Glass bead

Pythium insidiosum proteins were obtained as described by Rodrigues et al., with modifications [35 (link)]. The mycelial mass was frozen in liquid nitrogen and disrupted by grinding with a pestle until a fine powder was obtained. The powder mycelium was submitted to the Precellys instrument (2 cycles of 20 s; Bertin instruments, Montigny-le-Bretonneux, France) in 1 mL of Tris–Ca²⁺ buffer (20 mM Tris–HCl pH 8.8, 2 mM CaCl₂) containing a commercial cocktail of protease inhibitors (1:100; GE Healthcare, Chicago, IL, USA), RNase, and DNase enzymes (1:100; GE Healthcare); and glass beads (Sigma, St. Louis, MO, USA, 425–600 μm). Afterwards, the cell debris and glass beads were removed via centrifugation at 14,000× g for 10 min at 4 °C, and dithiothreitol (20 mM; Sigma, St. Louis, MO, USA) was added to the supernatant. The protein concentration was determined by the Bradford (Bio-Rad, Hercules, CA, USA) method [36 (link)], and the sample was kept at −80 °C.

Strains were grown in MRS/MRSf in the absence or presence of 5 ppm Se at 30°C for 24 h; cells were harvested by centrifugation and washed with 0.1 M sodium phosphate buffer (pH 7.5) with 1 mM EDTA. Then, microbial cells were mixed with the same buffer and glass beads (150–212 μm diameter; Sigma-Aldrich Chemical Co.) in a 1:2:1 ratio (cells:buffer:glass beads, w/v/w). Cells were lysed by mechanical beating after loading the vials in a Mini-Bead Beater-8 cellular disruptor (Biospec Products Inc., Bartlesville, OK, USA) with intermittent cooling on ice-bath (6 cycles of 1.5 min). The cell debris were removed by centrifugation and lysates were used for GR activity assay (Pophaly et al., 2017 (link)). The assay depends upon the transformation of GSSG to GSH with concomitant oxidation of 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB, Sigma-Aldrich Chemical Co.) to a colored compound. Briefly, the reaction mixture contained 150 μl of 0.6 mg/ml DTNB, 10 μl of 10 mg/ml NADPH (Sigma-Aldrich Chemical Co.), and 20 μl of cell-free extract. The reaction was initiated by adding 10 μl of 1 mg/ml GSSG (Sigma-Aldrich Chemical Co.). All solutions were made in 0.1 M sodium phosphate buffer (pH 7.5) with 1 mM EDTA. Absorbance was monitored at 405 nm over a period of 4 min in a 15-s time interval in a microplate reader (Versamax, Molecular Devices, USA).

ACSF spiked with different concentrations of Streptococcus pneumoniae or Cryptococcus neoformans culture was used to optimize the nucleic acid extraction method. The modified protocol of QIAamp cador pathogen Mini Kit was used. Briefly, 250 μL of each sample was added to the pathogen lysis tube, which contained 0.2 g glass beads with a particle size of 450 μm to 600 μm and 0.2 g glass beads with a particle size of 100 μm (Sigma, St. Louis, MO). The lysis tube was centrifuged for 5 min at 14,000×g, and then 200 μL of the supernatant was transferred to a 1.5 mL microcentrifuge tube containing 20 μL QIAGEN Protease K. A total of 400 μL lysis buffer was added into the pathogen lysis tube to re-suspend the pellet. The pathogen lysis was then extracted as the manual. Total nucleic acid was eluted in 100 μL AVE buffer. A blank control (H₂O) was included for each batch of extraction to monitor laboratory contaminations. If the blank control showed positive on the PM-TAC assay, the entire batch of total nucleic acid was discarded.