A functional genomics approach: Characterisation of two Non-ribosomal peptide synthetase genes, and investigation of an adaptive response towards alkylating DNA damage in Aspergillus fumigatus.

Aspergillus fumigatus is a serious opportunistic human pathogen. Availability of the complete genome sequence of A. fumigatus allows the identification and subsequent characterisation of genes which may encode virulence factors, or novel drug targets. Nonribosomal peptide (NRP) synthetases have been implicated in virulence of A. fumigatus and other fungi. The work presented here reports opposing roles for two previously uncharacterised NRP synthetases with respect to A. fumigatus virulence. The NRP synthetase Pes3 appears to encode a structural peptide necessary for fungal recognition by the innate immune system; deletion of pes3 resulted in hypervirulence in both insect (p < 0.001) and murine (p = 0.02) models of invasive aspergillosis, and increased susceptibility to voriconazole (p < 0.001). The NRP synthetase PesL was found to be essential for fumigaclavine C biosynthesis and deletion of pesL resulted in complete loss of fumigaclavine C accompanied by severely reduced virulence (p < 0.001), increased sensitivity of ΔpesL to H2O2 (> 1 mM) (p = 0.05), and increased sensitivity to the antifungal voriconazole (> 0.25 μg/ml) (p < 0.01) compared to wild-type. An adaptive response to alkylating agents (e.g. N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)) was identified in A. fumigatus. Afmpt is a transcriptional regulator for this response, and exposure to MNNG causes up-regulation of Afmpt and Afagt, an alkylguaninetransferase. Afmpt and Afagt functions were confirmed through targeted gene deletion, phenotypic and expression analyses, and yeast complementation studies. Identification of this response, which has no mammalian equivalent, makes this pathway an attractive anti-fungal drug target worthy of further investigation. Overall, this work further highlights the importance that NRPS plays in this serious human pathogen, and uncovered some interesting features such as possible secondary metabolite cluster cross-talk and NRP synthetase redundancy, themes which are beginning to emerge in the literature