Peptide Mimics of the Cysteine-Rich Regions of HapX and SreA Bind a [2Fe-2S] Cluster In Vitro.

HapX and SreA are transcription factors that regulate the response of the fungus Aspergillus fumigatus to the availability of iron. During iron starvation, HapX represses genes involved in iron consuming pathways and upon a shift to iron excess, HapX activates these same genes. SreA blocks the expression of genes needed for iron uptake during periods of iron availability. Both proteins possess cysteine-rich regions (CRR) that are hypothesized to be necessary for the sensing of iron levels. However, the contribution of each of these domains to the function of the protein has remained unclear. Here, the ability of peptide analogs of each CRR is determined to bind an iron-sulfur cluster in vitro. UV-vis and resonance Raman (RR) spectroscopies reveal that each CRR is capable of coordinating a [2Fe-2S] cluster with comparable affinities. The iron-sulfur cluster coordinated to the CRR-B domain of HapX displays particularly high stability. The data are consistent with HapX and SreA mediating responses to cellular iron levels through the direct coordination of [2Fe-2S] clusters. The high stability of the CRR-B peptide may also find use as a starting point for the development of new green catalysts.

Fonsecaea pedrosoi produces ferricrocin and can utilize different host iron sources.

The survival of living organisms depends on iron, one of the most abundant metals in the Earth's crust. Nevertheless, this micronutrient is poorly available in our aerobic atmosphere as well as inside the mammalian host. This problem is circumvented by the expression of high affinity iron uptake machineries, including the production of siderophores, in pathogenic fungi. Here we demonstrated that F. pedrosoi, the causative agent of the neglected tropical disease chromoblastomycosis, presents gene clusters for siderophore production. In addition, ten putative siderophore transporters were identified. Those genes are upregulated under iron starvation, a condition that induces the secretion of hydroxamates, as revealed by chrome azurol S assays. RP-HPLC and mass spectrometry analysis allowed the identification of ferricrocin as an intra- and extracellular siderophore. F. pedrosoi can grow in different iron sources, including the bacterial ferrioxamine B and the host proteins ferritin, hemoglobin and holotransferrin. Of note, addition of hemoglobin, lactoferrin and holotransferrin to the growth medium of macrophages infected with F. pedrosoi enhanced significantly fungal survival. The ability to produce siderophores in iron limited conditions added to the versatility to utilize different sources of iron are strategies that certainly may contribute to fungal survival inside the host.

HapX-mediated H2B deub1 and SreA-mediated H2A.Z deposition coordinate in fungal iron resistance.

Plant pathogens are challenged by host-derived iron starvation or excess during infection, but the mechanism through which pathogens counteract iron stress is unclear. Here, we found that Fusarium graminearum encounters iron excess during the colonization of wheat heads. Deletion of heme activator protein X (FgHapX), siderophore transcription factor A (FgSreA) or both attenuated virulence. Further, we found that FgHapX activates iron storage under iron excess by promoting histone H2B deubiquitination (H2B deub1) at the promoter of the responsible gene. Meanwhile, FgSreA is shown to inhibit genes mediating iron acquisition during iron excess by facilitating the deposition of histone variant H2A.Z and histone 3 lysine 27 trimethylation (H3K27 me3) at the first nucleosome after the transcription start site. In addition, the monothiol glutaredoxin FgGrx4 is responsible for iron sensing and control of the transcriptional activity of FgHapX and FgSreA via modulation of their enrichment at target genes and recruitment of epigenetic regulators, respectively. Taken together, our findings elucidated the molecular mechanisms for adaptation to iron excess mediated by FgHapX and FgSreA during infection in F. graminearum and provide novel insights into regulation of iron homeostasis at the chromatin level in eukaryotes.

Iron Starvation Induces Ferricrocin Production and the Reductive Iron Acquisition System in the Chromoblastomycosis Agent Cladophialophora carrionii.

Iron is a micronutrient required by almost all living organisms. Despite being essential, the availability of this metal is low in aerobic environments. Additionally, mammalian hosts evolved strategies to restrict iron from invading microorganisms. In this scenario, the survival of pathogenic fungi depends on high-affinity iron uptake mechanisms. Here, we show that the production of siderophores and the reductive iron acquisition system (RIA) are employed by Cladophialophora carrionii under iron restriction. This black fungus is one of the causative agents of chromoblastomycosis, a neglected subcutaneous tropical disease. Siderophore biosynthesis genes are arranged in clusters and, interestingly, two RIA systems are present in the genome. Orthologs of putative siderophore transporters were identified as well. Iron starvation regulates the expression of genes related to both siderophore production and RIA systems, as well as of two transcription factors that regulate iron homeostasis in fungi. A chrome azurol S assay demonstrated the secretion of hydroxamate-type siderophores, which were further identified via RP-HPLC and mass spectrometry as ferricrocin. An analysis of cell extracts also revealed ferricrocin as an intracellular siderophore. The presence of active high-affinity iron acquisition systems may surely contribute to fungal survival during infection.

The Siderophore Ferricrocin Mediates Iron Acquisition in Aspergillus fumigatus.

The opportunistic fungal pathogen Aspergillus fumigatus utilizes two high-affinity iron uptake mechanisms, termed reductive iron assimilation (RIA) and siderophore-mediated iron acquisition (SIA). The latter has been shown to be crucial for virulence of this fungus and is a target for development of novel strategies for diagnosis and treatment of fungal infections. So far, research on SIA in this mold focused mainly on the hyphal stage, revealing the importance of extracellular fusarinine-type siderophores in iron acquisition as well as of the siderophore ferricrocin in intracellular iron handling. The current study aimed to characterize iron acquisition during germination. High expression of genes involved in biosynthesis and uptake of ferricrocin in conidia and during germination, independent of iron availability, suggested a role of ferricrocin in iron acquisition during germination. In agreement, (i) bioassays indicated secretion of ferricrocin during growth on solid media during both iron sufficiency and limitation, (ii) ferricrocin was identified in the supernatant of conidia germinating in liquid media during both iron sufficiency and limitation, (iii) in contrast to mutants lacking all siderophores, mutants synthesizing ferricrocin but lacking fusarinine-type siderophores were able to grow under iron limitation in the absence of RIA, and (iv) genetic inactivation of the ferricrocin transporter Sit1 decreased germination in the absence of RIA. Taken together, this study revealed that ferricrocin has not only an intracellular role but also functions as an extracellular siderophore to support iron acquisition. The iron availability-independent ferricrocin secretion and uptake during early germination indicate developmental, rather than iron regulation. IMPORTANCE Aspergillus fumigatus is one of the most common airborne fungal pathogens for humans. Low-molecular-mass iron chelators, termed siderophores, have been shown to play a central role in iron homeostasis and, consequently, virulence of this mold. Previous studies demonstrated the crucial role of secreted fusarinine-type siderophores, such as triacetylfusarinine C, in iron acquisition, as well as of the ferrichrome-type siderophore ferricrocin in intracellular iron storage and transport. Here, we demonstrate that ferricrocin is also secreted to mediate iron acquisition during germination together with reductive iron assimilation. During early germination, ferricrocin secretion and uptake were not repressed by iron availability, indicating developmental regulation of this iron acquisition system in this growth phase.

Regulation of High-Affinity Iron Acquisition, Including Acquisition Mediated by the Iron Permease FtrA, Is Coordinated by AtrR, SrbA, and SreA in Aspergillus fumigatus.

Iron acquisition is crucial for virulence of the human pathogen Aspergillus fumigatus. Previous studies indicated that this mold regulates iron uptake via both siderophores and reductive iron assimilation by the GATA factor SreA and the SREBP regulator SrbA. Here, characterization of loss of function as well as hyperactive alleles revealed that transcriptional activation of iron uptake depends additionally on the Zn2Cys6 regulator AtrR, most likely via cooperation with SrbA. Mutational analysis of the promoter of the iron permease-encoding ftrA gene identified a 210-bp sequence, which is both essential and sufficient to impart iron regulation. Further studies located functional sequences, densely packed within 75 bp, that largely resemble binding motifs for SrbA, SreA, and AtrR. The latter, confirmed by chromatin immunoprecipitation (ChIP) analysis, is the first one not fully matching the 5'-CGGN12CCG-3' consensus sequence. The results presented here emphasize for the first time the direct involvement of SrbA, AtrR, and SreA in iron regulation. The essential role of both AtrR and SrbA in activation of iron acquisition underlines the coordination of iron homeostasis with biosynthesis of ergosterol and heme as well as adaptation to hypoxia. The rationale is most likely the iron dependence of these pathways along with the enzymatic link of biosynthesis of ergosterol and siderophores. IMPORTANCE Aspergillus fumigatus is the most common filamentous fungal pathogen infecting humans. Iron acquisition via siderophores has previously been shown to be essential for virulence of this mold species. Here, we demonstrate that AtrR, a transcription factor previously shown to control ergosterol biosynthesis, azole resistance, and adaptation to hypoxia, is essential for activation of iron acquisition, including siderophore biosynthesis and uptake. Dissection of an iron-regulated promoter identified binding motifs for AtrR and the two previously identified regulators of iron acquisition, SrbA and SreA. Altogether, this study identified a new regulator required for maintenance of iron homeostasis, revealed insights into promoter architecture for iron regulation, and emphasized the coordinated regulation of iron homeostasis ergosterol biosynthesis and adaptation to hypoxia.

Ambient Availability of Amino Acids, Proteins, and Iron Impacts Copper Resistance of Aspergillus fumigatus.

The transition metals iron and copper are required by virtually all organisms but are toxic in excess. Acquisition of both metals and resistance to copper excess have previously been shown to be important for virulence of the most common airborne human mold pathogen, Aspergillus fumigatus. Here we demonstrate that the ambient availability of amino acids and proteins increases the copper resistance of A. fumigatus wild type and particularly of the ΔcrpA mutant that lacks export-mediated copper detoxification. The highest-protecting activity was found for L-histidine followed by L-asparagine, L-aspartate, L-serine, L-threonine, and L-tyrosine. Other amino acids and proteins also displayed significant but lower protection. The protecting activity of non-proteinogenic D-histidine, L-histidine-mediated growth inhibition in the absence of high-affinity copper uptake, determination of cellular metal contents, and expression analysis of copper-regulated genes suggested that histidine inhibits low-affinity but not high-affinity copper acquisition by extracellular copper complexation. An increase in the cellular copper content was found to be accompanied by an increase in the iron content, and, in agreement, iron starvation increased copper susceptibility, which underlines the importance of cellular metal balancing. Due to the role of iron and copper in nutritional immunity, these findings are likely to play an important role in the host niche.

Structural insights into cooperative DNA recognition by the CCAAT-binding complex and its bZIP transcription factor HapX.

The heterotrimeric CCAAT-binding complex (CBC) is a fundamental eukaryotic transcription factor recognizing the CCAAT box. In certain fungi, like Aspergilli, the CBC cooperates with the basic leucine zipper HapX to control iron metabolism. HapX functionally depends on the CBC, and the stable interaction of both requires DNA. To study this cooperative effect, X-ray structures of the CBC-HapX-DNA complex were determined. Downstream of the CCAAT box, occupied by the CBC, a HapX dimer binds to the major groove. The leash-like N terminus of the distal HapX subunit contacts the CBC, and via a flexible polyproline type II helix mediates minor groove interactions that stimulate sequence promiscuity. In vitro and in vivo mutagenesis suggest that the structural and functional plasticity of HapX results from local asymmetry and its ability to target major and minor grooves simultaneously. The latter feature may also apply to related transcription factors such as yeast Hap4 and distinct Yap family members.

Peroxiredoxin Asp f3 Is Essential for Aspergillus fumigatus To Overcome Iron Limitation during Infection.

Aspergillus fumigatus is an important fungal pathogen that causes allergic reactions but also life-threatening infections. One of the most abundant A. fumigatus proteins is Asp f3. This peroxiredoxin is a major fungal allergen and known for its role as a virulence factor, vaccine candidate, and scavenger of reactive oxygen species. Based on the hypothesis that Asp f3 protects A. fumigatus against killing by immune cells, we investigated the susceptibility of a conditional aspf3 mutant by employing a novel assay. Surprisingly, Asp f3-depleted hyphae were killed as efficiently as the wild type by human granulocytes. However, we identified an unexpected growth defect of mutants that lack Asp f3 under low-iron conditions, which explains the avirulence of the Δaspf3 deletion mutant in a murine infection model. A. fumigatus encodes two Asp f3 homologues which we named Af3l (Asp f3-like) 1 and Af3l2. Inactivation of Af3l1, but not of Af3l2, exacerbated the growth defect of the conditional aspf3 mutant under iron limitation, which ultimately led to death of the double mutant. Inactivation of the iron acquisition repressor SreA partially compensated for loss of Asp f3 and Af3l1. However, Asp f3 was not required for maintaining iron homeostasis or siderophore biosynthesis. Instead, we show that it compensates for a loss of iron-dependent antioxidant enzymes. Iron supplementation restored the virulence of the Δaspf3 deletion mutant in a murine infection model. Our results unveil the crucial importance of Asp f3 to overcome nutritional immunity and reveal a new biological role of peroxiredoxins in adaptation to iron limitation. IMPORTANCE Asp f3 is one of the most abundant proteins in the pathogenic mold Aspergillus fumigatus. It has an enigmatic multifaceted role as a fungal allergen, virulence factor, reactive oxygen species (ROS) scavenger, and vaccine candidate. Our study provides new insights into the cellular role of this conserved peroxiredoxin. We show that the avirulence of a Δaspf3 mutant in a murine infection model is linked to a low-iron growth defect of this mutant, which we describe for the first time. Our analyses indicated that Asp f3 is not required for maintaining iron homeostasis. Instead, we found that Asp f3 compensates for a loss of iron-dependent antioxidant enzymes. Furthermore, we identified an Asp f3-like protein which is partially functionally redundant with Asp f3. We highlight an unexpected key role of Asp f3 and its partially redundant homologue Af3l1 in overcoming the host's nutritional immunity. In addition, we uncovered a new biological role of peroxiredoxins.

The bZIP Transcription Factor HapX Is Post-Translationally Regulated to Control Iron Homeostasis in Aspergillus fumigatus.

The airborne fungus Aspergillus fumigatus causes opportunistic infections in humans with high mortality rates in immunocompromised patients. Previous work established that the bZIP transcription factor HapX is essential for virulence via adaptation to iron limitation by repressing iron-consuming pathways and activating iron acquisition mechanisms. Moreover, HapX was shown to be essential for transcriptional activation of vacuolar iron storage and iron-dependent pathways in response to iron availability. Here, we demonstrate that HapX has a very short half-life during iron starvation, which is further decreased in response to iron, while siderophore biosynthetic enzymes are very stable. We identified Fbx22 and SumO as HapX interactors and, in agreement, HapX post-translational modifications including ubiquitination of lysine161, sumoylation of lysine242 and phosphorylation of threonine319. All three modifications were enriched in the immediate adaptation from iron-limiting to iron-replete conditions. Interfering with these post-translational modifications, either by point mutations or by inactivation, of Fbx22 or SumO, altered HapX degradation, heme biosynthesis and iron resistance to different extents. Consistent with the need to precisely regulate HapX protein levels, overexpression of hapX caused significant growth defects under iron sufficiency. Taken together, our results indicate that post-translational regulation of HapX is important to control iron homeostasis in A. fumigatus.

Fungal iron homeostasis with a focus on Aspergillus fumigatus.

To maintain iron homeostasis, fungi have to balance iron acquisition, storage, and utilization to ensure sufficient supply and to avoid toxic excess of this essential trace element. As pathogens usually encounter iron limitation in the host niche, this metal plays a particular role during virulence. Siderophores are iron-chelators synthesized by most, but not all fungal species to sequester iron extra- and intracellularly. In recent years, the facultative human pathogen Aspergillus fumigatus has become a model for fungal iron homeostasis of siderophore-producing fungal species. This article summarizes the knowledge on fungal iron homeostasis and its links to virulence with a focus on A. fumigatus. It covers mechanisms for iron acquisition, storage, and detoxification, as well as the modes of transcriptional iron regulation and iron sensing in A. fumigatus in comparison to other fungal species. Moreover, potential translational applications of the peculiarities of fungal iron metabolism for treatment and diagnosis of fungal infections is addressed.

Absent regulation of iron acquisition by the copper regulator Mac1 in A. fumigatus.

Aspergillus fumigatus is the most common cause of invasive aspergillosis, a life-threatening infection mainly affecting immunocompromised patients. The essential metals copper and iron play crucial roles in virulence of this mold. Recently, the copper-regulatory transcription factor Mac1 was reported to additionally be involved in the control of iron acquisition. However, in the current study, neither growth assays on solid and in liquid media, analysis of siderophore production nor expression analysis of genes involved in iron acquisition indicated the involvement of Mac1 in the regulation of iron uptake in A. fumigatus.

Novel intermicrobial molecular interaction: Pseudomonas aeruginosa Quinolone Signal (PQS) modulates Aspergillus fumigatus response to iron.

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af), the commonest bacterium and fungus in compromised host airways, compete for iron (Fe). The Pseudomonas quinolone signal (PQS), a Pa quorum sensing molecule, also chelates Fe, and delivers Fe to the Pa cell membrane using Pa siderophores. In models of Af biofilm formation or preformed biofilms, PQS inhibited Af in a low Fe environment. AfΔsidA (mutant unable to produce siderophores) biofilm was more sensitive to PQS inhibition than wild-type (WT), as was planktonic AfΔsidA growth. PQS decreased WT Af growth on agar. All these inhibitory actions were reversed by Fe. The Pa siderophore pyoverdin, or Af siderophore inhibitor celastrol, act cooperatively with PQS in Af inhibition. These findings all indicate PQS inhibition is owing to Fe chelation. Remarkably, in high Fe environments, PQS enhanced Af biofilm at 1/100 to 1/2000 Fe concentration required for Fe alone to enhance. Planktonic Af growth, and on agar, Af conidiation, were also enhanced by PQS+Fe compared to Fe alone. In contrast, neither AfΔsidA biofilm, nor planktonic AfΔsidA, were enhanced by PQS-Fe compared to Fe. When Af siderophore ferricrocin (FC),+PQS, were added to AfΔsidA, Af was then boosted more than by FC alone. Moreover, FC+PQS+Fe boosted AfΔsidA more than Fe, FC, FC+Fe, PQS+FC or PQS+Fe. Thus PQS-Fe maximal stimulation requires Af siderophores. PQS inhibits Af via chelation under low Fe conditions. In a high Fe environment, PQS paradoxically stimulates Af efficiently, and this involves Af siderophores. PQS production by Pa could stimulate Af in cystic fibrosis airways, where Fe homeostasis is altered and Fe levels increase, supporting fungal growth.

Comparison of 68Ga-labeled RGD mono- and multimers based on a clickable siderophore-based scaffold.

Cyclic pentapeptides containing the amino acid sequence arginine-glycine-aspartic (RGD) have been widely applied to target αvß3 integrin, which is upregulated in various tumors during tumor-induced angiogenesis. Multimeric cyclic RGD peptides have been reported to be advantageous over monomeric counterparts for angiogenesis imaging. Here, we prepared mono-, di-, and trimeric cyclic arginine-glycine-aspartic-D-phenylalanine-lysine (c (RGDfK)) derivatives by conjugation with the natural chelator fusarinine C (FSC) using click chemistry based on copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC). The αvß3 binding properties of 68Ga-labeled mono-, di-, and trimeric c(RGDfK) peptides were evaluated in vitro as well as in vivo and compared with the references monomeric [68Ga]GaNODAGA-c(RGDfK) and trimeric [68Ga]GaFSC(suc-c(RGDfK))3. All 68Ga-labeled c(RGDfK) peptides displayed hydrophilicity (logD = -2.96 to -3.80), low protein binding and were stable in phosphate buffered-saline (PBS) and serum up to 2 h. In vitro internalization assays with human melanoma M21 (αvß3-positive) and M21-L (αvß3-negative) cell lines showed specific uptake of all derivatives and increased in the series: mono- < di- < trimeric peptide. The highest tumor uptake, tumor-to-background ratios, and image contrast were found for the dimeric [68Ga]GaMAFC(c(RGDfK)aza)2. In conclusion, we developed a novel strategy for direct, straight forward preparation of mono-, di-, and trimeric c(RGDfK) conjugates based on the FSC scaffold. Interestingly, the best αvß3 imaging properties were found for the dimeric [68Ga]GaMAFC(c(RGDfK)aza)2.

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.

The monothiol glutaredoxin GrxD is essential for sensing iron starvation in Aspergillus fumigatus.

Efficient adaptation to iron starvation is an essential virulence determinant of the most common human mold pathogen, Aspergillus fumigatus. Here, we demonstrate that the cytosolic monothiol glutaredoxin GrxD plays an essential role in iron sensing in this fungus. Our studies revealed that (i) GrxD is essential for growth; (ii) expression of the encoding gene, grxD, is repressed by the transcription factor SreA in iron replete conditions and upregulated during iron starvation; (iii) during iron starvation but not iron sufficiency, GrxD displays predominant nuclear localization; (iv) downregulation of grxD expression results in de-repression of genes involved in iron-dependent pathways and repression of genes involved in iron acquisition during iron starvation, but did not significantly affect these genes during iron sufficiency; (v) GrxD displays protein-protein interaction with components of the cytosolic iron-sulfur cluster biosynthetic machinery, indicating a role in this process, and with the transcription factors SreA and HapX, which mediate iron regulation of iron acquisition and iron-dependent pathways; (vi) UV-Vis spectra of recombinant HapX or the complex of HapX and GrxD indicate coordination of iron-sulfur clusters; (vii) the cysteine required for iron-sulfur cluster coordination in GrxD is in vitro dispensable for interaction with HapX; and (viii) there is a GrxD-independent mechanism for sensing iron sufficiency by HapX; (ix) inactivation of SreA suppresses the lethal effect caused by GrxD inactivation. Taken together, this study demonstrates that GrxD is crucial for iron homeostasis in A. fumigatus.

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.

Triacetylfusarinine C: A urine biomarker for diagnosis of invasive aspergillosis.

OBJECTIVES: Early diagnosis of invasive aspergillosis (IA) remains challenging, with available diagnostics being limited by inadequate sensitivities and specificities. Triacetylfusarinine C, a fungal siderophore that has been shown to accumulate in urine in animal models, is a potential new biomarker for diagnosis of IA. METHODS: We developed a method allowing absolute and matrix-independent mass spectrometric quantification of TAFC. Urine TAFC, normalized to creatinine, was determined in 44 samples from 24 patients with underlying hematologic malignancies and probable, possible or no IA according to current EORTC/MSG criteria and compared to other established biomarkers measured in urine and same-day blood samples. RESULTS: TAFC/creatinine sensitivity, specificity, positive and negative likelihood ratio for probable versus no IA (cut-off ≥ 3) were 0.86, 0.88, 6.86, 0.16 per patient. CONCLUSION: For the first time, we provide proof for the occurrence of TAFC in human urine. TAFC/creatinine index determination in urine showed promising results for diagnosis of IA offering the advantages of non-invasive sampling. Sensitivity and specificity were similar as reported for GM determination in serum and bronchoalveolar lavage, the gold standard mycological criterion for IA diagnosis.