Inducible Cell Fusion Permits Use of Competitive Fitness Profiling in the Human Pathogenic Fungus Aspergillus fumigatus.

Antifungal agents directed against novel therapeutic targets are required for treating invasive, chronic, and allergic Aspergillus infections. Competitive fitness profiling technologies have been used in a number of bacterial and yeast systems to identify druggable targets; however, the development of similar systems in filamentous fungi is complicated by the fact that they undergo cell fusion and heterokaryosis. Here, we demonstrate that cell fusion in Aspergillus fumigatus under standard culture conditions is not predominately constitutive, as with most ascomycetes, but can be induced by a range of extracellular stressors. Using this knowledge, we have developed a barcode-free genetic profiling system that permits high-throughput parallel determination of strain fitness in a collection of diploid A. fumigatus mutants. We show that heterozygous cyp51A and arf2 null mutants have reduced fitness in the presence of itraconazole and brefeldin A, respectively, and a heterozygous atp17 null mutant is resistant to brefeldin A.

Identification of Functional and Druggable Sites in Aspergillus fumigatus Essential Phosphatases by Virtual Screening.

Fungal diseases are a serious health burden worldwide with drug resistance compromising efficacy of the limited arsenal of antifungals available. New drugs with novel mechanisms of action are desperately needed to overcome current challenges. The screening of the Aspergillus fumigatus genome identified 35 phosphatases, four of which were previously reported as essential for viability. In addition, we validated another three essential phosphatases. Phosphatases control critical events in fungi from cell wall integrity to cell cycle, thus they are attractive targets for drug development. We used VSpipe v1.0, a virtual screening pipeline, to evaluate the druggability of the seven essential phosphatases and identify starting points for drug discovery. Targeted virtual screening and evaluation of the ligand efficiency plots created by VSpipe, enabled us to define the most favourable chemical space for drug development and suggested different modes of inhibition for each phosphatase. Interestingly, the identified ligand binding sites match with functional sites (active site and protein interaction sites) reported for other yeast and human homologues. Thus, the VSpipe virtual screening approach identified both druggable and functional sites in these essential phosphatases for further experimental validation and antifungal drug development.

Correction: Mitochondrial Complex I Is a Global Regulator of Secondary Metabolism, Virulence and Azole Sensitivity in Fungi.

[This corrects the article DOI: 10.1371/journal.pone.0158724.].

A Nonredundant Phosphopantetheinyl Transferase, PptA, Is a Novel Antifungal Target That Directs Secondary Metabolite, Siderophore, and Lysine Biosynthesis in Aspergillus fumigatus and Is Critical for Pathogenicity.

Secondary metabolites are key mediators of virulence for many pathogens. Aspergillus fumigatus produces a vast array of these bioactive molecules, the biosynthesis of which is catalyzed by nonribosomal peptide synthetases (NRPSs) or polyketide synthases (PKSs). Both NRPSs and PKSs harbor carrier domains that are primed for acceptance of secondary metabolic building blocks by a phosphopantetheinyl transferase (P-pant). The A. fumigatus P-pant PptA has been shown to prime the putative NRPS Pes1 in vitro and has an independent role in lysine biosynthesis; however, its role in global secondary metabolism and its impact on virulence has not been described. Here, we demonstrate that PptA has a nonredundant role in the generation of the vast majority of detectable secondary metabolites in A. fumigatus, including the immunomodulator gliotoxin, the siderophores triacetylfusarinine C (TAFC) and ferricrocin (FC), and dihydroxy naphthalene (DHN)-melanin. We show that both the lysine and iron requirements of a pptA null strain exceed those freely available in mammalian tissues and that loss of PptA renders A. fumigatus avirulent in both insect and murine infection models. Since PptA lacks similarity to its mammalian orthologue, we assert that the combined role of this enzyme in both primary and secondary metabolism, encompassing multiple virulence determinants makes it a very promising antifungal drug target candidate. We further exemplify this point with a high-throughput fluorescence polarization assay that we developed to identify chemical inhibitors of PptA function that have antifungal activity.IMPORTANCE Fungal diseases are estimated to kill between 1.5 and 2 million people each year, which exceeds the global mortality estimates for either tuberculosis or malaria. Only four classes of antifungal agents are available to treat invasive fungal infections, and all suffer pharmacological shortcomings, including toxicity, drug-drug interactions, and poor bioavailability. There is an urgent need to develop a new class of drugs that operate via a novel mechanism of action. We have identified a potential drug target, PptA, in the fungal pathogen Aspergillus fumigatus PptA is required to synthesize the immunotoxic compound gliotoxin, DHN-melanin, which A. fumigatus employs to evade detection by host cells, the amino acid lysine, and the siderophores TAFC and FC, which A. fumigatus uses to scavenge iron. We show that strains lacking the PptA enzyme are unable to establish an infection, and we present a method which we use to identify novel antifungal drugs that inactivate PptA.

Mitochondrial Complex I Is a Global Regulator of Secondary Metabolism, Virulence and Azole Sensitivity in Fungi.

Recent estimates of the global burden of fungal disease suggest that that their incidence has been drastically underestimated and that mortality may rival that of malaria or tuberculosis. Azoles are the principal class of antifungal drug and the only available oral treatment for fungal disease. Recent occurrence and increase in azole resistance is a major concern worldwide. Known azole resistance mechanisms include over-expression of efflux pumps and mutation of the gene encoding the target protein cyp51a, however, for one of the most important fungal pathogens of humans, Aspergillus fumigatus, much of the observed azole resistance does not appear to involve such mechanisms. Here we present evidence that azole resistance in A. fumigatus can arise through mutation of components of mitochondrial complex I. Gene deletions of the 29.9KD subunit of this complex are azole resistant, less virulent and exhibit dysregulation of secondary metabolite gene clusters in a manner analogous to deletion mutants of the secondary metabolism regulator, LaeA. Additionally we observe that a mutation leading to an E180D amino acid change in the 29.9 KD subunit is strongly associated with clinical azole resistant A. fumigatus isolates. Evidence presented in this paper suggests that complex I may play a role in the hypoxic response and that one possible mechanism for cell death during azole treatment is a dysfunctional hypoxic response that may be restored by dysregulation of complex I. Both deletion of the 29.9 KD subunit of complex I and azole treatment alone profoundly change expression of gene clusters involved in secondary metabolism and immunotoxin production raising potential concerns about long term azole therapy.

Body size over-estimation in women with anorexia nervosa is not qualitatively different from female controls.

Over-estimation of body size is a cardinal feature of anorexia nervosa (AN), usually revealed by comparing individuals who have AN with non-AN individuals, the inference being that over-estimation is pathological. We show that the same result can be reproduced by sampling selectively from a single distribution of performance in body size judgement by comparing low BMI individuals with normal BMI individuals. Over-estimation of body size in AN is not necessarily pathological and can be predicted by normal psychophysical biases in magnitude estimation. We confirm this prediction in a dataset from a morphing study in which 30 women with AN and 137 control women altered a photograph of themselves to estimate their actual body size. We further investigated the relative contributions of sensory and attitudinal factors to body-size overestimation in a sample of 166 women. Our results suggest that both factors play a role, but their relative importance is task dependent.

The Aspergillus fumigatus dihydroxyacid dehydratase Ilv3A/IlvC is required for full virulence.

Dihydroxyacid dehydratase (DHAD) is a key enzyme in the branched-chain amino acid biosynthetic pathway that exists in a variety of organisms, including fungi, plants and bacteria, but not humans. In this study we identified four putative DHAD genes from the filamentous fungus Aspergillus fumigatus by homology to Saccharomyces cerevisiae ILV3. Two of these genes, AFUA_2G14210 and AFUA_1G03550, initially designated AfIlv3A and AfIlv3B for this study, clustered in the same group as S. cerevisiae ILV3 following phylogenetic analysis. To investigate the functions of these genes, AfIlv3A and AfIlv3B were knocked out in A. fumigatus. Deletion of AfIlv3B gave no apparent phenotype whereas the Δilv3A strain required supplementation with isoleucine and valine for growth. Thus, AfIlv3A is required for branched-chain amino acid synthesis in A. fumigatus. A recombinant AfIlv3A protein derived from AFUA_2G14210 was shown to have DHAD activity in an in vitro assay, confirming that AfIlv3A is a DHAD. In addition we show that mutants lacking AfIlv3A and ilv3B exhibit reduced levels of virulence in murine infection models, emphasising the importance of branched-chain amino acid biosynthesis in fungal infections, and hence the potential of targeting this pathway with antifungal agents. Here we propose that AfIlv3A/AFUA_2G2410 be named ilvC.