CELLWALLPATH

Identification of new molecules able to bypass the cell wall compensatory pathways in the pathogenic fungus Aspergillus fumigatus

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 Obiettivo del progetto (Objective)

'Aspergillus fumigatus is the most important air-borne fungal pathogen, causing severe infections with invasive growth in immunocompromised patients. For growth in all habitats, the fungus needs to be able to sense environmental stimuli and to transduce signals via specific signalling cascades. Calmodulin/calcineurin, Ras/cAMP, mTOR and general Mitogen-activated protein kinase (MAPK) signalling pathways are involved in the regulation of various cellular responses in eukaryotes. Previous studies revealed that the central core of the MAPK cell wall integrity signalling pathway in A. fumigatus is composed of three protein kinases. Deletion of these genes resulted in severe sensitivity of the mutants against cell wall-disturbing compounds and drastic alterations of the fungal morphology. Nevertheless, the mutants were still virulent in a murine infection model. Even more, therapeutic treatment with medical commercial cell wall-acting compounds, like echinocandins and azoles, did not result in effective treatment against aspergillosis, indicating the importance of cell wall assembling pathways compensating the inhibition caused by cell wall-disturbing compounds. Protein kinase activation requires phosphorylation. This process is reversible even in the continued presence of activating stimuli. The importance of protein phosphatases for signalling pathways and cross talk interactions is emerging. Ontology classification analysis revealed that the A. fumigatus genome contains 24 genes coding for putative protein phosphatases. A comprehensive and extensive study on protein phosphatases in filamentous fungi has never been attempted so far. Here, we propose to study protein phosphatase families in A. fumigatus. Mutant strains will be used to investigate cell wall compensatory pathways in order to identify new molecules for therapeutic intervention against aspergillosis.'

Introduzione (Teaser)

Aspergillosis is caused by fungal infection from Aspergillus fumigatus and most often affects patients with a weakened immune system such as those suffering from leukaemia. The EU-funded project CELLWALLPATH investigated biochemical pathways involved in cell wall maintenance and defence in the fungus to find therapies for the disease.

Descrizione progetto (Article)

Mitogen-activated protein kinase (MAPK) signalling pathways are important for maintaining cell wall integrity in A. fumigatus. Research has shown that three protein kinases play a key role here. Protein kinases are reversibly activated through phosphorylation and are essential in signalling pathways and crosstalk interactions in fungi.

Using ontology classification of protein phosphatases in A. fumigatus, CELLWALLPATH researchers investigated cell wall compensatory pathways. Previous research has shown that calmodulin/calcineurin, Ras/cAMP, mTOR and general MAPK signalling pathways help regulate cellular responses in fungi.

Project studies showed that the polyketide synthase (pksP) gene cluster is regulated by cAMP and MAPK signalling pathways present in A. fumigatus. This could prove to be a useful biomarker to study crosstalk interaction in MAPK signalling pathways.

Researchers also investigated the mechanism of action of Caspofungin (CAS), a cell wall-acting compound and tested its effectiveness through transcriptome analysis. CAS worked by affecting cellular and carbohydrate metabolism. In addition to cell wall stress, CAS affected cell membranes by inducing osmolarity stress. As such, CAS was deemed to be appropriate for use in combination therapy. Also, the peptide MDN-0010 was found to be 4.5 times stronger than CAS against these fungi.

To identify key protein phosphatases, a comparative blastp and tblastn analysis was performed and 33 putative protein phosphatases were identified. Phosphatase screening revealed that serine and threonine protein phosphatases are important for cell wall maintenance. Researchers successfully deleted 28 putative protein phosphatase genes and created five inducible mutant strains using fast cloning methodology and Saccharomyces cerevisiae.

Currently, there is no effective treatment against aspergillosis. The need for effective therapy has become increasingly important over the last two decades as this disease incidence has increased. Successful project outcomes could reveal novel drug compounds and provide options for effective therapeutic intervention.