Journal: Journal of Fungi

Article Title: Understanding the Role of Nitronate Monooxygenases in Virulence of the Human Fungal Pathogen Aspergillus fumigatus

doi: 10.3390/jof8070736

Figure Lengend Snippet: Phenotypic characterisation of wild-type, Δ nmo2, and Δ nmo5 A. fumigatus strains after being treated with various oxidative stressors. The sensitivity of A. fumigatus strains to different oxidative stress-inducing compounds was evaluated using inhibition zone ( A – D ) and droplet ( E ) assays. The inhibition zones in the diffusion assay were measured after adding a specific oxidative compound—( A ) H 2 O 2 , ( B ) menadione, ( C ) diamide, and ( D ) tert-butyl peroxide (TBH). Circular-shaped data points correspond to wild-type, square-shaped to Δ nmo2 , and triangular-shaped to Δ nmo5 . In the droplet assay ( E ), 0.1 mM tert-butyl peroxide was added to agar plates. Black arrows indicate the diameters of fungal colonies. Data were obtained from 5 independent experiments, and the statistical test used was the one-way ANOVA test.

Article Snippet: The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/jof8070736/s1 , Table S1: Primers used in this study to generate A. fumigatus mutant strains; Table S2: Primers used for validation of deletion strains; Table S3: Signal peptide and cellular localisation predictions for A. fumigatus Nmos; Table S4: Conserved protein domains in Nmo2 and Nmo5 of Magnaporthe oryzae , Metarhizium brunneum , and Aspergillus fumgatus ; Table S5: Global similarity (Blosum62) results of MAFFT alignment; Table S6: Identity results of MAFFT alignment; Table S7: Aspergillus fumigatus strains used in this study; Figure S1: Validation of nmo2 and nmo5 gene deletion; Figure S2: Protein sequence analysis of Nmos in A. fumigatus ; Figure S3: Interpro Scan results for Nmo2 and Nmo5 of Aspergillus fumigatus and Magnaporthe oryzae ; Figure S4: Maximum likelihood tree of Nmo1–5 in Aspergillus fumigatus , Magnaporthe oryzae , and Metarhizium brunneum ; Figure S5: Protein oxidation in infected BMDMs [ , , , , , , , , , , , ].

Techniques: Inhibition, Diffusion-based Assay

Journal: Journal of Fungi

Article Title: Understanding the Role of Nitronate Monooxygenases in Virulence of the Human Fungal Pathogen Aspergillus fumigatus

doi: 10.3390/jof8070736

Figure Lengend Snippet: Evaluation of the importance of Nmo2 and Nmo5 for protection against peroxynitrite and the enzymatic denitrification of nitropropane. ( A ) Percentage of metabolic activity of A. fumigatus strains after being treated with peroxynitrite at a concentration of 0.05 mM. Data were obtained from 2 independent experiments with 16 replicates in each group. The statistical test used was a one-way ANOVA test; * indicates p < 0.05. ( B ) Determination of enzymatic activity levels using proteins isolated from A. fumigatus strains in the presence of a specific substrate—nitropropane. The amount of accumulated product was measured spectroscopically as a change in absorbance, which was normalised to the total amount of protein in a reaction mix. Data were obtained from 4 independent experiments.

Article Snippet: The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/jof8070736/s1 , Table S1: Primers used in this study to generate A. fumigatus mutant strains; Table S2: Primers used for validation of deletion strains; Table S3: Signal peptide and cellular localisation predictions for A. fumigatus Nmos; Table S4: Conserved protein domains in Nmo2 and Nmo5 of Magnaporthe oryzae , Metarhizium brunneum , and Aspergillus fumgatus ; Table S5: Global similarity (Blosum62) results of MAFFT alignment; Table S6: Identity results of MAFFT alignment; Table S7: Aspergillus fumigatus strains used in this study; Figure S1: Validation of nmo2 and nmo5 gene deletion; Figure S2: Protein sequence analysis of Nmos in A. fumigatus ; Figure S3: Interpro Scan results for Nmo2 and Nmo5 of Aspergillus fumigatus and Magnaporthe oryzae ; Figure S4: Maximum likelihood tree of Nmo1–5 in Aspergillus fumigatus , Magnaporthe oryzae , and Metarhizium brunneum ; Figure S5: Protein oxidation in infected BMDMs [ , , , , , , , , , , , ].

Techniques: Activity Assay, Concentration Assay, Isolation

Journal: Journal of Fungi

Article Title: Understanding the Role of Nitronate Monooxygenases in Virulence of the Human Fungal Pathogen Aspergillus fumigatus

doi: 10.3390/jof8070736

Figure Lengend Snippet: Investigation of the virulence of A. fumigatus Δ nmo2 and Δ nmo5 . ( A ) Nitro-oxidative homeostasis is altered in infected phagocytes. Macrophages produce nitric oxide that reacts with NADPH-oxidase derived superoxide to form a highly reactive nitrogen intermediate, peroxynitrite. Peroxynitrate acts as signalling molecules and thus can affect immune responses. Nmos, in turn, can either directly or indirectly mitigate biological activities of peroxynitrite. ( B ) Percentage of fungal killing by BMDMs either with or without DPI pre-treatment. Data were obtained from 3 independent experiments. Circular-shaped data points correspond to wild-type, square-shaped to Δ nmo2 , and triangular-shaped to Δ nmo5 . The statistical test used was the two-way ANOVA test; * indicates p < 0.05 and ** indicates p < 0.01. ( C , D ) Kaplan–Meier survival curves of Galleria mellonella larvae infected with wild-type, Δ nmo2 , and Δ nmo5 A. fumigatus strains. Larvae were infected with ( A ) 1 × 10 5 conidia/mL or ( B ) 1 × 10 6 conidia/mL of A. fumigatus . The control group were injected with a 30 μL dose of PBS solution. Surveillance was conducted every 24 h over a period of 72 h. Twelve biological replicates were included in each group. The statistical test used was the Log-rank test; * indicates p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, respectively.

Article Snippet: The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/jof8070736/s1 , Table S1: Primers used in this study to generate A. fumigatus mutant strains; Table S2: Primers used for validation of deletion strains; Table S3: Signal peptide and cellular localisation predictions for A. fumigatus Nmos; Table S4: Conserved protein domains in Nmo2 and Nmo5 of Magnaporthe oryzae , Metarhizium brunneum , and Aspergillus fumgatus ; Table S5: Global similarity (Blosum62) results of MAFFT alignment; Table S6: Identity results of MAFFT alignment; Table S7: Aspergillus fumigatus strains used in this study; Figure S1: Validation of nmo2 and nmo5 gene deletion; Figure S2: Protein sequence analysis of Nmos in A. fumigatus ; Figure S3: Interpro Scan results for Nmo2 and Nmo5 of Aspergillus fumigatus and Magnaporthe oryzae ; Figure S4: Maximum likelihood tree of Nmo1–5 in Aspergillus fumigatus , Magnaporthe oryzae , and Metarhizium brunneum ; Figure S5: Protein oxidation in infected BMDMs [ , , , , , , , , , , , ].

Techniques: Infection, Derivative Assay, Control, Injection

Journal: Journal of Fungi

Article Title: Understanding the Role of Nitronate Monooxygenases in Virulence of the Human Fungal Pathogen Aspergillus fumigatus

doi: 10.3390/jof8070736

Figure Lengend Snippet: Assessment of cytokine secretion by bone-marrow-derived macrophages during infection with A. fumigatus , Δ nmo2 , and Δ nmo5 . BMDMs were exposed to swollen A. fumigatus conidia and the supernatant was collected after incubation overnight. Levels of TNF-α, IL-1β, and IL-10 in supernatants were analysed via ELISA. Data were obtained from 2 independent experiments. Rhombs and error bars indicate means and standard deviation.

Article Snippet: The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/jof8070736/s1 , Table S1: Primers used in this study to generate A. fumigatus mutant strains; Table S2: Primers used for validation of deletion strains; Table S3: Signal peptide and cellular localisation predictions for A. fumigatus Nmos; Table S4: Conserved protein domains in Nmo2 and Nmo5 of Magnaporthe oryzae , Metarhizium brunneum , and Aspergillus fumgatus ; Table S5: Global similarity (Blosum62) results of MAFFT alignment; Table S6: Identity results of MAFFT alignment; Table S7: Aspergillus fumigatus strains used in this study; Figure S1: Validation of nmo2 and nmo5 gene deletion; Figure S2: Protein sequence analysis of Nmos in A. fumigatus ; Figure S3: Interpro Scan results for Nmo2 and Nmo5 of Aspergillus fumigatus and Magnaporthe oryzae ; Figure S4: Maximum likelihood tree of Nmo1–5 in Aspergillus fumigatus , Magnaporthe oryzae , and Metarhizium brunneum ; Figure S5: Protein oxidation in infected BMDMs [ , , , , , , , , , , , ].

Techniques: Derivative Assay, Infection, Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation