Soluble Enolase 1 of Candida albicans and Aspergillus fumigatus Stimulates Human and Mouse B Cells and Monocytes.

Because of the growing numbers of immunocompromised patients, the incidence of life-threatening fungal infections caused by Candida albicans and Aspergillus fumigatus is increasing. We have recently identified enolase 1 (Eno1) from A. fumigatus as an immune evasion protein. Eno1 is a fungal moonlighting protein that mediates adhesion and invasion of human cells and also immune evasion through complement inactivation. We now show that soluble Eno1 has immunostimulatory activity. We observed that Eno1 from both C. albicans and A. fumigatus directly binds to the surface of lymphocytes, preferentially human and mouse B cells. Functionally, Eno1 upregulated CD86 expression on B cells and induced proliferation. Although the receptor for fungal Eno1 on B lymphocytes is still unknown, the comparison of B cells from wild-type and MyD88-deficient mice showed that B cell activation by Eno1 required MyD88 signaling. With respect to infection biology, we noted that mouse B cells stimulated by Eno1 secreted IgM and IgG2b. These Igs bound C. albicans hyphae in vitro, suggesting that Eno1-induced Ab secretion might contribute to protection from invasive fungal disease in vivo. Eno1 also triggered the release of proinflammatory cytokines from monocytes, particularly IL-6, which is a potent activator of B cells. Together, our data shed new light on the role of secreted Eno1 in infections with C. albicans and A. fumigatus. Eno1 secretion by these pathogenic microbes appears to be a double-edged sword by supporting fungal pathogenicity while triggering (antifungal) immunity.

Genetic validation of Aspergillus fumigatus phosphoglucomutase as a viable therapeutic target in invasive aspergillosis.

Aspergillus fumigatus is the causative agent of invasive aspergillosis, an infection with mortality rates of up to 50%. The glucan-rich cell wall of A. fumigatus is a protective structure that is absent from human cells and is a potential target for antifungal treatments. Glucan is synthesized from the donor uridine diphosphate glucose, with the conversion of glucose-6-phosphate to glucose-1-phosphate by the enzyme phosphoglucomutase (PGM) representing a key step in its biosynthesis. Here, we explore the possibility of selectively targeting A. fumigatus PGM (AfPGM) as an antifungal treatment strategy. Using a promoter replacement strategy, we constructed a conditional pgm mutant and revealed that pgm is required for A. fumigatus growth and cell wall integrity. In addition, using a fragment screen, we identified the thiol-reactive compound isothiazolone fragment of PGM as targeting a cysteine residue not conserved in the human ortholog. Furthermore, through scaffold exploration, we synthesized a para-aryl derivative (ISFP10) and demonstrated that it inhibits AfPGM with an IC50 of 2 µM and exhibits 50-fold selectivity over the human enzyme. Taken together, our data provide genetic validation of PGM as a therapeutic target and suggest new avenues for inhibiting AfPGM using covalent inhibitors that could serve as tools for chemical validation.

Crystal structure of Aspergillus fumigatus AroH, an aromatic amino acid aminotransferase.

Aspergillus fumigatus is a saprophytic ubiquitous fungus whose spores can trigger reactions such as allergic bronchopulmonary aspergillosis or the fatal invasive pulmonary aspergillosis. To survive in the lungs, the fungus must adapt to a hypoxic and nutritionally restrictive environment, exploiting the limited availability of aromatic amino acids (AAAs) in the best possible way, as mammals do not synthesize them. A key enzyme for AAAs catabolism in A. fumigatus is AroH, a pyridoxal 5'-phosphate-dependent aromatic aminotransferase. AroH was recently shown to display a broad substrate specificity, accepting L-kynurenine and α-aminoadipate as amino donors besides AAAs. Given its pivotal role in the adaptability of the fungus to nutrient conditions, AroH represents a potential target for the development of innovative therapies against A. fumigatus-related diseases. We have solved the crystal structure of Af-AroH at 2.4 Å resolution and gained new insight into the dynamics of the enzyme's active site, which appears to be crucial for the design of inhibitors. The conformational plasticity of the active site pocket is probably linked to the wide substrate specificity of AroH.

Host Lung Environment Limits Aspergillus fumigatus Germination through an SskA-Dependent Signaling Response.

Aspergillus fumigatus isolates display significant heterogeneity in growth, virulence, pathology, and inflammatory potential in multiple murine models of invasive aspergillosis. Previous studies have linked the initial germination of a fungal isolate in the airways to the inflammatory and pathological potential, but the mechanism(s) regulating A. fumigatus germination in the airways is unresolved. To explore the genetic basis for divergent germination phenotypes, we utilized a serial passaging strategy in which we cultured a slow germinating strain (AF293) in a murine-lung-based medium for multiple generations. Through this serial passaging approach, a strain emerged with an increased germination rate that induces more inflammation than the parental strain (herein named LH-EVOL for lung homogenate evolved). We identified a potential loss-of-function allele of Afu5g08390 (sskA) in the LH-EVOL strain. The LH-EVOL strain had a decreased ability to induce the SakA-dependent stress pathway, similar to AF293 ΔsskA and CEA10. In support of the whole-genome variant analyses, sskA, sakA, or mpkC loss-of-function strains in the AF293 parental strain increased germination both in vitro and in vivo. Since the airway surface liquid of the lungs contains low glucose levels, the relationship of low glucose concentration on germination of these mutant AF293 strains was examined; interestingly, in low glucose conditions, the sakA pathway mutants exhibited an enhanced germination rate. In conclusion, A. fumigatus germination in the airways is regulated by SskA through the SakA mitogen-activated protein kinase (MAPK) pathway and drives enhanced disease initiation and inflammation in the lungs. IMPORTANCE Aspergillus fumigatus is an important human fungal pathogen particularly in immunocompromised individuals. Initiation of growth by A. fumigatus in the lung is important for its pathogenicity in murine models. However, our understanding of what regulates fungal germination in the lung environment is lacking. Through a serial passage experiment using lung-based medium, we identified a new strain of A. fumigatus that has increased germination potential and inflammation in the lungs. Using this serially passaged strain, we found it had a decreased ability to mediate signaling through the osmotic stress response pathway. This finding was confirmed using genetic null mutants demonstrating that the osmotic stress response pathway is critical for regulating growth in the murine lungs. Our results contribute to the understanding of A. fumigatus adaptation and growth in the host lung environment.

Discovery of Octahydroisoindolone as a Scaffold for the Selective Inhibition of Chitinase B1 from Aspergillus fumigatus: In Silico Drug Design Studies.

Chitinases represent an alternative therapeutic target for opportunistic invasive mycosis since they are necessary for fungal cell wall remodeling. This study presents the design of new chitinase inhibitors from a known hydrolysis intermediate. Firstly, a bioinformatic analysis of Aspergillus fumigatus chitinase B1 (AfChiB1) and chitotriosidase (CHIT1) by length and conservation was done to obtain consensus sequences, and molecular homology models of fungi and human chitinases were built to determine their structural differences. We explored the octahydroisoindolone scaffold as a potential new antifungal series by means of its structural and electronic features. Therefore, we evaluated several synthesis-safe octahydroisoindolone derivatives by molecular docking and evaluated their AfChiB1 interaction profile. Additionally, compounds with the best interaction profile (1-5) were docked within the CHIT1 catalytic site to evaluate their selectivity over AfChiB1. Furthermore, we considered the interaction energy (MolDock score) and a lipophilic parameter (aLogP) for the selection of the best candidates. Based on these descriptors, we constructed a mathematical model for the IC50 prediction of our candidates (60-200 µM), using experimental known inhibitors of AfChiB1. As a final step, ADME characteristics were obtained for all the candidates, showing that 5 is our best designed hit, which possesses the best pharmacodynamic and pharmacokinetic character.

Characterization of a lytic polysaccharide monooxygenase from Aspergillus fumigatus shows functional variation among family AA11 fungal LPMOs.

The discovery of oxidative cleavage of recalcitrant polysaccharides by lytic polysaccharide monooxygenases (LPMOs) has affected the study and industrial application of enzymatic biomass processing. Despite being widespread in fungi, LPMOs belonging to the auxiliary activity (AA) family AA11 have been understudied. While these LPMOs are considered chitin active, some family members have little or no activity toward chitin, and the only available crystal structure of an AA11 LPMO lacks features found in bacterial chitin-active AA10 LPMOs. Here, we report structural and functional characteristics of a single-domain AA11 LPMO from Aspergillus fumigatus, AfAA11A. The crystal structure shows a substrate-binding surface with features resembling those of known chitin-active LPMOs. Indeed, despite the absence of a carbohydrate-binding module, AfAA11A has considerable affinity for α-chitin and, more so, ß-chitin. AfAA11A is active toward both these chitin allomorphs and enhances chitin degradation by an endoacting chitinase, in particular for α-chitin. The catalytic activity of AfAA11A on chitin increases when supplying reactions with hydrogen peroxide, showing that, like LPMOs from other families, AfAA11A has peroxygenase activity. These results show that, in stark contrast to the previously characterized AfAA11B from the same organism, AfAA11A likely plays a role in fungal chitin turnover. Thus, members of the hitherto rather enigmatic family of AA11 LPMOs show considerable structural and functional differences and may have multiple roles in fungal physiology.

Purification and characterization of ß-galactosidase from Aspergillus fumigatus PCSIR-2013.

Extra cellular ß-galactosidase enzyme was purified and characterized from Aspergillus fumigatus PCSIR- 2013. Estimated molecular mass of the enzyme was approximately 95 kDa. by native polyacrylamide gel electrophoresis. Initially, different fermentation parameters were optimized for maximum production of ß-galactosidase. The kinetic study of the partially purified enzyme exhibited that it remained active in broad range of temperature from 25°C to 70°C with an optimum of 60°C. The Km and Vmax were calculated as 9.95mmol/l and 51.78 U/ml/min, respectively. The optimum pH was 5.0, when reaction mixture was incubated for 30 min. The enzyme was very stable in the presence of different metal ions, although Na+ (16%) stimulates the activity at 10mM concentration. In contrast, Ba+2 and Hg+2 have negative effect on enzyme activity and activity decreased to 54% and 19%, respectively. Thermo stability study was revealed that the enzyme retained 72% of its activity at 50°C. Whereas, when enzyme was incubated at 60°C for 120 min, its residual activity was decreased to 42.0%. However, the enzyme was completely inactivated at 80°C after 120 min of pre-incubation. Among different surfactant which incorporated with enzyme, Tween 20 and Triton X-100 both have stimulatory effect and activity increased to 29% and 17%, respectively.

Pleiotropic Effects of the P5-Type ATPase SpfA on Stress Response Networks Contribute to Virulence in the Pathogenic Mold Aspergillus fumigatus.

Aspergillus fumigatus is a human-pathogenic mold that extracts nutrients from the environment or from host tissues by secreting hydrolytic enzymes. The ability of A. fumigatus to adjust secretion levels in proportion to demand relies on the assistance of the unfolded protein response (UPR), an adaptive stress response pathway that regulates the unique protein-folding environment of the endoplasmic reticulum (ER). The P5-type ATPase Spf1 has recently been implicated in a novel mechanism of ER homeostasis that involves correcting errors in ER-membrane protein targeting. However, the contribution of this protein to the biology of A. fumigatus is unknown. Here, we employed a gene knockout and RNA sequencing strategy to determine the functional role of the A. fumigatus gene coding for the orthologous P5 ATPase SpfA. The data reveal that the spfA gene is induced by ER stress in a UPR-dependent manner. In the absence of spfA, the A. fumigatus transcriptome shifts toward a profile of altered redox and lipid balance, in addition to a signature of ER stress that includes srcA, encoding a second P-type ATPase in the ER. A ΔspfA deletion mutant showed increased sensitivity to ER stress, oxidative stress, and antifungal drugs that target the cell wall or plasma membrane. The combined loss of spfA and srcA exacerbated these phenotypes and attenuated virulence in two animal infection models. These findings demonstrate that the ER-resident ATPases SpfA and SrcA act jointly to support diverse adaptive functions of the ER that are necessary for fitness in the host environment. IMPORTANCE The fungal UPR is an adaptive signaling pathway in the ER that buffers fluctuations in ER stress but also serves as a virulence regulatory hub in species of pathogenic fungi that rely on secretory pathway homeostasis for pathogenicity. This study demonstrates that the gene encoding the ER-localized P5-type ATPase SpfA is a downstream target of the UPR in the pathogenic mold A. fumigatus and that it works together with a second ER-localized P-type ATPase, SrcA, to support ER homeostasis, oxidative stress resistance, susceptibility to antifungal drugs, and virulence of A. fumigatus.

Triazolopyrimidine herbicides are potent inhibitors of Aspergillus fumigatus acetohydroxyacid synthase and potential antifungal drug leads.

Aspergillus fumigatus is a fungal pathogen whose effects can be debilitating and potentially fatal in immunocompromised patients. Current drug treatment options for this infectious disease are limited to just a few choices (e.g. voriconazole and amphotericin B) and these themselves have limitations due to potentially adverse side effects. Furthermore, the likelihood of the development of resistance to these current drugs is ever present. Thus, new treatment options are needed for this infection. A new potential antifungal drug target is acetohydroxyacid synthase (AHAS; EC 2.2.1.6), the first enzyme in the branched chain amino acid biosynthesis pathway, and a target for many commercial herbicides. In this study, we have expressed, purified and characterised the catalytic subunit of AHAS from A. fumigatus and determined the inhibition constants for several known herbicides. The most potent of these, penoxsulam and metosulam, have Ki values of 1.8 ± 0.9 nM and 1.4 ± 0.2 nM, respectively. Molecular modelling shows that these compounds are likely to bind into the herbicide binding pocket in a mode similar to Candida albicans AHAS. We have also shown that these two compounds inhibit A. fumigatus growth at a concentration of 25 µg/mL. Thus, AHAS inhibitors are promising leads for the development of new anti-aspergillosis therapeutics.

The OxrA Protein of Aspergillus fumigatus Is Required for the Oxidative Stress Response and Fungal Pathogenesis.

An efficient reactive oxygen species (ROS) detoxification system is vital for the survival of the pathogenic fungus Aspergillus fumigatus within the host high-ROS environment of the host. Therefore, identifying and targeting factors essential for oxidative stress response is one approach to developing novel treatments for fungal infections. The oxidation resistance 1 (Oxr1) protein is essential for protection against oxidative stress in mammals, but its functions in pathogenic fungi remain unknown. The present study aimed to characterize the role of an Oxr1 homolog in A. fumigatus. The results indicated that the OxrA protein plays an important role in oxidative stress resistance by regulating the catalase function in A. fumigatus, and overexpression of catalase can rescue the phenotype associated with OxrA deficiency. Importantly, the deficiency of oxrA decreased the virulence of A. fumigatus and altered the host immune response. Using the Aspergillus-induced lung infection model, we demonstrated that the ΔoxrA mutant strain induced less tissue damage along with decreased levels of lactate dehydrogenase (LDH) and albumin release. Additionally, the ΔoxrA mutant caused inflammation at a lower degree, along with a markedly reduced influx of neutrophils to the lungs and a decreased secretion of cytokine usually associated with recruitment of neutrophils in mice. These results characterize the role of OxrA in A. fumigatus as a core regulator of oxidative stress resistance and fungal pathogenesis. IMPORTANCE Knowledge of ROS detoxification in fungal pathogens is useful in the design of new antifungal drugs and could aid in the study of oxidative stress resistance mechanisms. In this study, we demonstrate that OxrA protein localizes to the mitochondria and functions to protect against oxidative damage. We demonstrate that OxrA contributes to oxidative stress resistance by regulating catalase function, and overexpression of catalase (CatA or CatB) can rescue the phenotype that is associated with OxrA deficiency. Remarkably, a loss of OxrA attenuated the fungal virulence in a mouse model of invasive pulmonary aspergillosis and altered the host immune response. Therefore, our finding indicates that inhibition of OxrA might be an effective approach for alleviating A. fumigatus infection. The present study is, to the best of our knowledge, a pioneer in reporting the vital role of Oxr1 protein in pathogenic fungi.

Engineering of a chitosanase fused to a carbohydrate-binding module for continuous production of desirable chitooligosaccharides.

Chitooligosaccharides (CHOS) with multiple biological activities are usually produced through enzymatic hydrolysis of chitosan or chitin. However, purification and recycling of the enzyme have largely limited the advancement of CHOS bioproduction. Here, we engineered a novel enzyme by fusing the native chitosanase Csn75 with a carbohydrate-binding module (CBM) that can specifically bind to curdlan. The recombinase Csn75-CBM was successfully expressed by Pichia pastoris and allowed one-step purification and immobilization in the chitosanase immobilized curdlan packed-bed reactor (CICPR), where a maximum adsorption capacity of 39.59 mg enzyme/g curdlan was achieved. CHOS with degrees of polymerization of 2-5 (a hydrolysis yield of 97.75%), 3-6 (75.45%), and 3-7 (73.2%) were continuously produced by adjusting the ratio of enzyme and chitosan or the flow rate of chitosan. Moreover, the CICPR exhibited good stability and reusability after several cycles. The recombinase Csn75-CBM has greatly improved the efficiency of the bioproduction of CHOS.

Loss of Septation Initiation Network (SIN) kinases blocks tissue invasion and unlocks echinocandin cidal activity against Aspergillus fumigatus.

Although considered effective treatment for many yeast fungi, the therapeutic efficacy of the echinocandin class of antifungals for invasive aspergillosis (IA) is limited. Recent studies suggest intense kinase- and phosphatase-mediated echinocandin adaptation in A. fumigatus. To identify A. fumigatus protein kinases required for survival under echinocandin stress, we employed CRISPR/Cas9-mediated gene targeting to generate a protein kinase disruption mutant library in a wild type genetic background. Cell wall and echinocandin stress screening of the 118 disruption mutants comprising the library identified only five protein kinase disruption mutants displaying greater than 4-fold decreased echinocandin minimum effective concentrations (MEC) compared to the parental strain. Two of these mutated genes, the previously uncharacterized A. fumigatus sepL and sidB genes, were predicted to encode protein kinases functioning as core components of the Septation Initiation Network (SIN), a tripartite kinase cascade that is necessary for septation in fungi. As the A. fumigatus SIN is completely uncharacterized, we sought to explore these network components as effectors of echinocandin stress survival. Our data show that mutation of any single SIN kinase gene caused complete loss of hyphal septation and increased susceptibility to cell wall stress, as well as widespread hyphal damage and loss of viability in response to echinocandin stress. Strikingly, mutation of each SIN kinase gene also resulted in a profound loss of virulence characterized by lack of tissue invasive growth. Through the deletion of multiple novel regulators of hyphal septation, we show that the non-invasive growth phenotype is not SIN-kinase dependent, but likely due to hyphal septation deficiency. Finally, we also find that echinocandin therapy is highly effective at eliminating residual tissue burden in mice infected with an aseptate strain of A. fumigatus. Together, our findings suggest that inhibitors of septation could enhance echinocandin-mediated killing while simultaneously limiting the invasive potential of A. fumigatus hyphae.

A Unique Dual-Readout High-Throughput Screening Assay To Identify Antifungal Compounds with Aspergillus fumigatus.

Treatment of invasive mold infections is limited by the lack of adequate drug options that are effective against these fatal infections. High-throughput screening of molds using traditional antifungal assays of growth is problematic and has greatly limited our ability to identify new mold-active agents. Here, we present a high-throughput screening platform for use with Aspergillus fumigatus, the most common causative agent of invasive mold infections, for the discovery of novel mold-active antifungals. This assay detects cell lysis through the release of the cytosolic enzyme adenylate kinase and, thus, is not dependent on changes in biomass or metabolism to detect antifungal activity. The ability to specifically detect cell lysis is a unique aspect of this assay that allows identification of molecules that disrupt fungal cell integrity, such as cell wall-active molecules. We also found that germinating A. fumigatus conidia release low levels of adenylate kinase and that a reduction in this background allowed us to identify molecules that inhibit conidial germination, expanding the potential for discovery of novel antifungal compounds. Here, we describe the validation of this assay and proof-of-concept pilot screens that identified a novel antifungal compound, PIK-75, that disrupts cell wall integrity. This screening assay provides a novel platform for high-throughput screens with A. fumigatus for the identification of anti-mold drugs. IMPORTANCE Fungal infections caused by molds have the highest mortality rates of human fungal infections. These devastating infections are hard to treat and available antifungal drugs are often not effective. Therefore, the identification of new antifungal drugs with mold activity is critical. Drug screening with molds is challenging and there are limited assays available to identify new antifungal compounds directly with these organisms. Here, we present an assay suitable for use for high-throughput screening with a common mold pathogen. This assay has exciting future potential for the identification of new drugs to treat these fatal infections.

Fungal and host protein persulfidation are functionally correlated and modulate both virulence and antifungal response.

Aspergillus fumigatus is a human fungal pathogen that can cause devastating pulmonary infections, termed "aspergilloses," in individuals suffering immune imbalances or underlying lung conditions. As rapid adaptation to stress is crucial for the outcome of the host-pathogen interplay, here we investigated the role of the versatile posttranslational modification (PTM) persulfidation for both fungal virulence and antifungal host defense. We show that an A. fumigatus mutant with low persulfidation levels is more susceptible to host-mediated killing and displays reduced virulence in murine models of infection. Additionally, we found that a single nucleotide polymorphism (SNP) in the human gene encoding cystathionine γ-lyase (CTH) causes a reduction in cellular persulfidation and correlates with a predisposition of hematopoietic stem cell transplant recipients to invasive pulmonary aspergillosis (IPA), as correct levels of persulfidation are required for optimal antifungal activity of recipients' lung resident host cells. Importantly, the levels of host persulfidation determine the levels of fungal persulfidation, ultimately reflecting a host-pathogen functional correlation and highlighting a potential new therapeutic target for the treatment of aspergillosis.

The Copper Chaperone CcsA, Coupled with Superoxide Dismutase SodA, Mediates the Oxidative Stress Response in Aspergillus fumigatus.

Superoxide dismutases (SODs) are important metalloenzymes that protect fungal pathogens against the toxic effects of reactive oxygen species (ROS) generated by host defense mechanisms during the infection process. The activation of Cu/Zn-SOD1 is found to be dependent on copper chaperone for SOD1 (Ccs1). However, the role of the Ccs1 ortholog in the human pathogen Aspergillus fumigatus and how these SODs coordinate to mediate oxidative stress response remain elusive. Here, we demonstrated that A. fumigatus CcsA, a Saccharomyces cerevisiae Ccs1 ortholog, is required for cells in response to oxidative response and the activation of Sod1. Deletion of ccsA resulted in increased ROS accumulation and enhanced sensitivity to oxidative stress due to the loss of SodA activity. Molecular characterization of CcsA revealed that the conserved CXC motif is required not only for the physical interaction with SodA but also for the oxidative stress adaption. Notably, addition of Mn2+ or overexpression of cytoplasmic Mn-SodC could rescue the defects of the ccsA or sodA deletion mutant, indicating the important role of Mn2+ and Mn-SodC in ROS detoxification; however, deletion of the CcsA-SodA complex could not affect A. fumigatus virulence. Collectively, our findings demonstrate that CcsA functions as a Cu/Zn-Sod1 chaperone that participates in the adaptation to oxidative stress in A. fumigatus and provide a better understanding of the CcsA-SodA complex-mediated oxidative stress response in filamentous fungi. IMPORTANCE Reactive oxygen species (ROS) produced by phagocytes have been reported to participate in the killing of fungal pathogens. Superoxide dismutases (SODs) are considered to be the first line of defense against superoxide anions. Characterizing the regulatory mechanisms of SOD activation is important for understanding how fungi adapt to oxidative stress in hosts. Our findings demonstrated that CcsA functions as a SodA chaperone in A. fumigatus and that the conserved CXC motif within CcsA is required for its interaction with SodA and the CcsA-SodA-mediated oxidative response. These data may provide new insights into how fungal pathogens adapt to oxidative stress via the CcsA-SodA complex.

Exo-inulinase production from Aspergillus fumigatus NFCCI 2426: purification, characterization, and immobilization for continuous fructose production.

Aspergillus fumigatus was found to produce thermostable exo-inulinase (EC 3.8.1.80; 38 U/ml) on inulin-rich infusions. Exo-inulinase (14.6 U/mg) was immobilized on glutaraldehyde activated Ca-alginate beads for continuous generation of fructose by hydrolyzing sucrose, chicory, and dandelion substrates. Immobilization of enzyme was confirmed by microscopic and spectroscopic techniques. The exo-inulinase was purified using ion-exchange (1.30-folds) and size-exclusion chromatography (2.71-folds). The purified exo-inulinase showed 64 kDa band on gel and was optimally active at 60 °C and pH 6.0. Kinetic constants, Km and Vmax of purified exo-inulinase, were 5.88 mM and 1.66 µM/min, respectively, and its relative activity was found to be enhanced (125.8%) in the presence of calcium ion. Immobilized preparation was utilized for continuous generation of fructose from chicory juice (26 to 70%) and dandelion root extracts (16 to 24%) by recycling upto five cycles, respectively. In comparison to other sweeteners, such as sucrose, fructose is considered as a healthy alternative. The present study demonstrated the use of immobilized exo-inulinase in continuous generation of fructose from some underutilized plant sources that can be used in food industry. PRACTICAL APPLICATION: Thermostable exo-inulinase produced by A. fumigatus was immobilized on calcium alginate matrix and was employed for continuous hydrolysis of chicory juice and dandelion root extract for generation of fructose syrup.

Discovery of fungal surface NADases predominantly present in pathogenic species.

Nicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host's NAD+ pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca2+-binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms.

Computational approach for identification, characterization, three-dimensional structure modelling and machine learning-based thermostability prediction of xylanases from the genome of Aspergillus fumigatus.

Identification of thermostable and alkaline xylanases from different fungal and bacterial species have gained an interest for the researchers because of its biotechnological relevance in many industries, such as pulp, paper, and bioethanol. In this study, we have identified and characterized xylanases from the genome of the thermophilic fungus of Aspergillus fumigatus by in silico analysis. Genome data mining revealed that the A fumigatus genome has six xylanase genes that belong to GH10, GH11, GH43 glycoside hydrolase families. In general, most of the bacterial and fungal GH11 xylanases are alkaline, and GH10 xylanases are acidic; however, we found that one identified xylanase from A fumigatus that belongs to the GH10 family is alkaline while the rest are acidic. Moreover, physicochemical properties also stated that most of the xylanases identified have lower molecular weight except one that belongs to the GH43 family. Structure prediction by homology modelling gave optimized structures of the xylanases. It suggests that GH10 family structure models adapt (ß∕α) 8 barrel type, GH11 homology models adapt ß-jelly type, and the GH43 family has a fivefold ß-propeller type structure. Molecular docking of identified xylanases with xylan revealed that GH11 xylanases have strong interaction (-9.6 kcal/mol) with xylan than the GH10 (-8.5 and -9.3 kcal/mol) and GH43 (-8.8 kcal/mol). We used the machine learning approach based TAXyl server to predict the thermostability of the xylanases. It revealed that two GH10 xylanases and one GH11 xylanase are thermo-active up to 75ᵒC. We have explored the physiochemical properties responsible for maintaining thermostability for bacterial and fungal GH10 and GH11 xylanases by comparing crystal structures. All the analyzed parameters specified that GH10 xylanases from both the fungi and bacteria are more thermostable due to higher hydrogen bonds, salt bridges, and helical content.

Integrated proteomics and metabolomics analysis of tea leaves fermented by Aspergillus niger, Aspergillus tamarii and Aspergillus fumigatus.

Post-fermented Pu-erh tea (PFPT) is a microbially-fermented tea with distinct sensory qualities and multiple health benefits. Aspergillus are the dominant fungi in the fermentation and the main contributors to the characteristics of PFPT, so their underlying functions warrant detailed study. Here, tea leaves were fermented by Aspergillus niger, Aspergillus tamarii and Aspergillus fumigatus, and resulting samples (designated as Asn, Ast and Asf, respectively) were analyzed by proteomic and metabolomic methods. Changes to the composition of flavonoids, glycerophospholipids, organo-oxygen compounds and fatty acids resulting from Aspergillus fermentation were observed. Carbohydrate-active enzymes, e.g., endoglucanases and cellulases, for degradation of cellulose, starch, lignin, pectin, xylan and xyloglucan were identified. Glycoside hydrolase, glycosyltransferases, tannase, laccases, vanillyl-alcohol oxidases and benzoquinone reductase were identified and hypothesized to catalyze hydrolysis, oxidation, polymerization and degradation of phenolic compounds. Together, functions of Aspergillius were demonstrated as production of enzymes to change concentrations and compositions of metabolites in tea leaves.

Identification and characterization of ß-d-galactofuranosidases from Aspergillus nidulans and Aspergillus fumigatus.

Although ß-d-galactofuranosidases (Galf-ases) that hydrolyze ß-d-galactofuranose (Galf)-containing oligosaccharides have been characterized in various organisms, to date no Galf-specific Galf-ase-encoding genes have been reported in Aspergillus fungi. Based on the amino acid sequences of previously identified bacterial Galf-ases, here we found two candidate Galf-specific Galf-ase genes AN2395 (gfgA) and AN3200 (gfgB) in the genome of Aspergillus nidulans. Indeed, recombinant GfgA and GfgB proteins exhibited Galf-specific Galf-ase activity, but no detectable α-l-arabinofuranosidase (Araf-ase) activity. Phylogenetic analysis of GfgA and GfgB orthologs indicated that there are two types of Aspergillus species: those containing one ortholog each for GfgA and GfgB; and those containing only one ortholog in total, among which Aspergillus fumigatus there is a representative with a single ortholog Galf-ase Afu2g14520. Unlike GfgA and GfgB, the recombinant Afu2g14520 protein showed higher Araf-ase activity than Galf-ase activity. An assay of substrate specificity revealed that although GfgA and GfgB are both exo-type Galf-ases and hydrolyze ß-(1,5) and ß-(1,6) linkages, GfgA hydrolyzes ß-(1,6)-linked Galf-oligosaccharide more effectively as compared with GfgB. Collectively, our findings indicate that Galf-ases in Aspergillus species may have a role in cooperatively degrading Galf-containing oligosaccharides depending on environmental conditions.