Genomic analyses of fungal genome structure have revealed the presence of physically-linked groups of genes, termed gene clusters, where collective functionality of encoded gene products serves a common biosynthetic purpose. In multiple fungal pathogens of humans and plants gene clusters have been shown to encode pathways for biosynthesis of secondary metabolites including metabolites required for pathogenicity. In the major mould pathogen of humans Aspergillus fumigatus, multiple clusters of co-ordinately upregulated genes were identified as having heightened transcript abundances, relative to laboratory cultured equivalents, during the early stages of murine infection. The aim of this study was to develop and optimise a methodology for manipulation of gene cluster architecture, thereby providing the means to assess their relevance to fungal pathogenicity. To this end we adapted a recombineering methodology which exploits lambda phage-mediated recombination of DNA in bacteria, for the generation of gene cluster deletion cassettes. By exploiting a pre-existing bacterial artificial chromosome (BAC) library of A. fumigatus genomic clones we were able to implement single or multiple intra-cluster gene replacement events at both subtelomeric and telomere distal chromosomal locations, in both wild type and highly recombinogenic A. fumigatus isolates. We then applied the methodology to address the boundaries of a gene cluster producing a nematocidal secondary metabolite, pseurotin A, and to address the role of this secondary metabolite in insect and mammalian responses to A. fumigatus challenge.

Author Summary Inhaled spores of the pathogenic mould Aspergillus fumigatus cause fungal lung infections in humans having immune defects. A. fumigatus spores germinate within the immunocompromised lung, producing invasively growing, elongated cells called hyphae. Hyphae degrade the surrounding pulmonary tissue, a process thought to be caused by secreted fungal enzymes; however, A. fumigatus mutants lacking one or more protease activities retain fully invasive phenotypes in mouse models of disease. Here we report the first discovery of a non-invasive A. fumigatus mutant, which lacks a pH-responsive transcription factor PacC. Using global transcriptional profiling of wild type and mutant isolates, and in vitro pulmonary invasion assays, we established that loss of PacC leads to a compound non-invasive phenotype characterised by deficits in both contact-mediated epithelial entry and protease expression. Consistent with an important role for epithelial entry in promoting invasive disease in mammalian tissues, PacC mutants remain surface-localised on mammalian epithelia, both in vitro and in vivo. Our study sets a new precedent for involvement of both host and pathogen activities in promoting epithelial invasion by A. fumigatus and supports a model wherein fungal protease activity acting subsequently to, or in parallel with, host-mediated epithelial entry provides the mechanistic basis for tissue invasion.

Aspergillus fumigatus is the most common airborne fungal pathogen for humans. In this mold, iron starvation induces production of the siderophore triacetylfusarinine C (TAFC). Here we demonstrate a link between TAFC and ergosterol biosynthetic pathways, which are both critical for virulence and treatment of fungal infections. Consistent with mevalonate being a limiting prerequisite for TAFC biosynthesis, we observed increased expression of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase (Hmg1) under iron starvation, reduced TAFC biosynthesis following lovastatin-mediated Hmg1 inhibition, and increased TAFC biosynthesis following Hmg1 overexpression. We identified enzymes, the acyl-CoA ligase SidI and the enoyl-CoA hydratase SidH, linking biosynthesis of mevalonate and TAFC, deficiency of which under iron starvation impaired TAFC biosynthesis, growth, oxidative stress resistance, and murine virulence. Moreover, inactivation of these enzymes alleviated TAFC-derived biosynthetic demand for mevalonate, as evidenced by increased resistance to lovastatin. Concordant with bilateral demand for mevalonate, iron starvation decreased the ergosterol content and composition, a phenotype that is mitigated in TAFC-lacking mutants.

Nonribosomal peptide synthesis (NRPS) is a documented virulence factor for the opportunistic pathogen Aspergillus fumigatus and other fungi. Secreted or intracellularly located NRP products include the toxic molecule gliotoxin and the iron-chelating siderophores triacetylfusarinine C and ferricrocin. No structural or immunologically relevant NRP products have been identified in the organism. We investigated the function of the largest gene in A. fumigatus, which encodes the NRP synthetase Pes3 (AFUA_5G12730), by targeted gene deletion and extensive phenotypic analysis. It was observed that in contrast to other NRP synthetases, deletion of pes3 significantly increases the virulence of A. fumigatus, whereby the pes3 deletion strain (A. fumigatus Δpes3) exhibited heightened virulence (increased killing) in invertebrate (P < 0.001) and increased fungal burden (P = 0.008) in a corticosteroid model of murine pulmonary aspergillosis. Complementation restored the wild-type phenotype in the invertebrate model. Deletion of pes3 also resulted in increased susceptibility to the antifungal, voriconazole (P < 0.01), shorter germlings, and significantly reduced surface β-glucan (P = 0.0325). Extensive metabolite profiling revealed that Pes3 does not produce a secreted or intracellularly stored NRP in A. fumigatus. Macrophage infections and histological analysis of infected murine tissue indicate that Δpes3 heightened virulence appears to be mediated by aberrant innate immune recognition of the fungus. Proteome alterations in A. fumigatus Δpes3 strongly suggest impaired germination capacity. Uniquely, our data strongly indicate a structural role for the Pes3-encoded NRP, a finding that appears to be novel for an NRP synthetase.

Carbon dioxide (CO(2)) and its hydration product bicarbonate (HCO(3)(-)) are essential molecules in various physiological processes of all living organisms. The reversible interconversion between CO(2) and HCO(3)(-) is in equilibrium. This reaction is slow without catalyst, but can be rapidly facilitated by Zn(2+)-metalloenzymes named carbonic anhydrases (CAs). To gain an insight into the function of multiple clades of fungal CA, we chose to investigate the filamentous fungi Aspergillus fumigatus and A. nidulans. We identified four and two CAs in A. fumigatus and A. nidulans, respectively, named cafA-D and canA-B. The cafA and cafB genes are constitutively, strongly expressed whereas cafC and cafD genes are weakly expressed but CO(2)-inducible. Heterologous expression of the A. fumigatus cafB, and A. nidulans canA and canB genes completely rescued the high CO(2)-requiring phenotype of a Saccharomyces cerevisiae Deltance103 mutant. Only the DeltacafA DeltacafB and DeltacanB deletion mutants were unable to grow at 0.033% CO(2), of which growth defects can be restored by high CO(2). Defects in the CAs can affect Aspergilli conidiation. Furthermore, A. fumigatus DeltacafA, DeltacafB, DeltacafC, DeltacafD and DeltacafA DeltacafB mutant strains are fully virulent in a low-dose murine infection.

Virulence of the fungal pathogen Aspergillus fumigatus is in part based on the saprophytic lifestyle that this mold has evolved. A crucial function for saprophytism resides in secreted proteases that allow assimilation of proteinaceous substrates. The impact of extracellular proteolytic activities on the pathogenesis of aspergillosis, however, remains controversial. In order to address this issue, characterization of a conserved regulatory factor, PrtT, that acts on expression of secreted proteases was pursued. Expression of PrtT appears to be regulated posttranscriptionally, and the existence of an mRNA leader sequence implies translational control via eIF2alpha kinase signaling. Phenotypic classification of a prtTDelta deletion mutant revealed that expression of several major extracellular proteases is PrtT dependent, resulting in the inability to utilize protein as a nutritional source. Certain genes encoding secreted proteases are not regulated by PrtT. Most strikingly, the deletant strain is not attenuated in virulence when tested in a leukopenic mouse model, which makes a strong case for reconsidering any impact of secreted proteases in pulmonary aspergillosis.

Aspergillus fumigatus is a common mould whose spores are a component of the normal airborne flora. Immune dysfunction permits developmental growth of inhaled spores in the human lung causing aspergillosis, a significant threat to human health in the form of allergic, and life-threatening invasive infections. The success of A. fumigatus as a pathogen is unique among close phylogenetic relatives and is poorly characterised at the molecular level. Recent genome sequencing of several Aspergillus species provides an exceptional opportunity to analyse fungal virulence attributes within a genomic and evolutionary context. To identify genes preferentially expressed during adaptation to the mammalian host niche, we generated multiple gene expression profiles from minute samplings of A. fumigatus germlings during initiation of murine infection. They reveal a highly co-ordinated A. fumigatus gene expression programme, governing metabolic and physiological adaptation, which allows the organism to prosper within the mammalian niche. As functions of phylogenetic conservation and genetic locus, 28% and 30%, respectively, of the A. fumigatus subtelomeric and lineage-specific gene repertoires are induced relative to laboratory culture, and physically clustered genes including loci directing pseurotin, gliotoxin and siderophore biosyntheses are a prominent feature. Locationally biased A. fumigatus gene expression is not prompted by in vitro iron limitation, acid, alkaline, anaerobic or oxidative stress. However, subtelomeric gene expression is favoured following ex vivo neutrophil exposure and in comparative analyses of richly and poorly nourished laboratory cultured germlings. We found remarkable concordance between the A. fumigatus host-adaptation transcriptome and those resulting from in vitro iron depletion, alkaline shift, nitrogen starvation and loss of the methyltransferase LaeA. This first transcriptional snapshot of a fungal genome during initiation of mammalian infection provides the global perspective required to direct much-needed diagnostic and therapeutic strategies and reveals genome organisation and subtelomeric diversity as potential driving forces in the evolution of pathogenicity in the genus Aspergillus.