The phytochemicals (ligands) from the Punica granatum were selected to analyze their inhibition ability to the functioning of the selected protein.…”

An array of signal-specific MoYpd1 isoforms determines full virulence in the pathogenic fungus Magnaporthe oryzae

“…In addition to its key role in the HOG signaling pathway, MoYPD1 appears to positively and negatively influence genes involved in other stress adaptation processes and virulence 6 . Studies on Ypd1p homologs in other fungi, such as Candida albicans and Aspergillus fumigatus , have shown that green fluorescent protein (GFP)-fused Ypd1p shuttles between the cytoplasm and the nucleus 28 , 29 . Therefore, it is conceivable that MoYpd1p interacts with targets in both the cytosol and nucleus.…”