The S-S bridge mutation between the A2 and A4 loops (T416C-I432C) of Cel7A of Aspergillus fumigatus enhances catalytic activity and thermostability.

Disulfide bonds are important for maintaining the structural conformation and stability of the protein. The introduction of the disulfide bond is a promising strategy to increase the thermostability of the protein. In this report, cysteine residues are introduced to form disulfide bonds in the Glycoside Hydrolase family GH 7 cellobiohydrolase (GH7 CBHs) or Cel7A of Aspergillus fumigatus. Disulfide by Design 2.0 (DbD2), an online tool is used for the detection of the mutation sites. Mutations are created (D276C-G279C; DSB1, D322C-G327C; DSB2, T416C-I432C; DSB3, G460C-S465C; DSB4) inside and outside of the peripheral loops but, not in the catalytic region. The introduction of cysteine in the A2 and A4 loop of DSB3 mutant showed higher thermostability (70% activity at 70°C), higher substrate affinity (Km = 0.081 mM) and higher catalytic activity (Kcat = 9.75 min-1; Kcat/Km = 120.37 mM min-1) compared to wild-type AfCel7A (50% activity at 70°C; Km = 0.128 mM; Kcat = 4.833 min-1; Kcat/Km = 37.75 mM min-1). The other three mutants with high B factor showed loss of thermostability and catalytic activity. Molecular dynamic simulations revealed that the mutation T416C-I432C makes the tunnel wider (DSB3: 13.6 Å; Wt: 5.3 Å) at the product exit site, giving flexibility in the entrance region or mobility of the substrate in the exit region. It may facilitate substrate entry into the catalytic tunnel and release the product faster than the wild type, whereas in other mutants, the tunnel is not prominent (DSB4), the exit is lost (DSB1), and the ligand binding site is absent (DSB2). This is the first report of the gain of function of both thermostability and enzyme activity of cellobiohydrolase Cel7A by disulfide bond engineering in the loop.IMPORTANCEBioethanol is one of the cleanest renewable energy and alternatives to fossil fuels. Cost efficient bioethanol production can be achieved through simultaneous saccharification and co-fermentation that needs active polysaccharide degrading enzymes. Cellulase enzyme complex is a crucial enzyme for second-generation bioethanol production from lignocellulosic biomass. Cellobiohydrolase (Cel7A) is an important member of this complex. In this work, we engineered (disulfide bond engineering) the Cel7A to increase its thermostability and catalytic activity which is required for its industrial application.

Histone acetyltransferase Sas3 contributes to fungal development, cell wall integrity, and virulence in Aspergillus fumigatus.

Histone acetyltransferase (HAT)-mediated epigenetic modification is essential for diverse cellular processes in eukaryotes. However, the functions of HATs in the human pathogen Aspergillus fumigatus remain poorly understood. In this study, we characterized the functions of MOZ, Ybf2/Sas3, Sas2, and Tip60 (MYST)-family histone acetyltransferase something about silencing (Sas3) in A. fumigatus. Phenotypic analysis revealed that loss of Sas3 results in significant impairments in colony growth, conidiation, and virulence in the Galleria mellonella model. Subcellular localization and Western blot analysis demonstrated that Sas3 localizes to nuclei and is capable of acetylating lysine 9 and 14 of histone H3 in vivo. Importantly, we found that Sas3 is critical for the cell wall integrity (CWI) pathway in A. fumigatus as evidenced by hypersensitivity to cell wall-perturbing agents, altered cell wall thickness, and abnormal phosphorylation levels of CWI protein kinase MpkA. Furthermore, site-directed mutagenesis studies revealed that the conserved glycine residues G641 and G643 and glutamate residue E664 are crucial for the acetylation activity of Sas3. Unexpectedly, only triple mutations of Sas3 (G641A/G643A/E664A) displayed defective phenotypes similar to the Δsas3 mutant, while double or single mutations did not. This result implies that the role of Sas3 may extend beyond histone acetylation. Collectively, our findings demonstrate that MYST-family HAT Sas3 plays an important role in the fungal development, virulence, and cell wall integrity in A. fumigatus. IMPORTANCE: Epigenetic modification governed by HATs is indispensable for various cellular processes in eukaryotes. Nonetheless, the precise functions of HATs in the human pathogen Aspergillus fumigatus remain elusive. In this study, we unveil the roles of MYST-family HAT Sas3 in colony growth, conidiation, virulence, and cell wall stress response in A. fumigatus. Particularly, our findings demonstrate that Sas3 can function through mechanisms unrelated to histone acetylation, as evidenced by site-directed mutagenesis experiments. Overall, this study broadens our understanding of the regulatory mechanism of HATs in fungal pathogens.

Alleviating the drought stress and improving the plant resistance properties of Triticum aestivum via biopriming with aspergillus fumigatus.

BACKGROUND: Wheat (Triticum aestivum L.) is one of the most widely grown and vital cereal crops, containing a high percentage of basic nutrients such as carbohydrates and proteins. Drought stress is one of the most significant limitations on wheat productivity. Due to climate change influences plant development and growth, physiological processes, grain quality, and yield. Drought stress has elicited a wide range of plant responses, namely physiological and molecular adaptations. Biopriming is one of the recent attempts to combat drought stress. Mitigating the harmful impact of abiotic stresses on crops by deploying extreme-habitat-adapted symbiotic microbes. The purpose of this study was to see how biopriming Triticum aestivum grains affected the effects of inoculating endophytic fungi Aspergillus fumigatus ON307213 isolated from stressed wheat plants in four model agricultural plants (Gemmiza-7, Sids-1, Sakha8, and Giza 168). And its viability in reducing drought stress through the use of phenotypic parameters such as root and shoot fresh and dry weight, shoot and root length, and so on. On a biochemical and physiological level, enzymatic parameters such as catalase and superoxidase dismutase are used. Total phenolics, flavonoids, and photosynthetic pigments are non-enzymatic parameters. Making use of molecular techniques such as reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: It has been found that using Aspergillus fumigatus as a biological biopriming tool can positively impact wheat plants experiencing drought stress. The total biomass of stressed wheat plants that had been bio-primed rose by more than 40% as compared to wheat plants that had not been bio-primed. A. fumigatus biopriming either increased or decreased the amount of enzymatic and non-enzymatic substances on biochemical scales, aside from the noticeable increase in photosynthetic pigment that occurs in plants that have been bio-primed and stressed. Drought-resistant genes show a biopriming influence in gene expression. CONCLUSIONS: This is the first paper to describe the practicality of a. fumigatus biopriming and its effect on minimizing the degrading effects of drought through water limitation. It suggests the potential applications of arid habitat-adapted endophytes in agricultural systems.

Aspergillus fumigatus ZrfC Zn(II) transporter scavengers zincophore-bound Zn(II).

Aspergillus fumigatus is an opportunistic pathogen that is able to invade and grow in the lungs of immunosuppressed patients and cause invasive pulmonary aspergillosis. The concentration of free Zn(II) in living tissues is much lower than that required for optimal fungal growth; thus, to obtain Zn(II) from the host, Aspergillus fumigatus uses highly specified Zn(II) transporters: ZrfA, ZrfB and ZrfC. The ZrfC transporter plays the main role in Zn(II) acquisition from the host in neutral and mildly alkaline environment via interacting with the secreted Aspf2 zincophore. Understanding the Aspf2-ZrfC interactions is therefore necessary for explaining the process of Zn(II) acquisition by Aspergillus fumigatus, and identifying Zn(II) binding sites in its transporter and describing the thermodynamics of such binding are the fundamental steps to achieve this goal. We focus on two probable ZrfC Zn(II) binding sites and show that the Ac-MNCHFHAGVEHCIGAGESESGSSQ-NH2 region binds Zn(II) with higher affinity than the Ac-TGCHSHGS-NH2 one and that this binding is much stronger than the binding of Zn(II) to the Aspf2 zincophore, allowing efficient Zn(II) transport from the Aspf2 zincophore to the ZrfC transporter. The same ZrfC fragments also able to bind Ni(II), another metal ion essential for fungi that could also compete with Zn(II) binding, with comparable affinity.

Aspergillus fumigatus mitogen-activated protein kinase MpkA is involved in gliotoxin production and self-protection.

Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. The Mitogen-Activated Protein kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. The sensor histidine kinase SlnASln1 is important for modulation of MpkA phosphorylation. Our work emphasizes the importance of MpkA and compartmentalization of cellular events for GT production and self-defense.

Functional, transcriptomic, and lipidomic studies of the choC gene encoding a phospholipid methyltransferase in Aspergillus fumigatus.

IMPORTANCE: This study explored the phospholipid metabolic pathway in A. fumigatus and its relationship with fungal growth, metabolism, and pathogenicity. ChoC, based on its critical roles in many aspects of the fungus and relatively conserved characteristics in filamentous fungi with low similarity with mammalian ones, can be a novel target of new antifungal drugs.

Discovery of antifungal secondary metabolites from an intestinal fungus Fusarium sp.

Intestinal fungi, which are important parts of the gut microbiota, have the ability to produce specialized metabolites that significantly contribute to maintaining the balance of the gut microbiota and promoting the health of the host organism. In the present study, two new glycosides, including fusintespyrone A (1) and cerevisterolside A (4), as well as ten known compounds were isolated from the intestinal fungus Fusarium sp. LE06. The structures of the new compounds were elucidated by a combination of spectroscopic methods, such as mass spectrometry (MS) and nuclear magnetic resonance (NMR), along with chemical reactions and calculations of NMR and ECD spectra. Compounds 1-3 showed significant growth inhibition against Aspergillus fumigatus, Fusarium oxysporum, and Verticillium dahliae with MIC values in the range of 1.56-6.25 µg ml-1.

Subinhibitory effects of 2,4-diacetylphloroglucinol on filamentous fungus Aspergillus fumigatus.

AIMS: Aspergillus fungi are common members of the soil microbiota. Some physiological and structural characteristics of Aspergillus species make them important participants in soil ecological processes. In this study, we aimed to evaluate the impact of 2,4-diacetylphloroglucinol (2,4-DAPG), a common metabolite of soil and rhizosphere bacteria, on the physiology of Aspergillus fumigatus. METHODS AND RESULTS: Integrated analysis using microscopy, spectrophotometry, and liquid chromatography showed the following effects of 2,4-DAPG on Aspergillus physiology. It was found that A. fumigatus in the biofilm state is resistant to high concentrations of 2,4-DAPG. However, experimental exposure led to a depletion of the extracellular polymeric substance, changes in the structure of the cell wall of the mycelium (increase in the content of α- and ß-glucans, chitin, and ergosterol), and conidia (decrease in the content of DHN-melanin). 2,4-DAPG significantly reduced the production of mycotoxins (gliotoxin and fumagillin) but increased the secretion of proteases and galactosaminogalactan. CONCLUSIONS: Overall, the data obtained suggest that 2,4-DAPG-producing Pseudomonas bacteria are unlikely to directly eliminate A. fumigatus fungi, as they exhibit a high level of resistance when in the biofilm state. However, at low concentrations, 2,4-DAPG significantly alters the physiology of aspergilli, potentially reducing the adaptive and competitive capabilities of these fungi.

Novel Calcium-Binding Motif Stabilizes and Increases the Activity of Aspergillus fumigatus Ecto-NADase.

Nicotinamide adenine dinucleotide (NAD) is an essential molecule in all kingdoms of life, mediating energy metabolism and cellular signaling. Recently, a new class of highly active fungal surface NADases was discovered. The enzyme from the opportunistic human pathogen Aspergillus fumigatus was thoroughly characterized. It harbors a catalytic domain that resembles that of the tuberculosis necrotizing toxin from Mycobacterium tuberculosis, which efficiently cleaves NAD+ to nicotinamide and ADP-ribose, thereby depleting the dinucleotide pool. Of note, the A. fumigatus NADase has an additional Ca2+-binding motif at the C-terminus of the protein. Despite the presence of NADases in several fungal divisions, the Ca2+-binding motif is uniquely found in the Eurotiales order, which contains species that have immense health and economic impacts on humans. To identify the potential roles of the metal ion-binding site in catalysis or protein stability, we generated and characterized A. fumigatus NADase variants lacking the ability to bind calcium. X-ray crystallographic analyses revealed that the mutation causes a drastic and dynamic structural rearrangement of the homodimer, resulting in decreased thermal stability. Even though the calcium-binding site is at a long distance from the catalytic center, the structural reorganization upon the loss of calcium binding allosterically alters the active site, thereby negatively affecting NAD-glycohydrolase activity. Together, these findings reveal that this unique calcium-binding site affects the protein fold, stabilizing the dimeric structure, but also mediates long-range effects resulting in an increased catalytic rate.

Distinct transcriptional responses to fludioxonil in Aspergillus fumigatus and its ΔtcsC and Δskn7 mutants reveal a crucial role for Skn7 in the cell wall reorganizations triggered by this antifungal.

BACKGROUND: Aspergillus fumigatus is a major fungal pathogen that causes severe problems due to its increasing resistance to many therapeutic agents. Fludioxonil is a compound that triggers a lethal activation of the fungal-specific High Osmolarity Glycerol pathway. Its pronounced antifungal activity against A. fumigatus and other pathogenic molds renders this agent an attractive lead substance for the development of new therapeutics. The group III hydride histidine kinase TcsC and its downstream target Skn7 are key elements of the multistep phosphorelay that represents the initial section of the High Osmolarity Glycerol pathway. Loss of tcsC results in resistance to fludioxonil, whereas a Δskn7 mutant is partially, but not completely resistant. RESULTS: In this study, we compared the fludioxonil-induced transcriptional responses in the ΔtcsC and Δskn7 mutant and their parental A. fumigatus strain. The number of differentially expressed genes correlates well with the susceptibility level of the individual strains. The wild type and, to a lesser extend also the Δskn7 mutant, showed a multi-faceted stress response involving genes linked to ribosomal and peroxisomal function, iron homeostasis and oxidative stress. A marked difference between the sensitive wild type and the largely resistant Δskn7 mutant was evident for many cell wall-related genes and in particular those involved in the biosynthesis of chitin. Biochemical data corroborate this differential gene expression that does not occur in response to hyperosmotic stress. CONCLUSIONS: Our data reveal that fludioxonil induces a strong and TcsC-dependent stress that affects many aspects of the cellular machinery. The data also demonstrate a link between Skn7 and the cell wall reorganizations that foster the characteristic ballooning and the subsequent lysis of fludioxonil-treated cells.

Aspergillus fumigatus cytochrome c impacts conidial survival during sterilizing immunity.

IMPORTANCE: Aspergillus fumigatus can cause a life-threatening infection known as invasive pulmonary aspergillosis (IPA), which is marked by fungus-attributable mortality rates of 20%-30%. Individuals at risk for IPA harbor genetic mutations or incur pharmacologic defects that impair myeloid cell numbers and/or function, exemplified by bone marrow transplant recipients, patients that receive corticosteroid therapy, or patients with chronic granulomatous disease (CGD). However, treatments for Aspergillus infections remain limited, and resistance to the few existing drug classes is emerging. Recently, the World Health Organization classified A. fumigatus as a critical priority fungal pathogen. Our cell death research identifies an important aspect of fungal biology that impacts susceptibility to leukocyte killing. Furthering our understanding of mechanisms that mediate the outcome of fungal-leukocyte interactions will increase our understanding of both the underlying fungal biology governing cell death and innate immune evasion strategies utilized during mammalian infection pathogenesis. Consequently, our studies are a critical step toward leveraging these mechanisms for novel therapeutic advances.

Optimizing Eco-Friendly Degradation of Polyvinyl Chloride (PVC) Plastic Using Environmental Strains of Malassezia Species and Aspergillus fumigatus.

Worldwide, huge amounts of plastics are being introduced into the ecosystem, causing environmental pollution. Generally, plastic biodegradation in the ecosystem takes hundreds of years. Hence, the isolation of plastic-biodegrading microorganisms and finding optimum conditions for their action is crucial. The aim of the current study is to isolate plastic-biodegrading fungi and explore optimum conditions for their action. Soil samples were gathered from landfill sites; 18 isolates were able to grow on SDA. Only 10 isolates were able to the degrade polyvinyl chloride (PVC) polymer. Four isolates displayed promising depolymerase activity. Molecular identification revealed that three isolates belong to genus Aspergillus, and one isolate was Malassezia sp. Three isolates showed superior PVC-biodegrading activity (Aspergillus-2, Aspergillus-3 and Malassezia) using weight reduction analysis and SEM. Two Aspergillus strains and Malassezia showed optimum growth at 40 °C, while the last strain grew better at 30 °C. Two Aspergillus isolates grew better at pH 8-9, and the other two isolates grow better at pH 4. Maximal depolymerase activity was monitored at 50 °C, and at slightly acidic pH in most isolates, FeCl3 significantly enhanced depolymerase activity in two Aspergillus isolates. In conclusion, the isolated fungi have promising potential to degrade PVC and can contribute to the reduction of environmental pollution in eco-friendly way.

Transcription factors SltA and CrzA reversely regulate calcium homeostasis under calcium-limited conditions.

IMPORTANCE: Calcium ions are ubiquitous intracellular signaling molecules for many signaling pathways regulating the fungal response to stress and antifungal drugs. The concentration of intracellular calcium is tightly regulated in its storage, release, and distribution. CrzA is the best-studied transcription factor that regulates this process under sufficient calcium or other external signals. However, CrzA was excluded from nuclei and then lost transcriptional activation under calcium-limited conditions. The regulators in the Ca2+ signaling pathway under calcium-limited conditions remain unclear. Here, we identified SltA as a key regulator in the Ca2+ signaling pathway under calcium-limited conditions, and the underlying mechanisms were further explored in Aspergillus fumigatus. These findings reveal a transcriptional control pathway that precisely regulates calcium homeostasis under calcium-limited conditions.

A20 ameliorates Aspergillus fumigatus keratitis by promoting autophagy and inhibiting NF-κB signaling.

Fungal keratitis (FK) is a serious, potentially sight-threatening corneal infection, which is associated with poor prognosis. A20, also called TNFAIP3, plays significant roles in the negative regulation of inflammation and immunity. However, the function of A20 in Aspergillus fumigatus (A. fumigatus) keratitis remains obscure. Herein, we found that the level of A20 is increased in human corneal epithelial cells (HCECs) and in mouse corneas with A. fumigatus infection, and that nuclear factor-κB (NF-κB) signaling is required for A20 upregulation. A20 overexpression inhibits A. fumigatus-mediated inflammatory responses, while A20 knockdown results in opposite effect. Mechanically, we showed that A20 inhibits NF-κB signaling and activates autophagy in infected HCECs. We also showed that inhibition of NF-κB signaling reverses the increased inflammatory responses in infected HCECs with A20 knockdown. Furthermore, autophagy blockage impedes the anti-inflammatory effect of A20 in A. fumigatus infected HCECs. Moreover, A20 ameliorates the corneal damage and inflammation in A. fumigatus infected mouse corneas. In conclusion, this study reveals that A20 alleviates A. fumigatus keratitis by activating autophagy and inhibiting NF-κB signaling. This suggests that exogenous use of A20 protein may be a potentially promising therapeutic strategy for FK treatment.

Use of a human small airway epithelial cell line to study the interactions of Aspergillus fumigatus with pulmonary epithelial cells.

During the initiation of invasive aspergillosis, inhaled Aspergillus fumigatus conidia are deposited on the epithelial cells lining the bronchi, terminal bronchioles, and alveoli. While the interactions of A. fumigatus with bronchial and type II alveolar cell lines have been investigated in vitro, little is known about the interactions of this fungus with terminal bronchiolar epithelial cells. Using the HSAEC1-KT human small airway epithelial (HSAE) cell line, we developed an in vitro model to study the interaction of two strains of A. fumigatus with these cells. We then compared the interactions of A. fumigatus with the A549 type II alveolar epithelial cell line and the HSAE cell line. We found that A. fumigatus conidia were poorly endocytosed by A549 cells, but avidly endocytosed by HSAE cells. A. fumigatus germlings invaded both cell types by induced endocytosis, but not by active penetration. A549 cell endocytosis of A. fumigatus was independent of fungal viability, more dependent on host microfilaments than microtubules, and induced by A. fumigatus CalA interacting with host cell integrin α5ß1. By contrast, HSAE cell endocytosis required fungal viability, was more dependent on microtubules than microfilaments, and did not require CalA or integrin α5ß1. HSAE cells were more susceptible than A549 cells to damage caused by direct contact with killed A. fumigatus germlings and by secreted fungal products. In response to A. fumigatus infection, A549 cells secreted a broader profile of cytokines and chemokines than HSAE cells. Taken together, these results demonstrate that studies of HSAE cells provide complementary data to A549 cells and thus represent a useful model for probing the interactions of A. fumigatus with bronchiolar epithelial cells in vitro. Importance During the initiation of invasive aspergillosis, Aspergillus fumigatus interacts with the epithelial cells that line the airways and alveoli. Previous studies of A. fumigatus-epithelial cell interactions in vitro used either large airway epithelial cell lines or the A549 type II alveolar epithelial cell line; the interactions of fungi with terminal bronchiolar epithelial cells were not investigated. Using the TERT-immortalized human small airway epithelial HSAEC1-KT (HSAE) cell line, we developed an in vitro model of the interactions of A. fumigatus with bronchiolar epithelial cells. We discovered that A. fumigatus invades and damages A549 and HSAE cell lines by distinct mechanisms. Also, the proinflammatory responses of the cell lines to A. fumigatus are different. These results provide insight into how A. fumigatus interacts with different types of epithelial cells during invasive aspergillosis and demonstrate that HSAE cells are useful in vitro model for investigating the interactions of this fungus with bronchiolar epithelial cells.

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.

Abnormal adenosine metabolism of neutrophils inhibits airway inflammation and remodeling in asthma model induced by Aspergillus fumigatus.

BACKGROUND: Neutrophils consume a large amount of energy when performing their functions. Compared with other white blood cells, neutrophils contain few mitochondria and mainly rely on glycolysis and gluconeogenesis to produce ATP. The inflammatory site is hypoxic and nutrient poor. Our aim is to study the role of abnormal adenosine metabolism of neutrophils in the asthmatic airway inflammation microenvironment. METHOD: In this study, an asthma model was established by intratracheal instillation of Aspergillus fumigatus extract in Ecto-5'-Nucleotidase (CD73) gene-knockout and wild-type mice. Multiple analyses from bronchoalveolar lavage fluid (BALF) were used to determine the levels of cytokines and chemokines. Immunohistochemistry was used to detect subcutaneous fibrosis and inflammatory cell infiltration. Finally, adenosine 5'-(α, ß-methylene) diphosphate (APCP), a CD73 inhibitor, was pumped subcutaneously before Aspergillus attack to observe the infiltration of inflammatory cells and subcutaneous fibrosis to clarify its therapeutic effect. RESULT: PAS staining showed that CD73 knockout inhibited pulmonary epithelial cell proliferation and bronchial fibrosis induced by Aspergillus extract. The genetic knockdownof CD73 significantly reduced the production of Th2 cytokines, interleukin (IL)-4, IL-6, IL-13, chemokine (C-C motif) ligand 5 (CCL5), eosinophil chemokine, neutrophil IL-17, and granulocyte colony-stimulating factor (G-CSF). In addition, exogenous adenosine supplementation increased airway inflammation. Finally, the CD73 inhibitor APCP was administered to reduce inflammation and subcutaneous fibrosis. CONCLUSION: Elevated adenosine metabolism plays an inflammatory role in asthma, and CD73 could be a potential therapeutic target for asthma.

Fungal siderophore metabolism with a focus on Aspergillus fumigatus: impact on biotic interactions and potential translational applications.

Iron is an essential trace element that is limiting in most habitats including hosts for fungal pathogens. Siderophores are iron-chelators synthesized by most fungal species for high-affinity uptake and intracellular handling of iron. Moreover, virtually all fungal species including those lacking siderophore biosynthesis appear to be able to utilize siderophores produced by other species. Siderophore biosynthesis has been shown to be crucial for virulence of several fungal pathogens infecting animals and plants revealing induction of this iron acquisition system during virulence, which offers translational potential of this fungal-specific system. The present article summarizes the current knowledge on the fungal siderophore system with a focus on Aspergillus fumigatus and its potential translational application including noninvasive diagnosis of fungal infections via urine samples, imaging of fungal infections via labeling of siderophores with radionuclides such as Gallium-68 for detection with positron emission tomography, conjugation of siderophores with fluorescent probes, and development of novel antifungal strategies.

The C2H2-Type Transcription Factor ZfpA, Coordinately with CrzA, Affects Azole Susceptibility by Regulating the Multidrug Transporter Gene atrF in Aspergillus fumigatus.

The incidence of invasive aspergillosis caused by Aspergillus fumigatus has risen steadily over the past few decades due to the limited effective treatment options and the emergence of antifungal-resistant isolates. In clinic-isolated A. fumigatus, the azole resistance mechanism is primarily caused by mutations of the drug target and/or overexpression of drug efflux pumps. However, knowledge about how drug efflux pumps are transcriptionally regulated is limited. In this study, we found that loss of a C2H2 transcription factor ZfpA (zinc finger protein) results in the marked upregulation of a series of drug efflux pump-encoding genes, especially atrF, which contributes to azole drug resistance in A. fumigatus. CrzA is a previously identified positive transcription factor for genes of drug efflux pumps, and ZfpA transcriptionally inhibits expressions of drug efflux pumps in a CrzA-dependent way. Under the treatment of azoles, both ZfpA and CrzA transfer to nuclei and coregulate the expression of multidrug transporters and then keep normal drug susceptibility in fungal cells. Findings in this study demonstrated that ZfpA is not only involved in fungal growth and virulence potential but also negatively regulates antifungal drug susceptibility. IMPORTANCE Conserved across all kingdoms of life, ABC transporters comprise one of the largest protein families. They are associated with multidrug resistance, affecting aspects such as resistance to antimicrobials or anticancer drugs. Despite the importance of ABC transporters in multidrug resistance, the understanding of their regulatory network is still limited in A. fumigatus. Here, we found that the loss of the transcription factor ZfpA induces the expression of the ABC transporter gene atrF, altering azole susceptibility in A. fumigatus. ZfpA, coordinately with CrzA, affects the azole susceptibility by regulating the expression of the ABC transporter gene atrF. These findings reveal the regulatory mechanism of the ABC transporter gene atrF in A. fumigatus.

Genome-wide identification and comparative in-silico characterization of ß-galactosidase (GH-35) in ascomycetes and its role in germ tube development of Aspergillus fumigatus via RNA-seq analysis.

ß-galactosidase (Lactase), an enzyme belonging to the glycoside hydrolase family causing the hydrolysis and trans-glycosylation of ß-D-galactosides, has a vital role in dairy industries. The current investigation emphasizes on in-silico identification and comparative analysis of different fungal lactases present in Aspergillus fumigatus, Aspergillus oryzae, Botrytis cinerea, and Fusarium fujikuroi. Prediction of motifs and domains, chromosomal positioning, gene structure, gene ontology, sub-cellular localization and protein modeling were performed using different bioinformatics tools to have an insight into the structural and functional characteristics of ß-galactosidases. Evolutionary and homology relationships were established by phylogenetic and synteny analyses. A total of 14 ß-gal genes (GH-35) were identified in these species. Identified lactases, having 5 domains, were predicted to be stable, acidic, non-polar and extracellularly localized with roles in polysaccharide catabolic process. Results showed variable exonic/intronic ratios of the gene structures which were randomly positioned on chromosomes. Moreover, synteny blocks and close evolutionary relationships were observed between Aspergillus fumigatus and Aspergillus oryzae. Structural insights allowed the prediction of best protein models based on the higher ERRAT and Q-MEAN values. And RNA-sequencing analysis, performed on A. fumigatus, elucidated the role of ß-gal in germ tube development. This study would pave the way for efficient fungal lactase production as it identified ß-gal genes and predicted their various features and also it would provide a road-way to further the understanding of A. fumigatus pathogenicity via the expression insights of ß-gal in germ tube development.