Chitinase

The reaction mixtures contained 15 μg/mL chitinase (from S. griseus, Sigma-Aldrich, St. Louis, MO, USA), 10 mg/mL chitin as the substrate in the presence or absence of 2.0 μM ScLPMO10G, with 1.0 mM ascorbic acid, and incubated in 50 mM Tris-HCl buffer (pH 8.0) at 37 °C according to our previously published study [37 (link)]. The samples were collected to measure the release of reducing sugars. The reducing sugars released from chitin by enzyme hydrolysis were determined using a 4-Hydroxybenzhydrazide (PAHBAH) colorimetry assay [38 (link)] at 410 nm. The concentration of reducing sugars was quantified by a standard curve with known concentrations of N-acetylglucosamine. All reactions were performed in triplicates.

The chitin contents of KU80, MoMih1-41, MoMih1-44 and MoMih1c were determined following the previous method (Yin et al., 2016 (link)). 5 mg freeze-dried fungal powder was mixed with 1 mL 6% KOH solution, water bathed at 80°C for 90 min, and then centrifuged (16 000g, 10 min). The sediment was washed by 1 mL PBS for three times, resuspended in 0.5 mL Mcilvine’s buffer (pH 6), and then added 100 μL chitinase (C6137, Sigma-Aldrich, Shanghai, China) in suspension. After reacting in water bath at 37°C for 16 h, 100 μL sodium borate solution (0.27 M, pH 9) was added in the tubes, then boiled at 100°C for 10 min. 1 mL diluted Ehrlich’s reagent solution (10-folds) was finally added to the reaction tubes, and then incubated at 37°C for 20 min. The absorbance value of each sample was determined at OD₅₈₅. A standard curve was prepared using the D-GlcNAc (A8625, Sigma-Aldrich, Shanghai, China). This experiment was repeated three times.

Cell walls from C. albicans were simultaneously digested with 60 units of chitinase (Sigma) and 100 units of zymolyase (Zymo Research) overnight at room temperature. This fraction was used as the enzymatic source in the TGase activity assay. Digested cell walls (200 μl) were mixed with 0.2 mm MDC, 1 mm PMSF, 1 μg/ml pepstatin A, with no CaCl₂ or EDTA, and with 10 mm CaCl₂ or 2 mm EDTA, and incubated for 1 h at 4 °C. Proteins were separated by non-denaturing 7.5% polyacrylamide gels, and fluorescence was visualized at 350 nm with the Gel Doc EZ Imager system (Bio-Rad). Finally, fluorescent bands were excised and submitted to mass spectrometry (MS/MS) analysis for protein identification to the Proteomics Division at the University of Florida Interdisciplinary Center for Biotechnology Research.

Susceptibility of the fungal cell wall to Lysing Enzymes (LE), a commercial preparation containing β-1,3-glucanase and chitinase (Sigma, St. Louis, MO, USA), was assayed as described previously (Yoshimi et al., 2013 (link)). Washed 1-day-old mycelia of the wild-type, AGΔ, GAGΔ, and AG-GAGΔ strains (30 mg fresh weight) grown in CDE medium at 30°C were suspended in 1 ml of 0.8 M NaCl in sodium phosphate buffer (10 mM, pH 6.0) containing 10 mg/ml LE and incubated for 1, 2, or 4 h at 30°C. The number of protoplasts generated from the mycelia was counted with a hemocytometer (A106, SLGC, Tokyo, Japan).

BE01 was cultured in SDY in a shake flask at 25°C and 110 rpm for 3 days, and then a single-layer Miracloth filter was used to collect mycelia. The mycelia were washed three times with sterile distilled water and then three times with 0.7 M NaCl. In parallel, the enzyme mixture (3 mg ml⁻¹ lysing enzyme (Sigma), 7 mg ml⁻¹ driselase (Sigma), and 0.5 mg ml⁻¹ chitinase (Sigma) added in 30 ml of 0.7 M NaCl solution) was prepared, and filtered through a 0.22 μm filter. The washed mycelia were added to the filtered enzyme mixture, and the mixture was incubated at 25°C and 60 rpm for 3 h. Then, the protoplasts were collected by filtration through a double-layer Miracloth filter. The filtrate containing protoplasts was centrifuged at 2000 rpm for 8 min at 4°C, and the supernatant was discarded. The protoplasts were washed with 0.7 M NaCl solution and then resuspended in STC buffer (sorbitol, 145.74 g; Tris–HCl, 6.06 g; CaCl_2, 5.55 g; 1,000 ml of sterile distilled water; pH 8.0). The initial recorded concentration was 1.31 × 10⁷ protoplasts ml⁻¹, which was adjusted to 1.0 × 10⁴ protoplasts ml⁻¹.