The response regulator Skn7 of Aspergillus fumigatus is essential for the antifungal effect of fludioxonil.

Aspergillus fumigatus is an important fungal pathogen that represents a major threat for severely immunocompromised patients. Cases of invasive aspergillosis are associated with a high mortality rate, which reflects the limited treatment options that are currently available. The development of novel therapeutic approaches is therefore an urgent task. An interesting compound is fludioxonil, a derivative of the bacterial secondary metabolite pyrrolnitrin. Both agents possess potent antimicrobial activity against A. fumigatus and trigger a lethal activation of the group III hybrid histidine kinase TcsC, the major sensor kinase of the High Osmolarity Glycerol (HOG) pathway in A. fumigatus. In the current study, we have characterized proteins that operate downstream of TcsC and analyzed their roles in the antifungal activity of fludioxonil and in other stress situations. We found that the SskA-SakA axis of the HOG pathway and Skn7 can independently induce an increase of the internal glycerol concentration, but each of these individual responses amounts for only half of the level found in the wild type. The lethal fludioxonil-induced ballooning occurs in the sskA and the sakA mutant, but not in the skn7-deficient strain, although all three strains show comparable glycerol responses. This indicates that an elevated osmotic pressure is necessary, but not sufficient and that a second, decisive and Skn7-dependent mechanism mediates the antifungal activity. We assume that fludioxonil triggers a reorganization in the fungal cell wall that reduces its rigidity, which in combination with the elevated osmotic pressure executes the lethal expansion of the fungal cells. Two findings link Skn7 to the cell wall of A. fumigatus: (1) the fludioxonil-induced massive increase in the chitin content depends on Skn7 and (2) the skn7 mutant is more resistant to the cell wall stressor Calcofluor white. In conclusion, our data suggest that the antifungal activity of fludioxonil in A. fumigatus relies on two distinct and synergistic processes: A high internal osmotic pressure and a weakened cell wall. The involvement of Skn7 in both processes most likely accounts for its particular importance in the antifungal activity of fludioxonil.

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.

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.

Functional comparison of the group III hybrid histidine kinases TcsC of Aspergillus fumigatus and NikA of Aspergillus nidulans.

In filamentous fungi, group III hybrid histidine kinases (HHKs) are major and nonredundant sensing proteins of the high osmolarity glycerol pathway. In this study, we have compared the biological functions of the two homologous group III HHKs TcsC of Aspergillus fumigatus and NikA of A. nidulans. As expected from previous studies, the corresponding mutants are severely impaired in their ability to adapt to hyperosmotic stress and are both resistant to the antifungal agent fludioxonil. However, our data also reveal novel phenotypes and differences between these mutants. Both TcsC and NikA are required for wild-type-like growth on Czapek-Dox medium and a normal resistance to certain oxidative stressors, whereas an increased resistance to the cell wall disturbing agents Congo red and Calcofluor white was found for the ΔtcsC but not for the ΔnikA mutant. With respect to the cell wall reorganizations that are triggered by fludioxonil in a TcsC/NikA-dependent manner, we observed similarities but also striking differences. Strains from seven Aspergillus species, including A. fumigatus and A. nidulans incorporated more chitin into their cell walls in response to fludioxonil. In contrast, fludioxonil treatment resulted in a shedding of surface accessible galactomannan and ß-1,3-glucan in all Aspergillus strains tested except A. nidulans. Hence, the fludioxonil-induced activation of NikA results in a distinct and apparently A. nidulans-specific pattern of cell wall reorganizations that is not due to NikA itself, but its integration into the A. nidulans signaling network.

Identification of a novel clade of group A rotaviruses in fatally diseased domestic pigeons in Europe.

Rotaviruses are well-known causative agents of enteric disorders in humans and other mammals, but little is known about their virulence and pathogenic role in pigeons and other birds. Starting in summer 2017, a series of outbreaks of an acute disease with high mortalities was reported in domestic pigeons in Germany, Belgium and Denmark. The clinical picture was characterized by diarrhoea, vomiting, hepatic necrosis and sudden fatalities. From these severe outbreaks, we discovered several previously unknown group A rotavirus (RVA) lineages of genotype G18P[17]-I4-R4-C4-M4-A4-T4-N4-E19-H4, which were closely related but not identical to an RVA variant identified in cases of fatal hepatic necrosis in Australian pigeon lofts in 2016. Retrospective analysis demonstrated that the predecessors of the highly virulent variants have circulated in Europe since at least 2010. Our data indicate that reassortment and intercontinental spread has led to the emergence of novel RVA variants, which may constitute a major threat to animal welfare and health of domestic pigeon populations worldwide.

Molecular characterization of Aspergillus fumigatus TcsC, a characteristic type III hybrid histidine kinase of filamentous fungi harboring six HAMP domains.

The type III hybrid histidine kinase (HHK) TcsC enables the pathogenic mold Aspergillus fumigatus to thrive under hyperosmotic conditions. It is, moreover, of particular interest, since it is the target of certain antifungal agents, such as fludioxonil. This study was aimed at a functional characterization of the domains that constitute the sensing and the kinase module of TcsC. The sensing module consists of six HAMP domains, an architecture that is commonly found in type III HHKs of filamentous fungi. To dissect the functional role of the individual domains, we have analyzed a set of truncated derivatives of TcsC with respect to their impact on fungal growth and their ability to respond to hyperosmotic stress and fludioxonil. Our data demonstrate that the TcsC kinase module per se is constitutively active and under the control of the sensing module. We furthermore found that the sixth HAMP domain alone is sufficient to arrest the kinase module in an inactive state. This effect can be partially lifted by the presence of the fifth HAMP domain. Constructs harboring more than these two HAMP domains are per se inactive and all six HAMP domains are required to enable a response to fludioxonil or hyperosmotic stress. When expressed in an A. fumigatus wild type strain, the construct harboring only the sixth HAMP domain exerts a strong dominant negative effect on the native TcsC. This effect is successively reduced in other constructs harboring increasing numbers of HAMP domains. To our knowledge, this is the first molecular characterization of a type III HHK containing six HAMP domains. Our data strongly suggest that TcsC is a positive regulator of its MAPK SakA and thereby differs fundamentally from the prototypic yeast type III HHK DhNik1 of Debaryomyces hansenii, which harbors only five HAMP domains and acts as a negative regulator of its MAPK.

Agents that activate the High Osmolarity Glycerol pathway as a means to combat pathogenic molds.

Treatment of invasive fungal infections often fails due to the limited number of therapeutic options. In this study, we have analyzed the impact of agents activating the High Osmolarity Glycerol (HOG) pathway on molds that cause infections in humans and livestock. We found that agents like fludioxonil and iprodione, have a clear anti-fungal activity against pathogenic Aspergillus, Lichtheimia, Rhizopus and Scedosporium species. Only A. terreus turned out to be resistant to fludioxonil, even though it is sensitive to iprodione and able to adapt to hyperosmotic conditions. Moreover, the A. terreus tcsC gene can fully complement an A. fumigatus ΔtcsC mutant, thereby also restoring its sensitivity to fludioxonil. The particular phenotype of A. terreus is therefore likely to be independent of its TcsC kinase. In a second part of this study, we further explored the impact of fludioxonil using A. fumigatus as a model organism. When applied in concentrations of 1-2µg/ml, fludioxonil causes an immediate growth arrest and, after longer exposure, a quantitative killing. Hyphae respond to fludioxonil by the formation of new septa and closure of nearly all septal pores. Mitosis occurs in all compartments and is accompanied by a re-localization of the NimA kinase to the cytoplasm. In the swollen compartments, the massive extension of the cell wall triggers a substantial reorganization resulting in an enhanced incorporation of chitin and, most strikingly, a massive loss of galactomannan. Hence, HOG-activating agents have dramatic cell biological consequences and may represent a valuable, future element in the armory that can be used to combat mold infections.