Iron regulation and virulence in Aspergillus fumigatus

Over the past few decades, around 1.52 million deaths have been reported worldwide because of fungal infections every year. Aspergillus fumigatus currently represents one of the main fungal pathogenic species for human immunocompromised patients. This pathogen can also affect immunocompetent hosts, generating various forms of respiratory diseases or allergies. Due to the development of fungal resistance to the commonly used antifungal drugs, the continuous search for new possible therapeutic targets is a must. It has been previously proven that iron homeostasis is a key factor for A. fumigatus survival and pathogenicity. Iron represents an essential cofactor for a variety of cellular process; at the same time, an excess of free iron is toxic for the cell. One specific cellular regulator discovered in Prof. Haas lab emerged as a key factor for the maintenance of iron homeostasis. This protein, named HapX, not only plays a central role during iron starvation conditions, activating fungal pathways to increase iron acquisition from the environment and repressing iron-consumption, but it has also been identified to be essential in the presence of iron excess, as it promotes iron storage into cellular compartments to prevent toxicity effects. A link between iron and pathogenicity is strongly suggested by the fact that HapX deletion, as well as deletion of several of the characterized genes involved in A. fumigatus adaptation to iron starvation, causes defects in virulence. However, despite the identification of more and more players in iron metabolism, the overall picture of how the fungus senses and adapts to different iron conditions is poorly understood. The proposed project aims to identify so far unknown iron-linked genes by screening different A. fumigatus deletion mutant libraries (i.e. kinase, phosphatase and transcription factor deletion mutant libraries). A selection of most interesting candidates out of the screening will be then further characterized to identify attractive phenotype in different iron conditions, verifying growth, biomass production, iron uptake, transcript levels of iron-regulated genes. The selected strains will be tested to verify their involvement in virulence. Pathogenicity assays will be performed first in vitro (to further reduce the number of strains to test) and then in vivo. This will allow the identification of new possible antifungal targets and will open the door for novel therapies.

This research project aimed to gain insights into iron regulation and metabolism in the pathogenic fungus Aspergillus fumigatus, one of the most important human fungal pathogens, with the goal of identifying new targets for antifungal therapies. The project focused on screening deletion mutant libraries, characterizing selected strains, and completing the library with conditional-lethal mutants. Additionally, the investigation aimed to identify genes involved in pathogenicity and explore the possibility of specific inhibitors. The project began with the development of reliable screening strategies to identify iron-related defects in A. fumigatus mutants. Three screening methods were optimized and about 40 strains showing iron-related defects were selected to be further characterized to understand their involvement in iron metabolism. The project also aimed to complete the kinase mutant library with conditional-lethal mutants. However, due to the need for a detailed study on available inducible promoters, the library was instead supplemented with histidine kinase deletion mutants. The study on inducible promoters led to the development of new molecular tools, including inducible selection markers and a modular inducible multigene expression system. Investigations into the pathogenicity potential involved preliminary cell damage assays and in vivo studies using the greater wax moth Galleria mellonella. The murine model for invasive aspergillosis was planned for subsequent stages if interesting strains were identified. Due to the reduced duration of the project, these experiments are still ongoing thanks to the collaborators support. Several significant results were obtained during the project, for example the study of inducible selection markers revealed previously unknown undesired effects when such markers are used, and led to the development of new selectable marker cassettes. Moreover, a modular inducible multigene expression system was optimized and can allow the independent use of up to three inducible promoters in a single strain, a tool that can be heavily implemented in the industrial area. Moreover, thanks to the developed screening strategies, several mutant strains have been identified and further characterized, and some of them are already reported in independent scientific papers as relevant for pathogenicity. In conclusion, this research project provided valuable insights into iron regulation and potential antifungal targets in A. fumigatus. The findings contribute to the understanding of fungal pathogenesis and open avenues for the development of novel antifungal therapies.