The leucine biosynthetic pathway is crucial for adaptation to iron starvation and virulence in Aspergillus fumigatus.

In contrast to mammalia, fungi are able to synthesize the branched-chain amino acid leucine de novo. Recently, the transcription factor LeuB has been shown to cross-regulate leucine biosynthesis, nitrogen metabolism and iron homeostasis in Aspergillus fumigatus, the most common human mold pathogen. Moreover, the leucine biosynthetic pathway intermediate α-isopropylmalate (α-IPM) has previously been shown to posttranslationally activate LeuB homologs in S. cerevisiae and A. nidulans. Here, we demonstrate that in A. fumigatus inactivation of both leucine biosynthetic enzymes α-IPM synthase (LeuC), which disrupts α-IPM synthesis, and α-IPM isomerase (LeuA), which causes cellular α-IPM accumulation, results in leucine auxotrophy. However, compared to lack of LeuA, lack of LeuC resulted in increased leucine dependence, a growth defect during iron starvation and decreased expression of LeuB-regulated genes including genes involved in iron acquisition. Lack of either LeuA or LeuC decreased virulence in an insect infection model, and inactivation of LeuC rendered A. fumigatus avirulent in a pulmonary aspergillosis mouse model. Taken together, we demonstrate that the lack of two leucine biosynthetic enzymes, LeuA and LeuC, results in significant phenotypic consequences indicating that the regulator LeuB is activated by α-IPM in A. fumigatus and that the leucine biosynthetic pathway is an attractive target for the development of antifungal drugs.

Intermicrobial interaction: Aspergillus fumigatus siderophores protect against competition by Pseudomonas aeruginosa.

Pseudomonas aeruginosa and Aspergillus fumigatus are pathogens frequently co-inhabiting immunocompromised patient airways, particularly in people with cystic fibrosis. Both microbes depend on the availability of iron, and compete for iron in their microenvironment. We showed previously that the P. aeruginosa siderophore pyoverdine is the main instrument in battling A. fumigatus biofilms, by iron chelation and denial of iron to the fungus. Here we show that A. fumigatus siderophores defend against anti-fungal P. aeruginosa effects. P. aeruginosa supernatants produced in the presence of wildtype A. fumigatus planktonic supernatants (Afsup) showed less activity against A. fumigatus biofilms than P. aeruginosa supernatants without Afsup, despite higher production of pyoverdine by P. aeruginosa. Supernatants of A. fumigatus cultures lacking the sidA gene (AfΔsidA), unable to produce hydroxamate siderophores, were less capable of protecting A. fumigatus biofilms from P. aeruginosa supernatants and pyoverdine. AfΔsidA biofilm was more sensitive towards inhibitory effects of pyoverdine, the iron chelator deferiprone (DFP), or amphothericin B than wildtype A. fumigatus biofilm. Supplementation of sidA-deficient A. fumigatus biofilm with A. fumigatus siderophores restored resistance to pyoverdine. The A. fumigatus siderophore production inhibitor celastrol sensitized wildtype A. fumigatus biofilms towards the anti-fungal activity of DFP. In conclusion, A. fumigatus hydroxamate siderophores play a pivotal role in A. fumigatus competition for iron against P. aeruginosa.

Aspergillus-Pseudomonas interaction, relevant to competition in airways.

In airways of immunocompromised patients and individuals with cystic fibrosis, Pseudomonas aeruginosa and Aspergillus fumigatus are the most common opportunistic bacterial and fungal pathogens. Both pathogens form biofilms and cause acute and chronic illnesses. Previous studies revealed that P. aeruginosa is able to inhibit A. fumigatus biofilms in vitro. While numerous P. aeruginosa molecules have been shown to affect A. fumigatus, there never has been a systematic approach to define the principal causative agent. We studied 24 P. aeruginosa mutants, with deletions in genes important for virulence, iron acquisition, or quorum sensing, for their ability to interfere with A. fumigatus biofilms. Cells, planktonic or biofilm culture filtrates of four P. aeruginosa mutants, pvdD-pchE-, pvdD-, lasR-rhlR-, and lasR-, inhibited A. fumigatus biofilm metabolism or planktonic A. fumigatus growth significantly less than P. aeruginosa wild type. The common defect of these four mutants was a lack in the production of the P. aeruginosa siderophore pyoverdine. Pure pyoverdine affected A. fumigatus biofilm metabolism, and restored inhibition by the above mutants. In lungs from cystic fibrosis patients, pyoverdine production and antifungal activity correlated. The key inhibitory mechanism for pyoverdine was iron-chelation and denial of iron to A. fumigatus. Further experiments revealed a counteracting, self-protective mechanism by A. fumigatus, based on A. fumigatus siderophore production.

Riboflavin and pantothenic acid biosynthesis are crucial for iron homeostasis and virulence in the pathogenic mold Aspergillus fumigatus.

BACKGROUND: Aspergillus fumigatus is the most prevalent airborne fungal pathogen, causing invasive fungal infections mainly in immunosuppressed individuals. Death rates from invasive aspergillosis remain high because of limited treatment options and increasing antifungal resistance. The aim of this study was to identify key fungal-specific genes participating in vitamin B biosynthesis in A. fumigatus. Because these genes are absent in humans they can serve as possible novel targets for antifungal drug development. METHODS: By sequence homology we identified, deleted and analysed four key A. fumigatus genes (riboB, panA, pyroA, thiB) involved respectively in the biosynthesis of riboflavin (vitamin B2), pantothenic acid (vitamin B5), pyridoxine (vitamin B6) and thiamine (vitamin B1). RESULTS: Deletion of riboB, panA, pyroA or thiB resulted in respective vitamin auxotrophy. Lack of riboflavin and pantothenic acid biosynthesis perturbed many cellular processes including iron homeostasis. Virulence in murine pulmonary and systemic models of infection was severely attenuated following deletion of riboB and panA, strongly reduced after pyroA deletion and weakly attenuated after thiB deletion. CONCLUSIONS: This study reveals the biosynthetic pathways of the vitamins riboflavin and pantothenic acid as attractive targets for novel antifungal therapy. Moreover, the virulence studies with auxotrophic mutants serve to identify the availability of nutrients to pathogens in host niches. ABBREVIATIONS: BPS: bathophenanthrolinedisulfonate; BSA: bovine serum albumin; CFU: colony forming unit; -Fe: iron starvation; +Fe: iron sufficiency; hFe: high iron; NRPSs: nonribosomal peptide synthetases; PKSs: polyketide synthaseses; wt: wild type.

Additional oxidative stress reroutes the global response of Aspergillus fumigatus to iron depletion.

BACKGROUND: Aspergillus fumigatus has to cope with a combination of several stress types while colonizing the human body. A functional interplay between these different stress responses can increase the chances of survival for this opportunistic human pathogen during the invasion of its host. In this study, we shed light on how the H2O2-induced oxidative stress response depends on the iron available to this filamentous fungus, using transcriptomic analysis, proteomic profiles, and growth assays. RESULTS: The applied H2O2 treatment, which induced only a negligible stress response in iron-replete cultures, deleteriously affected the fungus under iron deprivation. The majority of stress-induced changes in gene and protein expression was not predictable from data coming from individual stress exposure and was only characteristic for the combination of oxidative stress plus iron deprivation. Our experimental data suggest that the physiological effects of combined stresses and the survival of the fungus highly depend on fragile balances between economization of iron and production of essential iron-containing proteins. One observed strategy was the overproduction of iron-independent antioxidant proteins to combat oxidative stress during iron deprivation, e.g. the upregulation of superoxide dismutase Sod1, the thioredoxin reductase Trr1, and the thioredoxin orthologue Afu5g11320. On the other hand, oxidative stress induction overruled iron deprivation-mediated repression of several genes. In agreement with the gene expression data, growth studies underlined that in A. fumigatus iron deprivation aggravates oxidative stress susceptibility. CONCLUSIONS: Our data demonstrate that studying stress responses under separate single stress conditions is not sufficient to understand how A. fumigatus adapts in a complex and hostile habitat like the human body. The combinatorial stress of iron depletion and hydrogen peroxide caused clear non-additive effects upon the stress response of A. fumigatus. Our data further supported the view that the ability of A. fumigatus to cause diseases in humans strongly depends on its fitness attributes and less on specific virulence factors. In summary, A. fumigatus is able to mount and coordinate complex and efficient responses to combined stresses like iron deprivation plus H2O2-induced oxidative stress, which are exploited by immune cells to kill fungal pathogens.

Studies of Pseudomonas aeruginosa Mutants Indicate Pyoverdine as the Central Factor in Inhibition of Aspergillus fumigatus Biofilm.

Pseudomonas aeruginosa and Aspergillus fumigatus are common opportunistic bacterial and fungal pathogens, respectively. They often coexist in airways of immunocompromised patients and individuals with cystic fibrosis, where they form biofilms and cause acute and chronic illnesses. Hence, the interactions between them have long been of interest and it is known that P. aeruginosa can inhibit A. fumigatusin vitro We have approached the definition of the inhibitory P. aeruginosa molecules by studying 24 P. aeruginosa mutants with various virulence genes deleted for the ability to inhibit A. fumigatus biofilms. The ability of P. aeruginosa cells or their extracellular products produced during planktonic or biofilm growth to affect A. fumigatus biofilm metabolism or planktonic A. fumigatus growth was studied in agar and liquid assays using conidia or hyphae. Four mutants, the pvdD pchE, pvdD, lasR rhlR, and lasR mutants, were shown to be defective in various assays. This suggested the P. aeruginosa siderophore pyoverdine as the key inhibitory molecule, although additional quorum sensing-regulated factors likely contribute to the deficiency of the latter two mutants. Studies of pure pyoverdine substantiated these conclusions and included the restoration of inhibition by the pyoverdine deletion mutants. A correlation between the concentration of pyoverdine produced and antifungal activity was also observed in clinical P. aeruginosa isolates derived from lungs of cystic fibrosis patients. The key inhibitory mechanism of pyoverdine was chelation of iron and denial of iron to A. fumigatusIMPORTANCE Interactions between human pathogens found in the same body locale are of vast interest. These interactions could result in exacerbation or amelioration of diseases. The bacterium Pseudomonas aeruginosa affects the growth of the fungus Aspergillus fumigatus Both pathogens form biofilms that are resistant to therapeutic drugs and host immunity. P. aeruginosa and A. fumigatus biofilms are found in vivo, e.g., in the lungs of cystic fibrosis patients. Studying 24 P. aeruginosa mutants, we identified pyoverdine as the major anti-A. fumigatus compound produced by P. aeruginosa Pyoverdine captures iron from the environment, thus depriving A. fumigatus of a nutrient essential for its growth and metabolism. We show how microbes of different kingdoms compete for essential resources. Iron deprivation could be a therapeutic approach to the control of pathogen growth.