Table 1.
Class-specific substrates used to detect the protease class and activity in fungal culture supernatants.
Fig 1.
Growth and protease activity of S. aurantiacum WM 06.482 (clinical isolate) and WM 10.136 (environmental isolate) in different media.
(A, B) Change in the dry weight of mycelia over time in 50 ml cultures. The data of day 0 represent the dry weight of the same amount of freeze dried conidia used to inoculate each flask. Due to the precipitating casein on day one in the casein supplemented medium, the dry weight measured encompasses both dry mycelia and casein; (C, D) pH of the culture supernatant; (E, F) general secreted protease activity, normalized against dry biomass. Data represent mean ± SD (3 biological replicates). ● SCFM: Synthetic CF sputum medium, ○ SCFM+C (casein added); ▲ SCFM+M (mucin added).
Fig 2.
Inhibition of protease activity in day-4 culture supernatant of S. aurantiacum in three media.
Percentage changes in the protease activity in the supernatant with an inhibitor relative to the non-inhibited control supernatant, set as 100% for each culture (dotted line). Data represent mean ± SD (3 biological repeats). A. Clinical strain; B. Environmental strain. SCFM: Synthetic CF sputum medium; SCFM+C (casein added); SCFM+M (mucin added).
Fig 3.
Classes and activities of specific secreted proteases in three media.
Activities of specific proteases were expressed as the amount of ρ-NA or MCA released in one minute per mg of dry biomass. Substrates and test conditions are given in Table 1. Data represent mean ± SD (3 biological repeats). SCFM: Synthetic CF sputum medium; SCFM+C (casein added); SCFM+M (mucin added). C: Clinical strain, E: environmental strain.
Fig 4.
SDS-PAGE and zymogram analysis of S. aurantiacum culture supernatants.
A. Proteins from the non-concentrated culture supernatant separated by protein electrophoresis. B. Enzyme activity zymograms containing 0.1% (w/v) casein as substrate. For protein gels without substrate, the supernatant was heated at 70°C for 10 min and the electrophoresis was run at room temperature. For the zymogram analysis, the supernatant was not heated, the electrophoresis was run at 4°C and incubation was conducted in Tris-HCl buffer pH 7.5 at 37°C for 16 hours. All gels were stained by Coomassie Brilliant Blue G-250. SCFM: Synthetic CF sputum medium; SCFM+C (casein added); SCFM+M (mucin added). d2, d4, d6: culture days.
Fig 5.
SDS-PAGE of proteins concentrated from the S. aurantiacum culture supernatants.
20 μg of protein was loaded in each well. The gel was cut horizontally into 10 pieces and the sections were numbered as shown. Note that some sections did not contain visible protein bands. Peptides from each single section were analysed by MS. Numbering of the bands corresponds to the numbers in Table 2. C: clinical strain, E: environmental strain.
Table 2.
Proteases identified by LC-MS/MS from day-4 culture supernatants of S. aurantiacum WM 06.482 (clinical isolate) and WM 10.136 (environmental isolate) grown in mucin supplemented SCFM.
Full lists of identified proteins are given in supporting information S1 and S2 Tables.