Aspergillus terreus and the Interplay with Amphotericin B: from Resistance to Tolerance?

Aspergillus terreus is an opportunistic causative agent of invasive aspergillosis and, in most cases, it is refractory to amphotericin B (AMB) therapy. Notably, AMB-susceptible Aspergillus terreus sensu stricto (s.s.) representatives exist which are also associated with poor clinical outcomes. Such findings may be attributable to drug tolerance, which is not detectable by antifungal susceptibility testing. Here, we tested in vitro antifungal susceptibility (AFST) and the fungicidal activity of AMB against 100 clinical isolates of A. terreus species complex in RPMI 1640 and antibiotic medium 3 (AM3). MICs ranged from 0.5 to 16 µg/mL for RPMI 1640 and from 1 to >16 mg/L for AM3. AMB showed medium-dependent activity, with fungicidal effects only in antibiotic medium 3, not in RPMI 1640. Furthermore, the presence of AMB-tolerant phenotypes of A. terreus has been examined by assessing the minimum duration for killing 99% of the population (MDK99) and evaluating the data obtained in a Galleria mellonella infection model. A time-kill curve analysis revealed that A. terreus with AMB MICs of ≤1 mg/L (susceptible range) displayed AMB-tolerant phenotypes, exhibiting MDK99s at 18 and 36 h, respectively. Survival rates of infected G. mellonella highlighted that AMB was effective against susceptible A. terreus isolates, but not against tolerant or resistant isolates. Our analysis reveals that A. terreus isolates which are defined as susceptible based on MIC may comprise tolerant phenotypes, which may, in turn, explain the worse outcome of AMB therapy for phenotypically susceptible isolates.

The Siderophore Transporters Sit1 and Sit2 Are Essential for Utilization of Ferrichrome-, Ferrioxamine- and Coprogen-Type Siderophores in Aspergillus fumigatus.

Siderophore-mediated acquisition of iron has been shown to be indispensable for the virulence of several fungal pathogens, the siderophore transporter Sit1 was found to mediate uptake of the novel antifungal drug VL-2397, and siderophores were shown to be useful as biomarkers as well as for imaging of fungal infections. However, siderophore uptake in filamentous fungi is poorly characterized. The opportunistic human pathogen Aspergillus fumigatus possesses five putative siderophore transporters. Here, we demonstrate that the siderophore transporters Sit1 and Sit2 have overlapping, as well as unique, substrate specificities. With respect to ferrichrome-type siderophores, the utilization of ferrirhodin and ferrirubin depended exclusively on Sit2, use of ferrichrome A depended mainly on Sit1, and utilization of ferrichrome, ferricrocin, and ferrichrysin was mediated by both transporters. Moreover, both Sit1 and Sit2 mediated use of the coprogen-type siderophores coprogen and coprogen B, while only Sit1 transported the bacterial ferrioxamine-type xenosiderophores ferrioxamines B, G, and E. Neither Sit1 nor Sit2 were important for the utilization of the endogenous siderophores fusarinine C and triacetylfusarinine C. Furthermore, A. fumigatus was found to lack utilization of the xenosiderophores schizokinen, basidiochrome, rhizoferrin, ornibactin, rhodotorulic acid, and enterobactin. Taken together, this study characterized siderophore use by A. fumigatus and substrate characteristics of Sit1 and Sit2.

Inducible Selectable Marker Genes to Improve Aspergillus fumigatus Genetic Manipulation.

The hygromycin B phosphotransferase gene from Escherichia coli and the pyrithiamine resistance gene from Aspergillus oryzae are two dominant selectable marker genes widely used to genetically manipulate several fungal species. Despite the recent development of CRISPR/Cas9 and marker-free systems, in vitro molecular tools to study Aspergillus fumigatus, which is a saprophytic fungus causing life-threatening diseases in immunocompromised hosts, still rely extensively on the use of dominant selectable markers. The limited number of drug selectable markers is already a critical aspect, but the possibility that their introduction into a microorganism could induce enhanced virulence or undesired effects on metabolic behavior constitutes another problem. In this context, here, we demonstrate that the use of ptrA in A. fumigatus leads to the secretion of a compound that allows the recovery of thiamine auxotrophy. In this study, we developed a simple modification of the two commonly used dominant markers in which the development of resistance can be controlled by the xylose-inducible promoter PxylP from Penicillium chrysogenum. This strategy provides an easy solution to avoid undesired side effects, since the marker expression can be readily silenced when not required.

Aspergillus terreus Species Complex.

Infections due to Aspergillus species are an acute threat to human health; members of the Aspergillus section Fumigati are the most frequently occurring agents, but depending on the local epidemiology, representatives of section Terrei or section Flavi are the second or third most important. Aspergillus terreus species complex is of great interest, as it is usually amphotericin B resistant and displays notable differences in immune interactions in comparison to Aspergillus fumigatus. The latest epidemiological surveys show an increased incidence of A. terreus as well as an expanding clinical spectrum (chronic infections) and new groups of at-risk patients being affected. Hallmarks of these non-Aspergillus fumigatus invasive mold infections are high potential for tissue invasion, dissemination, and possible morbidity due to mycotoxin production. We seek to review the microbiology, epidemiology, and pathogenesis of A. terreus species complex, address clinical characteristics, and highlight the underlying mechanisms of amphotericin B resistance. Selected topics will contrast key elements of A. terreus with A. fumigatus. We provide a comprehensive resource for clinicians dealing with fungal infections and researchers working on A. terreus pathogenesis, aiming to bridge the emerging translational knowledge and future therapeutic challenges on this opportunistic pathogen.

The Environmental Spread of Aspergillus terreus in Tyrol, Austria.

Fungal infections due to Aspergillus species have become a major cause of morbidity and mortality among immunocompromised patients. At the Medical University of Innsbruck, A. terreus and related species are the second most common causative agents of aspergillosis. In this one-year study we collected environmental samples to investigate (i) the environmental distribution, (ii) the ecological niche of A. terreus in Tyrol, (iii) the genetic relatedness of environmental and clinical isolates and the correlation between those two groups of isolates, and (iv) the antifungal susceptibility patterns. A. terreus was present in 5.4% of 3845 environmental samples, with a significantly higher frequency during winter (6.8%) than summer (3.9%). An increased A. terreus abundance in Tyrol's Eastern part was detected which is in agreement with the proof of clinical cases. In total, 92% of environmental and 98% of clinical A. terreus isolates were amphotericin B resistant; 22.6% and 9.8% were resistant against posaconazole. Overall, 3.9% of clinical isolates were resistant against voriconazole. Short tandem repeat analysis identified three major genotypes persisting in Tyrol. Soil from agricultural cornfields seems to be an important source; the environmental frequency of A. terreus correlates with the high incidence of A. terreus infections in certain geographical areas.

Arginine Auxotrophy Affects Siderophore Biosynthesis and Attenuates Virulence of Aspergillus fumigatus.

Aspergillus fumigatus is an opportunistic human pathogen mainly infecting immunocompromised patients. The aim of this study was to characterize the role of arginine biosynthesis in virulence of A. fumigatus via genetic inactivation of two key arginine biosynthetic enzymes, the bifunctional acetylglutamate synthase/ornithine acetyltransferase (argJ/AFUA_5G08120) and the ornithine carbamoyltransferase (argB/AFUA_4G07190). Arginine biosynthesis is intimately linked to the biosynthesis of ornithine, a precursor for siderophore production that has previously been shown to be essential for virulence in A. fumigatus. ArgJ is of particular interest as it is the only arginine biosynthetic enzyme lacking mammalian homologs. Inactivation of either ArgJ or ArgB resulted in arginine auxotrophy. Lack of ArgJ, which is essential for mitochondrial ornithine biosynthesis, significantly decreased siderophore production during limited arginine supply with glutamine as nitrogen source, but not with arginine as sole nitrogen source. In contrast, siderophore production reached wild-type levels under both growth conditions in ArgB null strains. These data indicate that siderophore biosynthesis is mainly fueled by mitochondrial ornithine production during limited arginine availability, but by cytosolic ornithine production during high arginine availability via cytosolic arginine hydrolysis. Lack of ArgJ or ArgB attenuated virulence of A. fumigatus in the insect model Galleria mellonella and in murine models for invasive aspergillosis, indicating limited arginine availability in the investigated host niches.

The Lysine Deacetylase RpdA Is Essential for Virulence in Aspergillus fumigatus.

Current suboptimal treatment options of invasive fungal infections and emerging resistance of the corresponding pathogens urge the need for alternative therapy strategies and require the identification of novel antifungal targets. Aspergillus fumigatus is the most common airborne opportunistic mold pathogen causing invasive and often fatal disease. Establishing a novel in vivo conditional gene expression system, we demonstrate that downregulation of the class 1 lysine deacetylase (KDAC) RpdA leads to avirulence of A. fumigatus in a murine model for pulmonary aspergillosis. The xylP promoter used has previously been shown to allow xylose-induced gene expression in different molds. Here, we demonstrate for the first time that this promoter also allows in vivo tuning of A. fumigatus gene activity by supplying xylose in the drinking water of mice. In the absence of xylose, an A. fumigatus strain expressing rpdA under control of the xylP promoter, rpdA xylP , was avirulent and lung histology showed significantly less fungal growth. With xylose, however, rpdA xylP displayed full virulence demonstrating that xylose was taken up by the mouse, transported to the site of fungal infection and caused rpdA induction in vivo. These results demonstrate that (i) RpdA is a promising target for novel antifungal therapies and (ii) the xylP expression system is a powerful new tool for in vivo gene silencing in A. fumigatus.

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 Siderophore Transporter Sit1 Determines Susceptibility to the Antifungal VL-2397.

VL-2397 (previously termed ASP2397) is an antifungal, aluminum-chelating cyclic hexapeptide with a structure analogous to that of ferrichrome-type siderophores, whereby replacement of aluminum by iron was shown to decrease the antifungal activity of this compound. Here, we found that inactivation of an importer for ferrichrome-type siderophores, termed Sit1, renders Aspergillus fumigatus resistant to VL-2397. Moreover, expression of the endogenous sit1 gene under the control of a xylose-inducible promoter (to uncouple sit1 expression from iron repression) combined with C-terminal tagging with a fluorescent protein demonstrated localization of Sit1 in the plasma membrane and xylose-dependent VL-2397 susceptibility. This underlines that Sit1-mediated uptake is essential for VL-2397 susceptibility. Under xylose-induced sit1 expression, VL-2397 also retained antifungal activity after replacing aluminum with iron, which demonstrates that VL-2397 bears antifungal activity independent of cellular aluminum importation. Analysis of sit1 expression indicated that the reduced antifungal activity of the iron-chelated VL-2397 is caused by downregulation of sit1 expression by the imported iron. Furthermore, we demonstrate that defects in iron homeostatic mechanisms modulate the activity of VL-2397. In contrast to A. fumigatus and Candida glabrata, Saccharomyces cerevisiae displays intrinsic resistance to VL-2397 antifungal activity. However, expression of sit1 from A. fumigatus, or its homologue from C. glabrata, resulted in susceptibility to VL-2397, which suggests that the intrinsic resistance of S. cerevisiae is based on lack of uptake and that A. fumigatus, C. glabrata, and S. cerevisiae share an intracellular target for VL-2397.

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.

Siroheme Is Essential for Assimilation of Nitrate and Sulfate as Well as Detoxification of Nitric Oxide but Dispensable for Murine Virulence of Aspergillus fumigatus.

The saprophytic mold Aspergillus fumigatus is the most common airborne fungal pathogen causing severe invasive fungal infections in immunocompromised patients. Siroheme is a heme-like prosthetic group used by plants and microorganisms for sulfate and nitrate assimilation but is absent in higher eukaryotes. Here, we investigated the role of siroheme in A. fumigatus by deletion of the gene encoding the bifunctional dehydrogenase/ferrochelatase enzyme Met8. Met8-deficiency resulted in the inability to utilize sulfate and nitrate as sulfur and nitrogen sources, respectively. These results match previous data demonstrating that siroheme is an essential cofactor for nitrite and sulfite reductases. Moreover, Met8-deficiency caused significantly decreased resistance against nitric oxide (NO) underlining the importance of nitrite reductase in NO detoxification. Met8-deficiency did not affect virulence in murine models for invasive aspergillosis indicating that neither NO-detoxification nor assimilation of sulfate and nitrate play major roles in virulence in this host. Interestingly, Met8-deficiency resulted in mild virulence attenuation in the Galleria mellonella infection model revealing differences in interaction of A. fumigatus with G. mellonella and mouse.

Human MAIT cells are rapidly activated by Aspergillus spp. in an APC-dependent manner.

Mucosal associated invariant T cells (MAIT cells) are innate-like T cells (TC) which are known to be activated by several bacteria and viruses. However, activation of MAIT cells by moulds, such as the opportunistic human pathogen Aspergillus, is not well described. Stimulation of human PBMC with A. fumigatus, A. flavus, or A. terreus conidia revealed that in contrast to conventional CD4+ and CD8+ TC, MAIT cells responded already after 4 h of coincubation with upregulation of CD69. Furthermore, concurrent increase of CD107a expression and reduced intracellular expression of cytolytic proteins like perforin and granzyme indicated degranulation of intracellular vesicles. MAIT cell activation only occurred in the presence of APC and was dependent on cell-cell contact as separation of TC and APC abrogated MAIT cell activation. Furthermore, we observed that MAIT cell activation by moulds requires presentation of riboflavin metabolites and depends on TCR engagement as antibody blocking of MR1, the antigen presenting molecule for MAIT cells, prevented upregulation of CD69 and CD107a. In summary, we could demonstrate that MAIT cells are activated by Aspergillus conidia in a TCR-dependent manner by APC. These findings reveal MAIT cells as an interesting new target in antifungal defense.

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.

Systematic Identification of Anti-Fungal Drug Targets by a Metabolic Network Approach.

New antimycotic drugs are challenging to find, as potential target proteins may have close human orthologs. We here focus on identifying metabolic targets that are critical for fungal growth and have minimal similarity to targets among human proteins. We compare and combine here: (I) direct metabolic network modeling using elementary mode analysis and flux estimates approximations using expression data, (II) targeting metabolic genes by transcriptome analysis of condition-specific highly expressed enzymes, and (III) analysis of enzyme structure, enzyme interconnectedness ("hubs"), and identification of pathogen-specific enzymes using orthology relations. We have identified 64 targets including metabolic enzymes involved in vitamin synthesis, lipid, and amino acid biosynthesis including 18 targets validated from the literature, two validated and five currently examined in own genetic experiments, and 38 further promising novel target proteins which are non-orthologous to human proteins, involved in metabolism and are highly ranked drug targets from these pipelines.

Histidine biosynthesis plays a crucial role in metal homeostasis and virulence of Aspergillus fumigatus.

Aspergillus fumigatus is the most prevalent airborne fungal pathogen causing invasive fungal infections in immunosuppressed individuals. The histidine biosynthetic pathway is found in bacteria, archaebacteria, lower eukaryotes, and plants, but is absent in mammals. Here we demonstrate that deletion of the gene encoding imidazoleglycerol-phosphate dehydratase (HisB) in A. fumigatus causes (i) histidine auxotrophy, (ii) decreased resistance to both starvation and excess of various heavy metals, including iron, copper and zinc, which play a pivotal role in antimicrobial host defense, (iii) attenuation of pathogenicity in 4 virulence models: murine pulmonary infection, murine systemic infection, murine corneal infection, and wax moth larvae. In agreement with the in vivo importance of histidine biosynthesis, the HisB inhibitor 3-amino-1,2,4-triazole reduced the virulence of the A. fumigatus wild type and histidine supplementation partially rescued virulence of the histidine-auxotrophic mutant in the wax moth model. Taken together, this study reveals limited histidine availability in diverse A. fumigatus host niches, a crucial role for histidine in metal homeostasis, and the histidine biosynthetic pathway as being an attractive target for development of novel antifungal therapy approaches.

Identification and characterization of haemofungin, a novel antifungal compound that inhibits the final step of haem biosynthesis.

OBJECTIVES: During recent decades, the number of invasive fungal infections among immunosuppressed patients has increased significantly, whereas the number of effective systemic antifungal drugs remains low and unsatisfactory. The aim of this study was to characterize a novel antifungal compound, CW-8/haemofungin, which we previously identified in a screen for compounds affecting fungal cell wall integrity. METHODS: The in vitro characteristics of haemofungin were investigated by MIC evaluation against a panel of pathogenic and non-pathogenic fungi, bacteria and mammalian cells in culture. Haemofungin mode-of-action studies were performed by screening an Aspergillus nidulans overexpression genomic library for resistance-conferring plasmids and biochemical validation of the target. In vivo efficacy was tested in the Galleria mellonella and Drosophila melanogaster insect models of infection. RESULTS: We demonstrate that haemofungin causes swelling and lysis of growing fungal cells. It inhibits the growth of pathogenic Aspergillus, Candida, Fusarium and Rhizopus isolates at micromolar concentrations, while only weakly affecting the growth of mammalian cell lines. Genetic and biochemical analyses in A. nidulans and Aspergillus fumigatus indicate that haemofungin primarily inhibits ferrochelatase (HemH), the last enzyme in the haem biosynthetic pathway. Haemofungin was non-toxic and significantly reduced mortality rates of G. mellonella and D. melanogaster infected with A. fumigatus and Rhizopus oryzae, respectively. CONCLUSIONS: Further development and in vivo validation of haemofungin is warranted.