Functional Genomic Analysis of Natural Product Biosynthesis and Secretion in Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic pathogen which can cause severe disease in immunocompromised patients. A. fumigatus produces metabolites, through a non-ribosomal peptide synthesis mechanism, including iron-chelating molecules known as siderophores and other unusual compounds such as gliotoxin. These provide the fungus with a unique strategy for survival in the host, and contribute to pathogenicity. The aim of this project was to identify interactions between non-ribosomal peptide synthetase genes (termed pes1 and sidD) and to identify metabolites produced by these genes, which contribute to the virulence of A. fumigatus in immunocompromised patients. A. fumigatus pes1 mutants (pes1) were generated in three genetic backgrounds (akuB, ATCC46645 and sidD46645), confirmed by Southern blot and qRT-PCR analysis. A. fumigatus akuB:pes1 was significantly more sensitive to oxidative stress than wild-type (P < 0.01). It was found that, in vitro, either pes1 or sidD significantly improved fungal tolerance to anti-fungal drugs (amphotericin B and voriconazole, respectively) which strongly suggests that either gene may play a role in mediating drug resistance/tolerance in patients. It was found that under oxidative stress conditions that this double mutant was significantly less sensitive to ironlimiting conditions than A. fumigatus sidD, indicating that the peptide encoded via pes1 is involved in the response of siderophore-deficient A. fumigatus to low iron availability, such as during infection. Compared to wild-type A. fumigatus, sidD:pes1 was significantly less virulent than either single mutant in the Galleria mellonella virulence model system. Significantly, pes1 was found to be involved in the biosynthesis of the conidial metabolite, fumigaclavine C, as this metabolite was absent in both A. fumigatus akuB:pes1 and pes146645. In parallel studies, we identified a gene (gliK) responsible for gliotoxin biosynthesis and showed that gliK is involved in protecting A. fumigatus against oxidative stress, gliotoxin presence and secretion of gliotoxin from A. fumigatus. Overall, our findings may lead to improvements in therapy for patients suffering from invasive aspergillosis in the medium-to long-term