Modeling Approaches Reveal New Regulatory Networks in <i>Aspergillus fumigatus</i> Metabolism

Systems biology approaches are extensively used to model and reverse-engineer gene regulatory networks from experimental data. Indoleamine 2,3-dioxygenases (IDOs)—belonging in the heme dioxygenase family—degrade l-tryptophan to kynurenines. These enzymes are also responsible for the de novo synthesis of nicotinamide adenine dinucleotide (NAD+). As such, they are expressed by a variety of species, including fungi. Interestingly, Aspergillus may degrade l-tryptophan not only via IDO but also via alternative pathways. Deciphering the molecular interactions regulating tryptophan metabolism is particularly critical for novel drug target discovery designed to control pathogen determinants in invasive infections. Using continuous time Bayesian networks over a time-course gene expression dataset, we inferred the global regulatory network controlling l-tryptophan metabolism. The method unravels a possible novel approach to target fungal virulence factors during infection. Furthermore, this study represents the first application of continuous-time Bayesian networks as a gene network reconstruction method in Aspergillus metabolism. The experiment showed that the applied computational approach may improve the understanding of metabolic networks over traditional pathways

Contributions of Spore Secondary Metabolites to UV-C Protection and Virulence Vary in Different Aspergillus fumigatus Strains

Fungi are versatile organisms which thrive in hostile environments, including the International Space Station (ISS). Several isolates of the human pathogen Aspergillus fumigatus have been found contaminating the ISS, an environment with increased exposure to UV radiation. Secondary metabolites (SMs) in spores, such as melanins, have been shown to protect spores from UV radiation in other fungi. To test the hypothesis that melanin and other known spore SMs provide UV protection to A. fumigatus isolates, we subjected SM spore mutants to UV-C radiation. We found that 1,8-dihydroxynaphthalene (DHN)-melanin mutants of two clinical A. fumigatus strains (Af293 and CEA17) but not an ISS-isolated strain (IF1SW-F4) were more sensitive to UV-C than their respective wild-type (WT) strains. Because DHN-melanin has been shown to shield A. fumigatus from the host immune system, we examined all DHN mutants for virulence in the zebrafish model of invasive aspergillosis. Following recent studies highlighting the pathogenic variability of different A. fumigatus isolates, we found DHN-melanin to be a virulence factor in CEA17 and IF1SW-F4 but not Af293. Three additional spore metabolites were examined in Af293, where fumiquinazoline also showed UV-C-protective properties, but two other spore metabolites, monomethylsulochrin and fumigaclavine, provided no UV-C-protective properties. Virulence tests of these three SM spore mutants indicated a slight increase in virulence of the monomethylsulochrin deletion strain. Taken together, this work suggests differential roles of specific spore metabolites across Aspergillus isolates and by types of environmental stress

A Multifaceted Role of Tryptophan Metabolism and Indoleamine 2,3-Dioxygenase Activity in Aspergillus fumigatus–Host Interactions

Aspergillus fumigatus is the most prevalent filamentous fungal pathogen of humans, causing either severe allergic bronchopulmonary aspergillosis or often fatal invasive pulmonary aspergillosis (IPA) in individuals with hyper- or hypo-immune deficiencies, respectively. Disease is primarily initiated upon the inhalation of the ubiquitous airborne conidia—the initial inoculum produced by A. fumigatus—which are complete developmental units with an ability to exploit diverse environments, ranging from agricultural composts to animal lungs. Upon infection, conidia initially rely on their own metabolic processes for survival in the host’s lungs, a nutritionally limiting environment. One such nutritional limitation is the availability of aromatic amino acids (AAAs) as animals lack the enzymes to synthesize tryptophan (Trp) and phenylalanine and only produce tyrosine from dietary phenylalanine. However, A. fumigatus produces all three AAAs through the shikimate–chorismate pathway, where they play a critical role in fungal growth and development and in yielding many downstream metabolites. The downstream metabolites of Trp in A. fumigatus include the immunomodulatory kynurenine derived from indoleamine 2,3-dioxygenase (IDO) and toxins such as fumiquinazolines, gliotoxin, and fumitremorgins. Host IDO activity and/or host/microbe-derived kynurenines are increasingly correlated with many Aspergillus diseases including IPA and infections of chronic granulomatous disease patients. In this review, we will describe the potential metabolic cross talk between the host and the pathogen, specifically focusing on Trp metabolism, the implications for therapeutics, and the recent studies on the coevolution of host and microbe IDO activation in regulating inflammation, while controlling infection

Contributions of Spore Secondary Metabolites to UV-C Protection and Virulence Vary in Different Aspergillus fumigatus Strains

Fungal spores contain secondary metabolites that can protect them from a multitude of abiotic and biotic stresses. Conidia (asexual spores) of the human pathogen Aspergillus fumigatus synthesize several metabolites, including melanin, which has been reported to be important for virulence in this species and to be protective against UV radiation in other fungi. Here, we investigate the role of melanin in diverse isolates of A. fumigatus and find variability in its ability to protect spores from UV-C radiation or impact virulence in a zebrafish model of invasive aspergillosis in two clinical strains and one ISS strain. Further, we assess the role of other spore metabolites in a clinical strain of A. fumigatus and identify fumiquinazoline as an additional UV-C-protective molecule but not a virulence determinant. The results show differential roles of secondary metabolites in spore protection dependent on the environmental stress and strain of A. fumigatus. As protection from elevated levels of radiation is of paramount importance for future human outer space explorations, the discovery of small molecules with radiation-protective potential may result in developing novel safety measures for astronauts.Fungi are versatile organisms which thrive in hostile environments, including the International Space Station (ISS). Several isolates of the human pathogen Aspergillus fumigatus have been found contaminating the ISS, an environment with increased exposure to UV radiation. Secondary metabolites (SMs) in spores, such as melanins, have been shown to protect spores from UV radiation in other fungi. To test the hypothesis that melanin and other known spore SMs provide UV protection to A. fumigatus isolates, we subjected SM spore mutants to UV-C radiation. We found that 1,8-dihydroxynaphthalene (DHN)-melanin mutants of two clinical A. fumigatus strains (Af293 and CEA17) but not an ISS-isolated strain (IF1SW-F4) were more sensitive to UV-C than their respective wild-type (WT) strains. Because DHN-melanin has been shown to shield A. fumigatus from the host immune system, we examined all DHN mutants for virulence in the zebrafish model of invasive aspergillosis. Following recent studies highlighting the pathogenic variability of different A. fumigatus isolates, we found DHN-melanin to be a virulence factor in CEA17 and IF1SW-F4 but not Af293. Three additional spore metabolites were examined in Af293, where fumiquinazoline also showed UV-C-protective properties, but two other spore metabolites, monomethylsulochrin and fumigaclavine, provided no UV-C-protective properties. Virulence tests of these three SM spore mutants indicated a slight increase in virulence of the monomethylsulochrin deletion strain. Taken together, this work suggests differential roles of specific spore metabolites across Aspergillus isolates and by types of environmental stress

Binding of <i>Aspergillus</i> conidia to airway mucins is FleA dependent.

<p>(A) Z-stack confocal images showing binding of WT <i>A</i>. <i>fumigatus</i> conidia (TJMP 131.5) to mucin and that <i>ΔfleA</i> conidia have very limited binding. (B) Quantitative binding of WT <i>A</i>. <i>fumigatus</i> conidia to mucin or <i>ΔfleA</i> deletion mutants. (C) Quantitative binding of WT <i>A</i>. <i>flavus</i> conidia to mucin or <i>ΔfleA</i> deletion mutants. Note that <i>A</i>. <i>fumigatus</i>-mucin interactions were investigated using GFP labeled conidia whereas the <i>A</i>. <i>flavus</i>-mucin interactions were investigated using Calcofluor white-stained conidia (since the <i>A</i>. <i>flavus</i> conidia lack GFP). The data shown in panels B and C (27 replicates) reflects the mean ± SD of three independent experiments.</p

FleA Expression in <i>Aspergillus fumigatus</i> Is Recognized by Fucosylated Structures on Mucins and Macrophages to Prevent Lung Infection

<div><p>The immune mechanisms that recognize inhaled <i>Aspergillus fumigatus</i> conidia to promote their elimination from the lungs are incompletely understood. FleA is a lectin expressed by <i>Aspergillus fumigatus</i> that has twelve binding sites for fucosylated structures that are abundant in the glycan coats of multiple plant and animal proteins. The role of FleA is unknown: it could bind fucose in decomposed plant matter to allow <i>Aspergillus fumigatus</i> to thrive in soil, or it may be a virulence factor that binds fucose in lung glycoproteins to cause <i>Aspergillus fumigatus</i> pneumonia. Our studies show that FleA protein and <i>Aspergillus fumigatus</i> conidia bind avidly to purified lung mucin glycoproteins in a fucose-dependent manner. In addition, FleA binds strongly to macrophage cell surface proteins, and macrophages bind and phagocytose <i>fleA-</i>deficient <i>(∆fleA</i>) conidia much less efficiently than wild type (WT) conidia. Furthermore, a potent fucopyranoside glycomimetic inhibitor of FleA inhibits binding and phagocytosis of WT conidia by macrophages, confirming the specific role of fucose binding in macrophage recognition of WT conidia. Finally, mice infected with <i>ΔfleA</i> conidia had more severe pneumonia and invasive aspergillosis than mice infected with WT conidia. These findings demonstrate that FleA is not a virulence factor for <i>Aspergillus fumigatus</i>. Instead, host recognition of FleA is a critical step in mechanisms of mucin binding, mucociliary clearance, and macrophage killing that prevent <i>Aspergillus fumigatus</i> pneumonia.</p></div

Binding of <i>A</i>. <i>fumig</i>atus conidia to alveolar macrophages is FleA-dependent.

<p>FACS data showing binding of FleA (black) to (A) RAW264.7 or (B) primary human alveolar macrophages. 100mM fucose (dark grey) inhibits binding almost down to the background level (light grey). Z-stack confocal images show binding and phagocytosis of GFP expressing WT conidia by (C) RAW264.7 cells, (E) primary human alveolar macrophages and that <i>ΔfleA</i> conidia are not bound well or phagocytosed effectively. Cells were dyed with CellMask Deep red (red); internalized conidia express GFP (green); calcofluor white (non-internalized conidia) stain blue. Quantitative data (expressed as an index of total number of conidia internalized/total cell number) demonstrate how loss of FleA significantly inhibits binding and phagocytosis of <i>A</i>. <i>fumigatus</i> conidia by (D) RAW264.7 cells or (F) primary human alveolar macrophages. Calcofluor white stained conidia were excluded from these counts to reflect only internalized conidia. The data shown in D (6 replicates) reflects the mean ± SD of three independent experiments whereas the data in panel F (6 replicates) reflects the mean ± SD of three independent donors. (G) FACS plots showing that binding of Dectin-1 is not significantly different between WT (dark grey) and <i>ΔfleA</i> (black) conidia compared to control (light grey). (H) Binding and internalization of FleA coated microspheres by RAW264.7 cells is significantly higher than control and inhibited by 500mM fucose. *Denotes significantly different from control, p = <0.05. Scale bar is 10μM.</p

Inhibition of FleA by 2EHex or fucose results in a loss of mucin binding and greatly reduced phagocytosis by macrophages.

<p>(A) Structures of fucopyranoside compounds. (B) Amount of compound required to inhibit binding of labeled FleA to mucin by 50% (IC50 [μM]). Addition of a methyl or allyl group to the anomeric position of fucose (methyl α-L-fucopyranoside, allyl α-L-fucopyranoside) improves inhibition by 2–4 fold but inclusion of a longer (6-carbon) unsaturated chain improves inhibition by 3 orders of magnitude. Removing the double bond or extending the carbon chain beyond 6 carbons (hexyl α-L-fucopyranoside, (2E)-octenyl α-L-fucopyranoside) markedly decreases inhibition. (2E)-hexenyl α-D-galactopyranoside has no effect on FleA binding to mucin. (C) Amount of compound required to inhibit PAIIL binding to mucin by 50% (IC50 [μM]). 2EHex does not have a strong inhibitory effect on PAIIL-mucin interactions. (D) Inhibiting WT conidia with 10mM 2EHex or 100mM fucose significantly reduced binding of conidia to mucin. WT conidia were poorly phagocytosed in the presence of 2EHex or fucose by RAW264.7 cells (E) or primary human macrophages (F). The data shown in D and E (6 replicates) reflects the mean ± SD of three independent experiments whereas the data in panel F (6 replicates) reflects the mean ± SD of three independent donors. *Denotes significantly different from control, p = <0.05.</p