Figure 1.
Overall strategy to assess the toxicity of spore borne metabolites.
Figure 2.
HPLC-chromatograms at 270 nm of A. fumigatus extracts.
A) conidiation-deficient A. fumigatus strain (ΔbrlA) and B) conidiation-restored strain (ΔbrlA::brlA). The fungi were cultured at 25°C on PDA for 13 days in the dark as described in the Materials and Methods.
Figure 3.
Identification and structures of the 5 major compounds isolated from A. fumigatus conidia extracts.
MS spectra (left) and UV-Vis spectra (right). A) tryptoquivaline F/J, B) fumiquinazoline C, C) trypacidin, D) monomethylsulochrin, E) questin.
Figure 4.
HPLC-DAD chromatograms from Aspergillus fumigatus (NRRL 35693) when grown in condition repressing (A) and inducing (B) conidiogenesis.
The NRRL 35693 strain was cultured on Czapek-Glucose medium, at 25°C for 13 days in the dark under shaking (180 r.p.m.). Every day, the culture (mycelia and medium) was removed from the flask and placed in a new sterile flask in order to avoid that mycelium adhering to glass and sporulating in contact with the air. After 13 days, culture medium was harvested and analyzed by DAD-HPLC. The remaining pellets of mycelia were exposed to daylight without medium at 25°C in order to induce conidiogenesis. The secondary metabolites were extracted from pellets as described in the Materials and Methods.
Figure 5.
Representative HPLC-DAD and LC-MS chromatograms of conidial extract from A. fumigatus.
(A) total scan photo diode array (PDA) chromatogram. Chromatogram of the ion at (B) m/z 403 in positive ionization mode (tryptoquivaline F), (C) m/z 444 in positive ionization mode (fumiquinazoline C), (D) m/z 345 in positive ionization mode (trypacidin), (E) m/z 345 in negative ionization mode (monomethylsulochrin), (F) m/z 283 in negative ionization mode (questin).
Figure 6.
Outline biosynthesic pathway for trypacidin.
Table 1.
Comparison of LC-MS analysis of the fractions obtained from flash chromatography and their effect on the A549 cell viability.
Table 2.
Comparison of LC-MS analysis of the F16 sub-fractions obtained from the RP-HPLC and their effect on the A549 cell viability.
Figure 7.
Toxicity effect of the trypacidin pathway metabolites on A549 cells.
Cell viability and cell lysis were measured using respectively MTT and LDH assay. The cells were exposed for 24 h in culture conditions to each metabolite before measuring cell viability and cell lysis as described in the Materials and Methods. The graph shows the mean values of three independent experiments, excepted for trypacidin for which five experiments were carried out. Negative and positive control corresponds respectively to 1% DMSO and gliotoxin (10 µM for MTT assay and 50 µM for LDH assay). Trypacidin was tested at 50 µM and the other metabolites at 50 and 100 µM. Significant difference from the negative control was analysed with the t-test (* for p<0.05; ** for p<0.001).
Table 3.
Inhibition of A549 cell viability by trypacidin.
Figure 8.
Flow cytometry analysis of ROS and RNS production.
A549 cells were seeded at 3×104 cells/cm2 and cultured for 24 h. Then, cells were exposed to trypacidin (green area) 10 µM and 50 µM or DMSO 1% (red line) for 1 h, 2 h and 24 h. H2O2 production was assessed using H2-DCF-DA, O2•− using DHE, NO using DAF-FM. Menadione was used as control for H2-DCF-DA and DHE, SNAP for DAF-FM (blue line). Peaks are representative of three independent experiments.
Figure 9.
Flow cytometry analysis of H2O2 and NO related to PI staining.
A549 cells were seeded at 3×104 cells/cm2 and cultured for 24 h. Then, cells were exposed to trypacidin 50 µM or DMSO 1% for 2 h and 24 h. H2O2 and NO production were measured using respectively H2-DCF-DA, and DAF-FM. IP was used to stain dead cells. Dot-blots are representative of three independent experiments.
Figure 10.
Flow cytometry analysis of the cell cycle.
A549 and HBEpC were exposed to 1, 10, 30 and 50 µM trypacidin for 24 h in culture conditions. In the negative control (0 µM trypacidin), cells were exposed to 1% DMSO. Gliotoxin 5 µM was used as positive control to induce sub-G1 particles. The cells were then harvested and analysed for cell cycle as described in the Materials and Methods. The figure shows mean values for 3 independent experiments.
Figure 11.
A549 cells were exposed 2 h or 24 h to 10 µM (green line) and 50 µM (blue line) trypacidin or with solely 1% DMSO as negative control (red line). After treatment, cells were stained with DiOC6 for 20 min. at 37°C. Cells incubated with 40 µM FCCP were used as positive control (brown line). The figure is representative of 3 independent experiments.