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The importance of natural glycoconjugates containing furanoside residues causes a continued demand for the development of efficient methods for the synthesis of corresponding oligosaccharide derivatives to be used as molecular probes in glycobiological studies. Currently, the chemical synthesis of furanose-containing oligosaccharides often represents a significant challenge because of the lack of short, efficient, and reliable methods for the preparation of selectively substituted furanoside blocks. Herein, we report an easy protocol toward galactofuranose-containing molecules based on the unusual equilibrium between pyranoside and furanoside forms observed for a series of substituted galactosides. The method's utility is illustrated by the syntheses of furanoside-containing oligosaccharides related to the antigenic polysaccharides of Aspergillus fumigatus and Klebsiella pneumoniae O2ac.

Invasive aspergillosis poses a significant threat to immunocompromised patients, leading to high mortality rates associated with these infections. Targeting the biosynthesis of cell wall carbohydrates is a promising strategy for antifungal drug development and will be advanced by a molecular-level understanding of the native structures of polysaccharides within their cellular context. Solid-state NMR spectroscopy has recently provided detailed insights into the cell wall organization of Aspergillus fumigatus, but genetic and biochemical evidence highlights species-specific differences among Aspergillus species. In this study, we employed a combination of 13 C, 15 N, and 1 H-detection solid-state NMR, supplemented by Dynamic Nuclear Polarization (DNP), to compare the structural organization of cell wall polymers and their assembly in the cell walls of A. fumigatus and A. nidulans, both of which are key model organisms and human pathogens. The two species exhibited a similar rigid core architecture, consisting of chitin, α-glucan, and β-glucan, which contributed to comparable cell wall properties, including polymer dynamics, water retention, and supramolecular organization. However, differences were observed in the chitin, galactosaminogalactan, protein, and lipid content, as well as in the dynamics of galactomannan and the structure of the glucan matrix., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The pathogenic fungus Aspergillus fumigatus utilizes a cyclic ferrioxamine E (FOXE) siderophore to acquire iron from the host. Biomimetic FOXE analogues were labeled with gallium-68 for molecular imaging with PET. [ 68 Ga]Ga(III)-FOXE analogues were internalized in A. fumigatus cells via Sit1. Uptake of [ 68 Ga]Ga(III)-FOX 2-5, the most structurally alike analogue to FOXE, was high by both A. fumigatus and bacterial Staphylococcus aureus . However, altering the ring size provoked species-specific uptake between these two microbes: ring size shortening by one methylene unit (FOX 2-4) increased uptake by A. fumigatus compared to that by S. aureus , whereas lengthening the ring (FOX 2-6 and 3-5) had the opposite effect. These results were consistent both in vitro and in vivo , including PET imaging in infection models. Overall, this study provided valuable structural insights into the specificity of siderophore uptake and, for the first time, opened up ways for selective targeting and imaging of microbial pathogens by siderophore derivatization.

Tuberculosis necrotizing toxin (TNT) is a protein domain discovered on the outer membrane of Mycobacterium tuberculosis (Mtb), and the fungal pathogen Aspergillus fumigatus. TNT domains have pure NAD(P) hydrolytic activity, setting them apart from other NAD-cleaving domains such as ADP-ribosyl cyclase and Toll/interleukin-1 receptor homology (TIR) domains which form a wider set of products. Importantly, the Mtb TNT domain has been shown to be involved in immune evasion via depletion of the intracellular NAD pool of macrophages. Therefore, an intriguing hypothesis is that TNT domains act as "NAD killers" in host cells facilitating pathogenesis. Here, we explore the phylogenetic distribution of TNT domains and detect their presence solely in bacteria and fungi. Within fungi, we discerned six TNT clades. In addition, X-ray crystallography and AlphaFold2 modeling unveiled clade-specific strategies to promote homodimer stabilization of the fungal enzymes, namely, Ca 2+ binding, disulfide bonds, or hydrogen bonds. We show that dimer stabilization is a requirement for NADase activity and that the group-specific strategies affect the active site conformation, thereby modulating enzyme activity. Together, these findings reveal the evolutionary lineage of fungal TNT enzymes, corroborating the hypothesis of them being pure extracellular NAD (eNAD) cleavers, with possible involvement in microbial warfare and host immune evasion., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The cellular surface of the pathogenic filamentous fungus Aspergillus fumigatus is enveloped in a mannose layer, featuring well-established fungal-type galactomannan and O -mannose-type galactomannan. This study reports the discovery of cell wall component in A. fumigatus mycelium, which resembles N -glycan outer chains found in yeast. The glycosyltransferases involved in its biosynthesis in A. fumigatus were identified, with a focus on two key α-(1→2)-mannosyltransferases, Mnn2 and Mnn5, and two α-(1→6)-mannosyltransferases, Mnn9 and Van1. In vitro examination revealed the roles of recombinant Mnn2 and Mnn5 in transferring α-(1→2)-mannosyl residues. Proton nuclear magnetic resonance ( 1 H-NMR) analysis of cell wall extracts from the ∆ mnn2 ∆ mnn5 strain indicated the existence of an α-(1→6)-linked mannan backbone in the A. fumigatus mycelium, with Mnn2 and Mnn5 adding α-(1→2)-mannosyl residues to this backbone. The α-(1→6)-linked mannan backbone was absent in strains where mnn9 or van1 was disrupted in the parental ∆ mnn2 ∆ mnn5 strain in A. fumigatus . Mnn9 and Van1 functioned as α-(1→6)-linked mannan polymerases in heterodimers when co-expressed in Escherichia coli , indicating their crucial role in biosynthesizing the α-(1→6)-linked mannan backbone. Disruptions of these mannosyltransferases did not affect fungal-type galactomannan biosynthesis. This study provides insights into the complexity of fungal cell wall architecture and a better understanding of mannan biosynthesis in A. fumigatus ., Importance: This study unravels the complexities of mannan biosynthesis in A. fumigatus , a key area for antifungal drug discovery. It reveals the presence of α-(1→6)-linked mannan structures resembling yeast N-glycan outer chains in A. fumigatus mycelium, offering fresh insights into the fungal cell wall's design. Key enzymes, Mnn2, Mnn5, Mnn9, and Van1, are instrumental in this process, with Mnn2 and Mnn5 adding specific mannose residues and Mnn9 and Van1 assembling the α-(1→6)-linked mannan structures. Although fungal-type galactomannan's presence in the cell wall is known, the existence of an α-(1→6)-linked mannan adds a new dimension to our understanding. This intricate web of mannan biosynthesis opens avenues for further exploration and enhances our understanding of fungal cell wall dynamics, paving the way for targeted drug development., Competing Interests: The authors declare no conflict of interest.

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., (© 2024 The Authors. Advanced Biology published by Wiley‐VCH GmbH.)

The endophyte Nemania primolutea, inhibited the growth of Penicillium chrysogenum in the coculture system. Four new compounds, nemmolutines A-B (1-2), and penigenumin (3) from N. primolutea, penemin (4) from P. chrysogenum were isolated from the coculture. On the other hand, P. chrysogenum inhibited the Aspergillus fumigatus in the coculture. Induced metabolites (13-16) with monasone naphthoquinone scaffolds including a new one from P. chrysogenum were produced by the coculture of P. chrysogenum, and A. fumigatus. Interesting, cryptic metabolites penicichrins A-B isolated from wild P. chrysogenum induced by host Ziziphus jujuba medium were also found in induced P. chrysogenum cultured in PDB ordinary medium. So the induction of penicichrin production by supplementing with host extract occurred in the fungus P. chrysogenum not the host medium. The productions of penicichrins were the spontaneous metabolism, and the metabolites (13-16) were the culture driven. Compounds 4, 6, 8, 10, 11, 14, and 15 showed significant antifungal activities against the phytopathogen Alternaria alternata with MIC S of 1-8 μg/mL, and compounds 7, 9, and 12 indicated significant antifeedant activities against silkworms with feeding deterrence indexes (FDIs) of 92 %, 66 %, and 64 %. The carboxy group in 4-(2-hydroxybutynoxy)benzoic acid derivatives, and xylabisboeins; the hydroxy group in mellein derivatives; and the quinoid in monasone naphthoquinone increased the antifungal activities., (© 2023 Wiley-VHCA AG, Zurich, Switzerland.)

Chemical investigation of endophytic fungus Aspergillus fumigatus HQD24, isolated from the flower of Rhizophora mucronata led to the isolation of eight alkaloids, including pyripyropene A ( 1 ), 1,11-dideacetyl-pyripyropene A ( 2 ), pyripyropene E ( 3 ), chaetominine ( 4 ), tryptoquivaline J ( 5 ), fumitremorgin C ( 6 ), 1-acetyl-β-carboline ( 7 ), and nicotinic acid ( 8 ). Their structures were unambiguously elucidated on the basis of extensive spectroscopic data and comparison with the data of literature. Compound 2 was known as a synthetic product and isolated as a natural product for the first time. The immunosuppressive and cytotoxic activities of all isolated compounds were evaluated.

Chemical epigenetic modifiers applied on a plant endophytic fungus Aspergillus fumigatus isolated from a healthy stem of terrestrial plant Cynodon dactylon , significantly changed of metabolic profile and resulted in the isolation of nineteen compounds, including ten alkaloids ( 1 - 10 ), six polyketides ( 11 - 16 ), and three benzene derivatives ( 17 - 19 ). This is the first report of 14 , 18 and 19 being isolated from this fungal species. And compound 14 was known as a synthetic product and isolated as a natural product for the first time. HPLC profiles of the control and treated samples indicated that compounds 11, 16, 18 are belonged to the newly induced secondary metabolites. Their structures were elucidated on the basis of extensive NMR spectroscopic and mass spectrometric analyses. The immunosuppressive and cytotoxic activities of all isolated compounds were evaluated.

In Argentina there are no reports on Aspergillus fumigatus fumagillin-producing strains. In this study we describe the isolation and mycotoxin production capacity of ten A. fumigatus strains isolated from farm and clinical samples. Farm strains were isolated from milk samples taken from dairy cows in Córdoba province, some of which were associated with subclinical mastitis. A culture medium was defined to optimize fumagillin production and a detection method was developed by HPLC chromatography. It is known that in addition to the host immune status, strain virulence is a fundamental characteristic that will determine its pathogenicity and, in this sense, fumagillin is considered to be among the virulence factors. In the present work, all the strains tested for the production of fumagillin were able to synthesize it, highlighting that the strain A. fumigatus RC2243, from a milk sample from a cow with clinical mastitis, was the most productive. The existence of fumagillin-producing strains represents a potential risk of mycotoxins being transferred to raw milk, constituting a public health risk., (Copyright © 2022 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

The COVID-19 pandemic and its continuing emerging variants emphasize the need to discover appropriate treatment, where vaccines alone have failed to show complete protection against the new variants of the virus. Therefore, treatment of the infected cases is critical. This paper discusses the bio-guided isolation of three indole diketopiperazine alkaloids, neoechinulin A ( 1 ), echinulin ( 2 ), and eurocristatine ( 3 ), from the Red Sea-derived Aspergillus fumigatus MR2012. Neoechinulin A ( 1 ) exhibited a potent inhibitory effect against SARS-CoV-2 M pro with IC 50 value of 0.47 μM, which is comparable to the reference standard GC376. Despite the structural similarity between the three compounds, only 1 showed a promising effect. The mechanism of inhibition is discussed in light of a series of extensive molecular docking, classical and steered molecular dynamics simulation experiments. This paper sheds light on indole diketopiperazine alkaloids as a potential structural motif against SARS-CoV-2 M pro . Additionally, it highlights the potential of different molecular docking and molecular dynamics simulation approaches in the discrimination between active and inactive structurally related M pro inhibitors.

Filamentous fungi form multicellular hyphae, which generally form pellets in liquid shake cultures, during the vegetative growth stage. Because of these characteristics, growth-monitoring methods commonly used in bacteria and yeast have not been applied to filamentous fungi. We have recently revealed that the cell wall polysaccharide α-1,3-glucan and extracellular polysaccharide galactosaminogalactan (GAG) contribute to hyphal aggregation in Aspergillus oryzae. Here, we tested whether Aspergillus fumigatus shows dispersed growth in liquid media that can be quantitatively monitored, similar to that of yeasts. We constructed a double disruptant mutant of both the primary α-1,3-glucan synthase gene ags1 and the putative GAG synthase gene gtb3 in A. fumigatus AfS35 and found that the hyphae of this mutant were fully dispersed. Although the mutant lost α-1,3-glucan and GAG, its growth and susceptibility to antifungal agents were not different from those of the parental strain. Mycelial weight of the mutant in shake-flask cultures was proportional to optical density for at least 18 h. We were also able to quantify the dose response of hyphal growth to antifungal agents by measuring optical density. Overall, we established a convenient strategy to monitor A. fumigatus hyphal growth. Our method can be directly used for screening for novel antifungals against Aspergillus species. IMPORTANCE Filamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi so that we may apply the methods used for unicellular organisms such as yeast and bacteria. In the present study, by using the fungal pathogen Aspergillus fumigatus strain with modified hyphal surface polysaccharides, we succeeded in monitoring the hyphal growth quantitatively by optical density. The principle of this easy measurement by optical density could lead to a novel standard of hyphal quantification such as those that have been used for yeasts and bacteria. Dose response of hyphal growth by antifungal agents could also be monitored. This method could be useful for screening for novel antifungal reagents against Aspergillus species.

The rapid spread of bacterial infection caused by Staphylococcus aureus has become a problem to public health despite the presence of past trials devoted to controlling the infection. Thus, the current study aimed to explore the chemical composition of the extract of endophytic fungus Aspergillus fumigatus , isolated from Albizia lucidior leaves, and investigate the antimicrobial activity of isolated metabolites and their probable mode of actions. The chemical investigation of the fungal extract via UPLC/MS/MS led to the identification of at least forty-two metabolites, as well as the isolation and complete characterization of eight reported metabolites. The antibacterial activities of isolated metabolites were assessed against S. aureus using agar disc diffusion and microplate dilution methods. Compounds ergosterol, helvolic acid and monomethyl sulochrin-4-sulphate showed minimal inhibitory concentration (MIC) values of 15.63, 1.95 and 3.90 µg/mL, respectively, compared to ciprofloxacin. We also report the inhibitory activity of the fungal extract on DNA gyrase and topoisomerase IV, which led us to perform molecular docking using the three most active compounds isolated from the extract against both enzymes. These active compounds had the required structural features for S. aureus DNA gyrase and topoisomerase IV inhibition, evidenced via molecular docking.

Strain SZW01 was isolated from sea sediment collected from Shenzhen in Guangdong province, China, and was later identified as Aspergillus fumigatus by16S rDNA sequence analysis. Various chromatographic processes led to the isolation and purification of three compounds from the fermentation culture of SZW01, including a new compound, 2,6'-dihydroxy-2,4'dimethoxy-8'-methyl-6-methoxy-acyl-ethyl-diphenylmethanone ( 1 ), and two known compounds: fumigaclavine C ( 2 ) and alternarosin A ( 3 ), as characterised by UV, IR, 1 D, 2 D-NMR and MS data. The antioxidant and α-glucosidase inhibitory activities of these compounds were evaluated. The result illustrated that compound 1 exhibited a moderate antioxidant activity and stronger α-glucosidase inhibitory activity than acarbose.

Invasive pulmonary aspergillosis (IPA) is a severe life-threatening condition. Diagnosis of fungal disease in general, and especially that caused by Aspergillus fumigatus is problematic. A. fumigatus secretes siderophores to acquire iron during infection, which are also essential for virulence. We describe the chemoacetylation of ferrated fusarinine C to diacetylated fusarinine C (DAFC), followed by protein conjugation, which facilitated triacetylfusarinine C (TAFC)-specific monoclonal antibody production with specific recognition of the ferrated form of TAFC. A single monoclonal antibody sequence was ultimately elucidated by a combinatorial strategy involving protein LC-MS/MS, cDNA sequencing and RNAseq. The resultant murine IgG2 a monoclonal antibody was secreted in, and purified from, mammalian cell culture (5 mg) and demonstrated to be highly specific for TAFC detection by competitive ELISA (detection limit: 15 nM) and in a lateral flow test system (detection limit: 3 ng), using gold nanoparticle conjugated- DAFC-bovine serum albumin for competition. Overall, this work reveals for the first time a recombinant TAFC-specific monoclonal antibody with diagnostic potential for IPA diagnosis in traditional and emerging patient groups (e.g., COVID-19) and presents a useful strategy for murine Ig sequence determination, and expression in HEK293 cells, to overcome unexpected limitations associated with aberrant or deficient murine monoclonal antibody production., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The current study was conducted to evaluate the antiproliferative and oxidative damage protection potential of endophytic fungi Aspergillus fumigatus and Chaetomium globosum isolated from Moringa oleifera. The chloroformic extract (CE) of both the fungi showed dose dependent antiproliferative activity against human prostate adenocarcinoma (PC-3) cell line with (IC 50 ) value of 0.055 mg/ml and 0.008 mg/ml, respectively. Further, CE of both the fungi was studied for their ability to induce apoptosis in PC-3 cell line. Various deformities in the cancerous cells treated with CE of both the fungi have been observed by confocal microscopy which indicates the cell death by apoptosis. Further apoptosis inducing ability of CE of both the fungi was observed using various flow cytometric studies. The chloroformic extract of both the fungi showed slight increase in the level of reactive oxygen species to induce apoptosis. It also showed arrest of cancerous cells at G0/G1 phase of cell cycle to induce apoptosis. The externalization of phosphatidylserine (PS) to induce apoptosis was also observed when analysed using Annexin V-FITC/PI double staining assay where the CE of A. fumigatus and C. globosum showed the total apoptosis of 94.2% and 90.3%, respectively, at the highest tested concentration of GI 70 . The CE of both the fungi further showed the protective behaviour for plasmid DNA pBR322, when tested for their effect against the oxidative stress caused by the Fenton's reagent. Thus, the studies demonstrated a good antiproliferative and oxidative damage protection potential of the endophytic fungi., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The fungus is a great source of secondary metabolites which may possess various biological activities for the treatment of human diseases and disorders. The present study aimed to identify the secondary metabolites from Aspergillus fumigatus strain MF-1 using analytical techniques (chromatographic and spectroscopic). The instruments includes 1 H-NMR, 13 C-NMR, DEPT, COSY, HMQC, FTIR and UV-Vis spectrophotometer were employed to study the extracted compound and the produced compound is 2,5-dioxocyclopentylamino-7-oxohepta-1,3,5-trienyl-2,5-dihydroxy-3-chlorophenyl-2,4,6-trimethyldeca-2,4-dienamide. Furthermore, the four fractions were tested over HeLa cell lines for their cytotoxicity performance and found that fraction 4 exhibited potent cytotoxic effect with IC 50 value of 74.38 ± 0.31 µg/mL on HeLa cell lines than other fractions., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aspergillus fumigatus is an important fungal pathogen and the main etiological agent of aspergillosis, a disease characterized by a noninvasive process that can evolve to a more severe clinical manifestation, called invasive pulmonary aspergillosis (IPA), in immunocompromised patients. The antifungal arsenal to threat aspergillosis is very restricted. Azoles are the main therapeutic approach to control IPA, but the emergence of azole-resistant A. fumigatus isolates has significantly increased over recent decades. Therefore, new strategies are necessary to combat aspergillosis, and drug repurposing has emerged as an efficient and alternative approach for identifying new antifungal drugs. Here, we used a screening approach to analyze A. fumigatus in vitro susceptibility to 1,127 compounds. A. fumigatus was susceptible to 10 compounds, including miltefosine, a drug that displayed fungicidal activity against A. fumigatus. By screening an A. fumigatus transcription factor null library, we identified a single mutant, which has the smiA ( s ensitive to mi ltefosine) gene deleted, conferring a phenotype of susceptibility to miltefosine. The transcriptional profiling (RNA-seq) of the wild-type and Δ smiA strains and chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-Seq) of an SmiA-tagged strain exposed to miltefosine revealed genes of the sphingolipid pathway that are directly or indirectly regulated by SmiA. Sphingolipid analysis demonstrated that the mutant has overall decreased levels of sphingolipids when growing in the presence of miltefosine. The identification of SmiA represents the first genetic element described and characterized that plays a direct role in miltefosine response in fungi. IMPORTANCE The filamentous fungus Aspergillus fumigatus causes a group of diseases named aspergillosis, and their development occurs after the inhalation of conidia dispersed in the environment. Very few classes of antifungal drugs are available for aspergillosis treatment, e.g., azoles, but the emergence of global resistance to azoles in A. fumigatus clinical isolates has increased over recent decades. Repositioning or repurposing drugs already available on the market is an interesting and faster opportunity for the identification of novel antifungal agents. By using a repurposing strategy, we identified 10 different compounds that impact A. fumigatus survival. One of these compounds, miltefosine, demonstrated fungicidal activity against A. fumigatus. The mechanism of action of miltefosine is unknown, and, aiming to get more insights about it, we identified a transcription factor, SmiA ( s ensitive to mi ltefosine), important for miltefosine resistance. Our results suggest that miltefosine displays antifungal activity against A. fumigatus, interfering in sphingolipid biosynthesis.

During the systematic screening of bioactive compounds from our marine natural product library, crude extract of the marine-derived fungus strain Aspergillus fumigatus MF029 exhibited moderate bioactivities against Bacillus subtilis , S taphylococcus aureus , methicillin-resistant S. aureus , and Mycobacterium bovis bacillus Calmette-Guérin (BCG). Further chemical investigation resulted in the identification of two new compounds, chaetominine A ( 1 ) and sphingofungin I ( 2 ), together with four known compounds, emodin ( 3 ), chaetominine ( 4 ), sphingofungin D ( 5 ) and trypacidin ( 6 ). Trypacidin displayed potential antitubercular activity with MIC value of 1.25 μg/mL.

A new epimer of azaphilone derivative pinophilin B, epi -pinophilin B ( 1 ), and three known analogues ( 2 - 4 ) were obtained from the culture of the gorgonian-derived fungus Aspergillus fumigatus 14-27. The structures of 1 - 4 , including their relative configurations were determined by extensive spectroscopic analysis and comparing with literature data. The absolute configuration of 1 was determined by electronic circular dichroism (ECD) and optical rotatory (OR) calculations methods. Compounds 1 - 4 were isolated from A. fumigatus for the first time. Their antibacterial and cytotoxic activities were also evaluated.

The research on bioactive secondary metabolites from Aspergillus fumigatus afforded six compounds, which were identified by mass spectrometer (MS) and nuclear magnetic resonance (NMR) spectroscopic analysis as cyclopyazonic acid (1), trypacidin A (2), asterric acid (3), methyl asterrate (4), demethylcitreoviranol (5), as well as (5-hydroxy-2-oxo-2H-pyran-4-yl) methyl acetate (6). Cyclopyazonic acid (1) was found to have potent antibacterial effects, especially against Bacillus licheniformis with minimal inhibitory concentration (MIC) value of 3.7μg/mL. Its antibacterial effects were possibly related to the olefinic acid group in the structure. Phenyl ether derivatives 3 and 4, and trypacidin A (2) also exhibited antimicrobial effects. In addition, compound 6 showed significant antioxidant effects with half maximal effective concentration (EC 50 ) value of 10.2μM in the ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) assay, which was better than the positive control.

Aspergillus fumigatus is a pathogenic fungus infecting the respiratory system and responsible for a variety of life-threatening lung diseases. A fucose-binding lectin named FleA which has a controversial role in A. fumigatus pathogenesis was recently identified. New chemical probes with high affinity and enzymatic stability are needed to explore the role of FleA in the infection process. In this study, we developed potent FleA antagonists based on optimized and non-hydrolysable thiofucoside ligands. We first synthesized a set of monovalent sugars showing micromolar affinity for FleA by isothermal titration calorimetry. The most potent derivative was co-crystallized with FleA to gain insights into the binding mode in operation. Its chemical multimerization on a cyclodextrin scaffold led to an hexavalent compound with a significantly enhanced binding affinity (K d = 223 ± 21 nM) thanks to a chelate binding mode. The compound could probe the role of bronchial epithelial cells in a FleA-mediated response to tissue invasion.

The maintenance of sufficient but nontoxic pools of metal micronutrients is accomplished through diverse homeostasis mechanisms in fungi. Siderophores play a well established role for iron homeostasis; however, no copper-binding analogs have been found in fungi. Here we demonstrate that, in Aspergillus fumigatus , xanthocillin and other isocyanides derived from the xan biosynthetic gene cluster (BGC) bind copper, impact cellular copper content, and have significant metal-dependent antimicrobial properties. xan BGC-derived isocyanides are secreted and bind copper as visualized by a chrome azurol S (CAS) assay, and inductively coupled plasma mass spectrometry analysis of A. fumigatus intracellular copper pools demonstrated a role for xan cluster metabolites in the accumulation of copper. A. fumigatus coculture with a variety of human pathogenic fungi and bacteria established copper-dependent antimicrobial properties of xan BGC metabolites, including inhibition of laccase activity. Remediation of xanthocillin-treated Pseudomonas aeruginosa growth by copper supported the copper-chelating properties of xan BGC isocyanide products. The existence of the xan BGC in several filamentous fungi suggests a heretofore unknown role of eukaryotic natural products in copper homeostasis and mediation of interactions with competing microbes., Competing Interests: The authors declare no competing interest.

Aspergillus fumigatus is a well-known opportunistic pathogen that causes invasive aspergillosis (IA) infections with high mortality in immunosuppressed individuals. Morphogenesis, including hyphal growth, conidiation, and cell wall biosynthesis is crucial in A. fumigatus pathogenesis. Based on a previous random insertional mutagenesis library, we identified the putative polysaccharide synthase gene Afcps1 and its para-log Afcps2. Homologs of the cps gene are commonly found in the genomes of most fungal and some bacterial pathogens. Afcps1/cpsA is important in sporulation, cell wall composition, and virulence. However, the precise regulation patterns of cell wall integrity by Afcps1/cpsA and further effects on the immune response are poorly understood. Specifically, our in-depth study revealed that Afcps1 affects cell-wall stability, showing an increased resistance of ΔAfcps1 to the chitinmicrofibril destabilizing compound calcofluor white (CFW) and susceptibility of ΔAfcps1 to the β-(1,3)-glucan synthase inhibitor echinocandin caspofungin (CS). Additionally, deletion of Afcps2 had a normal sporulation phenotype but caused hypersensitivity to Na + stress, CFW, and Congo red (CR). Specifically, quantitative analysis of cell wall composition using high-performance anion exchange chromatography-pulsed amperometric detector (HPAEC-PAD) analysis revealed that depletion of Afcps1 reduced cell wall glucan and chitin contents, which was consistent with the down-regulation of expression of the corresponding biosynthesis genes. Moreover, an elevated immune response stimulated by conidia of the ΔAfcps1 mutant in marrow-derived macrophages (BMMs) during phagocytosis was observed. Thus, our study provided new insights into the function of polysaccharide synthase Cps1, which is necessary for the maintenance of cell wall stability and the adaptation of conidia to the immune response of macrophages in A. fumigatus.

G-protein coupled receptors (GPCRs) are extracellular signaling receptors that sense environmental cues. Fungi sense their environment primarily through GPCR-mediated signaling pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. Aspergillus fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that present a wide range of clinical manifestations. Here, we investigate in detail the role of the GPCRs GprM and GprJ in growth and gene expression. GprM and GprJ are important for melanin production and the regulation of the cell wall integrity (CWI) pathway. Overexpression of gprM and gprJ causes a 20 and 50% reduction in growth rate compared to the wild-type (WT) strain and increases sensitivity to cell wall-damaging agents. Phosphorylation of the CWI protein kinase MpkA is increased in the Δ gprM and Δ gprJ strains and decreased in the overexpression mutants compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. Transcriptome sequencing suggests that GprM and GprJ negatively regulate genes encoding secondary metabolites (SMs). Mass spectrometry analysis confirmed that the production of fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, and fumitremorgin is reduced in the Δ gprM and Δ gprJ strains, at least partially through the activation of MpkA. Overexpression of grpM also resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signaling. Finally, we show that the Δ gprM and Δ gprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis. IMPORTANCE A. fumigatus is the main etiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immunocompromised humans. Withstanding the host environment is essential for A. fumigatus virulence, and sensing of extracellular cues occurs primarily through G-protein coupled receptors (GPCRs) that activate signal transduction pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. The A. fumigatus genome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GPCRs GprM and GprJ regulate the phosphorylation of the mitogen-activated protein kinase MpkA and thus control the regulation of the cell wall integrity pathway. GprM and GprJ are also involved in the regulation of the production of the secondary metabolites fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, melanin, and fumitremorgin, and this regulation partially occurs through the activation of MpkA. Furthermore, GprM and GprJ are important for virulence in the insect model Galleria mellonella This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial for A. fumigatus virulence., (Copyright © 2020 Filho et al.)

Even though both cellular and humoral immunities contribute to host defense, the role played by humoral immunity against the airborne opportunistic fungal pathogen Aspergillus fumigatus has been underexplored. In this study, we aimed at deciphering the role of the complement system, the major humoral immune component, against A. fumigatus Mass spectrometry analysis of the proteins extracted from A. fumigatus conidial (asexual spores and infective propagules) surfaces opsonized with human serum indicated that C3 is the major complement protein involved. Flow cytometry and immunolabeling assays further confirmed C3b (activated C3) deposition on the conidial surfaces. Assays using cell wall components of conidia indicated that the hydrophobin RodAp, β-(1,3)-glucan (BG) and galactomannan (GM) could efficiently activate C3. Using complement component-depleted sera, we showed that while RodAp activates C3 by the alternative pathway, BG and GM partially follow the classical and lectin pathways, respectively. Opsonization facilitated conidial aggregation and phagocytosis, and complement receptor (CR3 and CR4) blockage on phagocytes significantly inhibited phagocytosis, indicating that the complement system exerts a protective role against conidia by opsonizing them and facilitating their phagocytosis mainly through complement receptors. Conidial opsonization with human bronchoalveolar lavage fluid (BALF) confirmed C3 to be the major complement protein interacting with conidia. Nevertheless, complement C2 and mannose-binding lectin (MBL), the classical and lectin pathway components, respectively, were not identified, indicating that BALF activates the alternative pathway on the conidial surface. Moreover, the cytokine profiles were different upon stimulation of phagocytes with serum- and BALF-opsonized conidia, highlighting the importance of studying interaction of conidia with complement proteins in their biological niche., (Copyright © 2020 American Society for Microbiology.)

Aspergillus fumigatus is one of the most common airborne molds capable of causing mycoses and allergies in humans. During infection, fungal surface proteins mediate the first contact with the human immune system to evade immune responses or to induce hypersensitivity. Several methods have been established for surface proteomics (surfomics). Biotinylation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of peptides is a particularly efficient method to identify the surface-exposed regions of proteins that potentially mediate interaction with the host. After biotinylation of surface proteins during spore germination, we detected 231 different biotinylated surface proteins (including several well-known proteins such as RodA, CcpA, and DppV; allergens; and heat shock proteins [HSPs]), as well as some previously undescribed surface proteins. The dynamic change of the surface proteome was illustrated by detection of a relatively high number of proteins exclusively at one developmental stage. Using immunofluorescence microscopy, we confirmed the surface localization of several HSPs of the HSP70 family, which may have moonlighting functions. Collectively, by comparing our data with data representative of previously published A. fumigatus surface proteomes, our study generated a comprehensive data set corresponding to the A. fumigatus surfome and uncovered the surface-exposed regions of many proteins on the surface of conidia or hyphae. These surface-exposed regions are candidates for direct interaction with host cells and may represent antigenic epitopes that either induce protective immune responses or mediate immune evasion. Thus, our data sets provided and compiled here represent reasonable immunotherapy and diagnostic targets for future investigations. IMPORTANCE Aspergillus fumigatus is the most important airborne human-pathogenic mold, capable of causing both life-threatening invasive pulmonary aspergillosis in immunocompromised patients and allergy-inducing infections in individuals with atopic allergy. Despite its obvious medical relevance, timely diagnosis and efficient antifungal treatment of A. fumigatus infection remain major challenges. Proteins on the surface of conidia (asexually produced spores) and mycelium directly mediate host-pathogen interaction and also may serve as targets for diagnosis and immunotherapy. However, the similarity of protein sequences between A. fumigatus and other organisms, sometimes even including the human host, makes selection of targets for immunological-based studies difficult. Here, using surface protein biotinylation coupled with LC-MS/MS analysis, we identified hundreds of A. fumigatus surface proteins with exposed regions, further defining putative targets for possible diagnostic and immunotherapeutic design., (Copyright © 2020 Jia et al.)

Galactosaminogalactan (GAG) is a prominent cell wall component of the opportunistic fungal pathogen Aspergillus fumigatus. GAG is a heteropolysaccharide composed of α-1,4-linked galactose, galactosamine and N-acetylgalactosamine residues. To enable biochemical studies, a library of GAG-fragments was constructed featuring specimens containing α-galactose-, α-galactosamine and α-N-acetyl galactosamine linkages. Key features of the synthetic strategy include the use of di-tert-butylsilylidene directed α-galactosylation methodology and regioselective benzoylation reactions using benzoyl-hydroxybenzotriazole (Bz-OBt). Structural analysis of the Gal, GalN and GalNAc oligomers by a combination of NMR and MD approaches revealed that the oligomers adopt an elongated, almost straight, structure, stabilized by inter-residue H-bonds, one of which is a non-conventional C-H⋅⋅⋅O hydrogen bond between H5 of the residue (i+1) and O3 of the residue (i). The structures position the C-2 substituents almost perpendicular to the oligosaccharide main chain axis, pointing to the bulk solvent and available for interactions with antibodies or other binding partners., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Invasive fungal diseases are associated with significant morbidity and mortality, particularly in immunocompromised individuals. Early and accurate diagnosis is crucial for effective treatment. Despite traditional methods such as microbiological culture, histopathology, radiology and direct microscopy are available, antigen/antibody-based diagnostics are emerging for diagnosis of invasive fungal infections (IFI). Fungal cell wall is a unique structure composed of polysaccharides that are well correlated with fungal burden during fungal infections. Based on this feature, cell wall polysaccharides have been explored as antigens in IFIs diagnostics such as the galactomannan assay, mannan test, β-glucan assay and cryptococcal CrAg test. Herein, we provide an overview on the cell wall polysaccharides from three opportunistic pathogens: Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans, and their applications for IFIs diagnosis. The clinical outcome of newly developed cell wall polysaccharides-based diagnostics is also discussed., (© 2020 Blackwell Verlag GmbH.)

Diverting aminoacyl-transfer RNAs (tRNAs) from protein synthesis is a well-known process used by a wide range of bacteria to aminoacylate membrane constituents. By tRNA-dependently adding amino acids to glycerolipids, bacteria change their cell surface properties, which intensifies antimicrobial drug resistance, pathogenicity, and virulence. No equivalent aminoacylated lipids have been uncovered in any eukaryotic species thus far, suggesting that tRNA-dependent lipid remodeling is a process restricted to prokaryotes. We report here the discovery of ergosteryl-3β- O -l-aspartate (Erg-Asp), a conjugated sterol that is produced by the tRNA-dependent addition of aspartate to the 3β-OH group of ergosterol, the major sterol found in fungal membranes. In fact, Erg-Asp exists in the majority of "higher" fungi, including species of biotechnological interest, and, more importantly, in human pathogens like Aspergillus fumigatus We show that a bifunctional enzyme, ergosteryl-3β- O -l-aspartate synthase (ErdS), is responsible for Erg-Asp synthesis. ErdS corresponds to a unique fusion of an aspartyl-tRNA synthetase-that produces aspartyl-tRNA Asp (Asp-tRNA Asp )-and of a Domain of Unknown Function 2156 , which actually transfers aspartate from Asp-tRNA Asp onto ergosterol. We also uncovered that removal of the Asp modifier from Erg-Asp is catalyzed by a second enzyme, ErdH, that is a genuine Erg-Asp hydrolase participating in the turnover of the conjugated sterol in vivo. Phylogenomics highlights that the entire Erg-Asp synthesis/degradation pathway is conserved across "higher" fungi. Given the central roles of sterols and conjugated sterols in fungi, we propose that this tRNA-dependent ergosterol modification and homeostasis system might have broader implications in membrane remodeling, trafficking, antimicrobial resistance, or pathogenicity., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The human fungal pathogen Aspergillus fumigatus causes life-threatening invasive aspergillosis in immunocompromised individuals. Adaptation to the host environment is integral to survival of A. fumigatus and requires the coordination of short- and long-distance vesicular transport to move essential components throughout the fungus. We previously reported the importance of MyoE, the only class V myosin, for hyphal growth and virulence of A. fumigatus. Class V myosins are actin-based, cargo-carrying motor proteins that contain unique binding sites for specific cargo. Specific cargo carried by myosin V has not been identified in any fungus, and previous studies have only identified single components that interact with class V myosins. Here we utilized a mass spectrometry-based whole proteomic approach to identify MyoE interacting proteins in A. fumigatus for the first time. Several proteins previously shown to interact with myosin V through physical and genetic approaches were confirmed, validating our proteomic analysis. Importantly, we identified novel MyoE-interacting proteins, including members of the cytoskeleton network, cell wall synthesis, calcium signaling and a group of coat protein complex II (COPII) proteins involved in the endoplasmic reticulum (ER) to Golgi transport. Furthermore, we analyzed the localization patterns of the COPII proteins, UsoA (Uso1), SrgE (Sec31), and SrgF (Sec23), which suggested a potential role for MyoE in ER to Golgi trafficking., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aspergillus fumigatus is an opportunistic and allergenic pathogenic fungus, responsible for fungal infections in humans. A. fumigatus infections are usually treated with polyenes, azoles, or echinocandins. Echinocandins, such as caspofungin, can inhibit the biosynthesis of the β-1,3-glucan polysaccharide, affecting the integrity of the cell wall and leading to fungal death. In some A. fumigatus strains, caspofungin treatment at high concentrations induces an increase of fungal growth, a phenomenon called the c aspofungin p aradoxical e ffect (CPE). Here, we analyze the proteome and phosphoproteome of the A. fumigatus wild-type strain and of mitogen-activated protein kinase (MAPK) mpkA and sakA null mutant strains during CPE (2 μg/ml caspofungin for 1 h). The wild-type proteome showed 75 proteins and 814 phosphopeptides (corresponding to 520 proteins) altered in abundance in response to caspofungin treatment. The Δ mpkA (Δ mpkA caspofungin/wild-type caspofungin) and Δ sakA (Δ sakA caspofungin/wild-type caspofungin) strains displayed 626 proteins and 1,236 phosphopeptides (corresponding to 703 proteins) and 101 proteins and 1,217 phosphopeptides (corresponding to 645 proteins), respectively, altered in abundance. Functional characterization of the phosphopeptides from the wild-type strain exposed to caspofungin showed enrichment for transcription factors, protein kinases, and cytoskeleton proteins. Proteomic analysis of the Δ mpkA and Δ sakA mutants indicated that control of proteins involved in metabolism, such as in production of secondary metabolites, was highly represented in both mutants. Results of functional categorization of phosphopeptides from both mutants were very similar and showed a high number of proteins with decreased phosphorylation of proteins involved in transcriptional control, DNA/RNA binding, cell cycle control, and DNA processing. This report reveals novel transcription factors involved in caspofungin tolerance. IMPORTANCE Aspergillus fumigatus is an opportunistic human-pathogenic fungus causing allergic reactions or systemic infections, such as invasive pulmonary aspergillosis in immunocompromised patients. Caspofungin is an echinocandin that impacts the construction of the fungal cell wall by inhibiting the biosynthesis of the β-1,3-glucan polysaccharide. Caspofungin is a fungistatic drug and is recommended as a second-line therapy for treatment of aspergillosis. Treatment at high concentrations induces an increase of fungal growth, a phenomenon called the c aspofungin p aradoxical e ffect (CPE). Collaboration between the mitogen-activated protein kinases (MAPK) of the cell wall integrity (MapkA) and high-osmolarity glycerol (SakA) pathways is essential for CPE. Here, we investigate the global proteome and phosphoproteome of A. fumigatus wild-type, Δ mpkA , and Δ sakA strains upon CPE. This study showed intense cross talk between the two MAPKs for the CPE and identified novel protein kinases and transcription factors possibly important for CPE. Increased understanding of how the modulation of protein phosphorylation may affect the fungal growth in the presence of caspofungin represents an important step in the development of new strategies and methods to combat the fungus inside the host., (Copyright © 2020 Mattos et al.)

The human-pathogenic fungus Aspergillus fumigatus is a ubiquitous saprophyte that causes fatal lung infections in immunocompromised individuals. Following inhalation, conidia are ingested by innate immune cells and can arrest phagolysosome maturation. How this virulence trait could have been selected for in natural environments is unknown. Here, we found that surface exposure of the green pigment 1,8-dihydroxynaphthalene-(DHN)-melanin can protect conidia from phagocytic uptake and intracellular killing by the fungivorous amoeba Protostelium aurantium and delays its exocytosis from the nonfungivorous species Dictyostelium discoideum To elucidate the antiphagocytic properties of the surface pigment, we followed the antagonistic interactions of A. fumigatus conidia with the amoebae in real time. For both amoebae, conidia covered with DHN-melanin were internalized at far lower rates than were seen with conidia lacking the pigment, despite high rates of initial attachment to nonkilling D. discoideum When ingested by D. discoideum , the formation of nascent phagosomes was followed by transient acidification of phagolysosomes, their subsequent neutralization, and, finally, exocytosis of the conidia. While the cycle was completed in less than 1 h for unpigmented conidia, the process was significantly prolonged for conidia covered with DHN-melanin, leading to an extended intracellular residence time. At later stages of this cellular infection, pigmented conidia induced enhanced damage to phagolysosomes and infected amoebae failed to recruit the ESCRT ( e ndosomal s orting c omplex r equired for t ransport) membrane repair machinery or the canonical autophagy pathway to defend against the pathogen, thus promoting prolonged intracellular persistence in the host cell and the establishment of a germination niche in this environmental phagocyte. IMPORTANCE Infections with Aspergillus fumigatus are usually acquired by an inhalation of spores from environmental sources. How spores of a saprophytic fungus have acquired abilities to withstand and escape the phagocytic attacks of innate immune cells is not understood. The fungal surface pigment dihydroxynaphtalene-melanin has been shown to be a crucial factor for the delay in phagosome maturation. Here, we show that this pigment also has a protective function against environmental phagocytes. Pigmented conidia escaped uptake and killing by the fungus-eating amoeba Protostelium aurantium When ingested by the nonfungivorous phagocyte Dictyostelium discoideum , the pigment attenuated the launch of cell autonomous defenses against the fungal invader, such as membrane repair and autophagy, leading to prolonged intracellular retention. Membrane damage and cytoplasmic leakage may result in an influx of nutrients and thus may further promote intracellular germination of the fungus, indicating that A. fumigatus has acquired some of the basic properties of intracellular pathogens., (Copyright © 2020 Ferling et al.)

Maintaining appropriate levels of trace elements during infection of a host is essential for microbial pathogenicity. Here we compared the uptake of 10 trace elements from 3 commonly-used laboratory media by 3 pathogens, Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, and a model yeast, Saccharomyces cerevisiae. The trace element composition of the yeasts, C. albicans, C. neoformans and S. cerevisiae, grown in rich (YPD) medium, differed primarily in P, S, Fe, Zn and Co. Speciation analysis of the intracellular fraction, which indicates the size of the organic ligands with which trace elements are complexed, showed that the ligands for S were similar in the three fungi but there were significant differences in binding partners for Fe and Zn between C. neoformans and S.cerevisiae. The profile for Cu varied across the 3 yeast species. In a comparison of C. albicans and A. fumigatus hyphae, the former showed higher Fe, Cu, Zn and Mn, while A. fumigatus contained higher P, S Ca and Mo. Washing C. albicans cells with the cell-impermeable chelator, EGTA, depleted 50-90 % of cellular Ca, suggesting that a large proportion of this cation is stored in the cell wall. Treatment with the cell wall stressor, Calcofluor White (CFW), alone had little effect on the elemental profile whilst combined Ca + CFW stress resulted in high cellular Cu and very high Ca. Together our data enhance our understanding of trace element uptake by pathogenic fungi and provide evidence for the cell wall as an important storage organelle for Ca., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Microbial secondary metabolites (SMs) have long been viewed as a significant source of novel pharmaceutical and agrochemical molecules. With the increasing availability of genomic data, numerous biosynthetic gene clusters (BGCs) have been discovered. Despite the presence of tens of thousands of BGCs that can theoretically produce extremely diverse SMs, many gene clusters remain in a silent state under axenic culture conditions. Co-culture is a promising research approach as it stimulates the expression of cryptic BGCs to produce novel metabolites and also mimics natural interspecies interactions in a laboratory environment. In recent years, the roles of SMs in microbial communication have caught the attention of researchers and our understanding of microbes and their production of remarkable SMs has improved. SMs may be extensively involved in a variety of communication events among microorganisms. We herein summarize certain representative findings in the field of chemical communication involving SMs in co-culture systems., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tuning the electrocatalytic properties of high surface area porous metallic frameworks like Nanoporous Gold (NPG) by tailoring the structure is a convenient strategy to design electrochemical sensors. Accordingly, an NPG-based sensitive, selective and robust electroanalytical platform was designed for the detection of ascorbic acid (AA) in acidic extracts of Aspergillus fumigatus fungus and Arabidopsis thaliana leaves. NPG films were electrodeposited on a gold microelectrode by potentiostatic electrodeposition and characterized by electron microscopy techniques, which confirmed the morphology and highly porous structure resembling nanowires-type pure gold fractals. The electrodeposition parameters, particularly deposition potential and time, were optimized to achieve large and selective amperometric detection of AA on the NPG modified electrodes. Faster electron transfer kinetics was manifested on the 0.3 V shift in overpotential and remarkable enhancement of the oxidation peak current as compared with bare gold electrode. Amperometric measurements were performed at 0.3 V vs. Ag/AgCl (sat. KCl) in the highly acidic electrolyte solution employed to extract ascorbate from biological samples and minimize its autoxidation. The sensitivity of conventional Au-microelectrodes was increased about one thousand-fold upon modification with NPG film, reaching 2 nA μmol -1  L -1 . The detection limit for AA based on a linear current-concentration calibration plot was found to be 2 μmol L -1 . The NPG-based microsensor was demonstrated to be selective, reproducible and stable, and was employed for determinations of AA concentration in highly acidic biological extracts., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Methodologies to identify epitopes or ligands of the fungal cell wall polysaccharides influencing the immune response of human pathogens have to date been imperfect. Using the galactomannan (GM) of Aspergillus fumigatus as a model, we have shown that synthetic oligosaccharides of distinct structures representing key fragments of cell wall polysaccharides are the most precise tools to study the serological and immunomodulatory properties of a fungal polysaccharide., (Copyright © 2020 Wong et al.)

Fumagillin is a mycotoxin produced, above all, by the saprophytic filamentous fungus Aspergillus fumigatus . This mold is an opportunistic pathogen that can cause invasive aspergillosis, a disease that has high mortality rates linked to it. Its ability to adapt to environmental stresses through the production of secondary metabolites, including several mycotoxins (gliotoxin, fumagillin, pseurotin A, etc.) also seem to play an important role in causing these infections. Since the discovery of the A. fumigatus fumagillin in 1949, many studies have focused on this toxin and in this review we gather all the information currently available. First of all, the structural characteristics of this mycotoxin and the different methods developed for its determination are given in detail. Then, the biosynthetic gene cluster and the metabolic pathway involved in its production and regulation are explained. The activity of fumagillin on its target, the methionine aminopeptidase type 2 (MetAP2) enzyme, and the effects of blocking this enzyme in the host are also described. Finally, the applications that this toxin and its derivatives have in different fields, such as the treatment of cancer and its microsporicidal activity in the treatment of honeybee hive infections with Nosema spp., are reviewed. Therefore, this work offers a complete review of all the information currently related to the fumagillin mycotoxin secreted by A. fumigatus , important because of its role in the fungal infection process but also because it has many other applications, notably in beekeeping, the treatment of infectious diseases, and in oncology., Competing Interests: The authors declare no conflicts of interest.

Purpose: Elastase, produced by Aspergillus fumigatus and A. flavus, is an important pathogenic factor in pulmonary aspergillosis. We investigated the possibility of using A. fumigatus-derived A. fumigatus elastase inhibitor (AFUEI) as a therapeutic agent. As native-AFUEI (N-AFUEI) has an extremely low yield, we generated a synthetic-AFUEI (S-AFUEI) and investigated whether S-AFUEI has a biological activity against A. fumigatus elastase (AFUE) and inhibits cytotoxicity., Methodology: A. fumigatus was cultured in Yeast Carbon Base (YCB) -elastin culture medium for 3-7 days, and AFUE was purified by chromatography using DE52 cellulose and Sephadex G-75 column. Elastolytic activity was examined using Glt-Ala-Ala-Pro-Leu-pNA (GAAPLNA) as the substrate. The hydrolytic activity of AFUE was determined using the characteristic substrates, fibrinogen and collagen (Type IV), and human cell cytotoxicity was measured colorimetrically. Furthermore, the inhibitory effect of S-AFUEI on these activities was examined., Results: We confirmed that S-AFUEI demonstrated elastase inhibitory activity and heat stability equivalent to that demonstrated by N-AFUEI, and inhibited human collagen hydrolytic activity and human fibrinogen hydrolytic activity. Further, S-AFUEI inhibited cytotoxicity in AFUE human pulmonary artery endothelial cells (HPAEC), human small airway epithelial cells (HSAEC), and human pulmonary alveolar epithelial cells (HPAEpiC)., Conclusion: As S-AFUEI strongly inhibited cytotoxicity induced by elastase in human-derived cells, it could prove beneficial for the treatment of pulmonary aspergillosis., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Complement system and dendritic cells (DCs) form - beside neutrophils and macrophages - the first line of defense to combat fungal infections. Therefore, we here studied interactions of these first immune elements with Aspergillus fumigatus lacking ß-1,3-glucans ( fks1 tetOn rep under repressed conditions) to mechanistically explain the mode of action of echinocandins in more detail. Echinocandins are cell wall active agents blocking β-glucan synthase, making the A. fumigatus fks1 tetOn mutant a good model to study immune-modulatory actions of these drugs. We now demonstrate herein, that complement was activated to significantly higher levels by the fks1- deficient strain compared to its respective wild type. This enhanced covalent linking of complement fragments to the A. fumigatus fks1 tetOn rep mutant further resulted in enhanced DC binding and internalization of the fungus. Additionally, we found that fks1 tetOn rep induced a Th1-/Th17-polarizing cytokine profile program in DCs. The effect was essentially dependent on massive galactomannan shedding, since blocking of DC-SIGN significantly reduced the fks1 tetOn rep - mediated induction of an inflammatory cytokine profile.Our data demonstrate that lack of ß-1,3-glucan, also found under echinocandin therapy, results in improved recognition of Aspergillus fumigatus by complement and DCs and therefore not only directly affects the fungus by its fungistatic actions, but also is likely to exert indirect antifungal mechanisms by strengthening innate host immune mechanisms. Abbreviations : C: complement; CR:complement receptor; DC: dendritic cell; iDC: immature dendritic cell; DC-SIGN: Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; ERK: extracellular signal-regulated kinases; JNK : c-Jun N-terminal kinases; MAPK: mitogen-activated protein kinase; NHS: normal human serum; PRR: pattern recognition receptor; Th :T helper; TLR :Toll-like receptor; WT: wild type.

This research was carried out to study the secondary metabolites of endophytic fungus Aspergillosis fumigatus from Euphorbia royleana. The endophytic fungus A. fumigatus was fermented by solid fermentation,and purified by various chromatographic methods after extraction. The structures of the compounds were identified by1 H-NMR,13 C-NMR and HSQC,HMBC spectra and physicchemical properties. Three compounds were isolated and their structures were identified as 3-( 3,4-dihydroxybenzoyl)-5-( 3,4-dihydroxyphenyl)-6-methyl-5,6-dihydro-2 H-pyran-2-one( 1),hydroxysydonic acid( 2) and 11-hydroxysydonic acid( 3). Compound 1 is a new compound.

Oxygenated polyunsaturated fatty acids (PUFAs)play an outstanding role in the physiological and pathological regulation of several biological processes. These oxygenated metabolites can be produced both enzimatically, yielding almost pure enantiomers, and non-enzymatically. The free radical-mediated non-enzymatic oxidation commonly produces racemic mixtures which are used as biomarkers of oxidative stress and tissue damage. The biological activity of oxygenated PUFAs is often associated with only one enantiomer, making it necessary of availing of lipidomics platforms allowing to disclose the role of single enantiomers in health and disease. Polysaccharide-based chiral stationary phases (CSPs) play a dominating part in this setting. As for the cellulose backbone, 4-methylbenzoate derivatives exhibit very high chiral recognition ability towards this class of compounds. Concerning the phenylcarbamate derivatives of cellulose and amylose, the tris(3,5-dimethylphenylcarbamate) variants show the best enantioresolving ability for a variety of oxygenated PUFAs. Moreover, also the amylose tris(5-chloro-2-methylphenylcarbamate)-based selector produces relevant chromatographic performances. The extreme versatility of those CSPs mostly depends on their compatibility with the most relevant elution modes: normal- and reversed-phase, as well as polar organic/ionic-mode. In this review article, a selection of enantioseparation studies of different oxygenated PUFAs is reported, with both tris(benzoates) and tris(phenylcarbamates) of cellulose and amylose., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Galactosaminogalactan (GAG) is an insoluble aminosugar polymer produced by Aspergillus fumigatus and has anti-inflammatory properties. Here, the minimum glycosidic sequences required for the induction of IL-1Ra by peripheral blood mononuclear cells (PBMCs) was investigated. Using chemical degradation of native GAG to isolate soluble oligomers, we have found that the de- N -acetylation of galactosamine residues and the size of oligomer are critical for the in vitro immune response. A minimal oligomer size of 20 galactosamine residues is required for the anti-inflammatory response but the presence of galactose residues is not necessary. In a Dextran sulfate induced colitis mouse model, a fraction of de- N -acetylated oligomers of 13 < dp < 20 rescue inflammatory damage like the native GAG polymer in an IL-1Ra dependent pathway. Our results demonstrate the therapeutic suitability of water-soluble GAG oligosaccharides in IL-1 mediated hyper-inflammatory diseases and suggest that α-1,4-galactosamine oligomers chemically synthesized could represent new anti-inflammatory glycodrugs., (Copyright © 2019 Gressler, Heddergott, N'Go, Renga, Oikonomou, Moretti, Coddeville, Gaifem, Silvestre, Romani, Latgé and Fontaine.)

The cell walls of fungi are composed of glycoproteins, chitin, and α- and β-glucans. Although there are many reports on β-glucans, α-glucan polysaccharides are not yet fully understood. This review characterizes the physicochemical properties and functions of (1→3)-α-d-glucans. Particular attention has been paid to practical application and the effect of glucans in various respects, taking into account unfavourable effects and potential use. The role of α-glucans in plant infection has been proven, and collected facts have confirmed the characteristics of Aspergillus fumigatus infection associated with the presence of glucan in fungal cell wall. Like β-glucans, there are now evidence that α-glucans can also stimulate the immune system. Moreover, α-d-glucans have the ability to induce mutanases and can thus decompose plaque.

UV and gamma irradiation mutagenesis was applied on Aspergillus fumigatus and Alternaria tenuissima in order to improve their producing ability of paclitaxel. Among the screened mutants, two stable strains (designated TXD105-GM6 and TER995-GM3) showed the maximum paclitaxel production. Paclitaxel titers of the two respective mutants were dramatically intensified to 1.22- and 1.24-fold, as compared by their respective parents. Immobilization using five different entrapment carriers of calcium alginate, agar-agar, Na-CMC, gelatin, and Arabic gum was successfully applied for production enhancement of paclitaxel by the two mutants. The immobilized cultures were superior to free-cell cultures and paclitaxel production by the immobilized mycelia was much higher than that of the immobilized spores using all the tried carriers. Moreover, calcium alginate gel beads were found the most conductive and proper entrapment carrier for maximum production of paclitaxel. The feasibility of the paclitaxel production by the immobilized mycelia as affected by incubation period, medium volume, and number of beads per flask was adopted. Under the favorable immobilization conditions, the paclitaxel titers were significantly intensified to 1.31- and 1.88-fold by the respective mutants, as compared by their free cultures. The obtained paclitaxel titers by the immobilized mycelia of the respective mutants (694.67 and 388.65 μg L -1 ) were found promising in terms of fungal production of paclitaxel. Hence, these findings indicate the future possibility to reduce the cost of producing paclitaxel and suggest application of the immobilization technique for the biotechnological production of paclitaxel at an industrial scale.

Here we report the first evaluation of isavuconazole inhibition of Aspergillus fumigatus CYP51 and thus sterol biosynthesis in the fungus. Voriconazole and isavuconazole both bound tightly to recombinant A. fumigatus CYP51 isoenzymes A and B (AfCYP51A and AfCYP51B) isolated in Escherichia coli membranes. CYP51 reconstitution assays confirmed that AfCYP51A and AfCYP51B as well as three AfCYP51A mutants known to confer azole resistance (G54W, L98H and M220K) were strongly inhibited by both triazoles. Voriconazole bound relatively weakly to purified Homo sapiens CYP51 (HsCYP51), unlike isavuconazole that bound tightly. However, isavuconazole was a relatively poor inhibitor of HsCYP51 activity, with an IC 50 value (half-maximal inhibitory concentration) of 25 µM, which was 55- to 120-fold greater than those observed for the A. fumigatus CYP51 enzymes, albeit not as poor an inhibitor of HsCYP51 as voriconazole with an IC 50 value of 112 µM. Sterol analysis of triazole-treated A. fumigatus Af293 cells confirmed that isavuconazole and voriconazole both inhibited cellular CYP51 activity with the accumulation of 14-methylated sterol substrates and depletion of ergosterol levels. Isavuconazole elicited a stronger perturbation of the sterol composition in A. fumigatus Af293 than voriconazole at 0.0125 µg/mL, indicating increased potency. However, complementation studies in Saccharomyces cerevisiae using strains containing AfCYP51A and AfCYP51B showed isavuconazole to be equally as effective at inhibiting CYP51 activity as voriconazole. These in vitro studies suggest that isavuconazole is an effective alternative to voriconazole as an antifungal agent against the target CYP51 in A. fumigatus., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It is composed mainly of polysaccharides that are synthetized by protein complexes. At the cell wall level, enzyme activities are involved in postsynthesis polysaccharide modifications such as cleavage, elongation, branching, and cross-linking. Glycosylphosphatidylinositol (GPI)-anchored proteins have been shown to participate in cell wall biosynthesis and specifically in polysaccharide remodeling. Among these proteins, the DFG family plays an essential role in controlling polar growth in yeast. In the filamentous fungus and opportunistic human pathogen Aspergillus fumigatus , the DFG gene family contains seven orthologous DFG genes among which only six are expressed under in vitro growth conditions. Deletions of single DFG genes revealed that DFG3 plays the most important morphogenetic role in this gene family. A sextuple-deletion mutant resulting from the deletion of all in vitro expressed DFG genes did not contain galactomannan in the cell wall and has severe growth defects. This study has shown that DFG members are absolutely necessary for the insertion of galactomannan into the cell wall of A. fumigatus and that the proper cell wall localization of the galactomannan is essential for correct fungal morphogenesis in A. fumigatus IMPORTANCE The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It is composed mainly of polysaccharides that are synthetized by protein complexes. Enzymes involved in postsynthesis polysaccharide modifications, such as cleavage, elongation, branching, and cross-linking, are essential for fungal life. Here, we investigated in Aspergillus fumigatus the role of the members of the Dfg family, one of the 4 GPI-anchored protein families common to yeast and molds involved in cell wall remodeling. Molecular and biochemical approaches showed that DFG members are required for filamentous growth, conidiation, and cell wall organization and are essential for the life of this fungal pathogen., (Copyright © 2019 Muszkieta et al.)

Four previously undescribed isochromanes were isolated from the fermentation broth of an endophytic fungus Aspergillus fumigatus , which was obtained from the fruiting body of Cordyceps sinensis . Their structures were elucidated through extensive spectroscopic analyses. One racemic isochromane was further purified by chiral HPLC to yield a pair of enantiomers and their absolute configurations were determined by quantum chemical ECD calculations. These isolated compounds were evaluated for cytotoxicity against two cell lines (MV4-11 and MDA-ME-231) and the result showed that compounds 1a and 2 exhibited moderate growth inhibition against MV4-11 cell line.

β-(1,3)-Glucan is one of the antigenic components of the bacterial as well as fungal cell wall. We designed microcapsules (MCs) ligated with β-(1,3)-glucan, to study its immunomodulatory effect. The MCs were obtained by interfacial polycondensation between diacyl chloride (sebacoyl chloride and terephtaloyl chloride) and diethylenetriamine in organic and aqueous phases, respectively. Planar films were first designed to optimize monomer compositions and to examine the kinetics of film formation. MCs with aqueous fluorescent core were then obtained upon controlled emulsification-polycondensation reactions using optimized monomer compositions and adding fluorescein into the aqueous phase. The selected MC-formulation was grafted with Curdlan, a linear β-(1,3)-glucan from  Agrobacterium species or branched β-(1,3)-glucan isolated from the cell wall of Aspergillus fumigatus. These β-(1,3)-glucan grafted MCs were phagocytosed by human monocyte-derived macrophages, and stimulated cytokine secretion. Moreover, the blocking of dectin-1, a β-(1,3)-glucan recognizing receptor, did not completely inhibit the phagocytosis of these β-(1,3)-glucan grafted MCs, suggesting the involvement of other receptors in the recognition and uptake of β-(1,3)-glucan. Overall, grafted MCs are a useful tool for the study of the mechanism of phagocytosis and immunomodulatory effect of the microbial polysaccharides.