Figure 4.

AGXX® causes protein S-bacillithiolation, protein aggregation and an oxidative shift in the BSH redox potential in S. aureus. (A) Non-reducing BSH-specific Western blot analysis showed increasing of S-bacillithiolated GapDH protein in S. aureus USA300 strain under AGXX® stress. The Coomassie-stained SDS-PAGE is shown as loading control below the BSH Western blot. (B) To analyze protein aggregates, S. aureus cells were grown in RPMI medium to OD₅₀₀ of 0.5 and treated with 4–10 μg/ml AGXX® for 30 min. The insoluble protein fractions of protein aggregates were obtained by centrifugation of the cell lysates and repeated centrifugation of the NP-40-dissolved pellet as described in the Methods section. The insoluble protein fractions were analyzed by SDS-PAGE and visualized with Coomassie staining (C) S. aureus COL expressing Brx-roGFP2 was treated with the sub-lethal amount of 4 μg/ml AGXX® at OD₅₀₀ of 0.5 in RPMI medium and the biosensor oxidation degree (OxD) was measured at different times after stress. The Brx-roGFP2 biosensor is rapidly oxidized by AGXX®. The OxD values of the Brx-roGFP2 biosensor measurements with and without AGXX® were calibrated to fully reduced (DTT-treated) and oxidized (diamide-treated) control samples. The OxD values of fully reduced and oxidized controls were defined as “0” and “1,” respectively. The data were calculated as mean values from 3 biological replicate experiments. Error bars represent the SD and p-values are calculated using a Student's unpaired two-tailed t-test by the graph prism software. Symbols are defined as follows: **p ≤ 0.01; ***p ≤ 0.001 and ****p ≤ 0.0001 (p < 0.0003 for control/AGXX stress at 5 min; p = 0.0002 at 10 min; p < 0.0001 at 20 min; p = 0.0013 at 30 min; p = 0.0001 at 60 min).