Fig. 4

H₂O₂ and HOCl impair SpeB secretion by GAS. A GAS 5448 strain was exposed to indicated concentration of hydrogen peroxide (H₂O₂) and bacterial numbers were determined 1 h post stimulations. Each dot represents one independent experiment. Lines denote mean values (n = 5). B Assessment of SpeB positivity/negativity of 5448 strain post H₂O₂ treatment as displayed in A. Mean percentage from five independent experiments is shown (n = 5). C GAS 5448 strain was exposed to indicated concentration of HOCl and bacterial numbers were determined 1 h post stimulations. Each dot represents one independent experiment. Lines denote mean values (n = 5). D Assessment of SpeB positivity/negativity of 5448 strain post HOCl treatment as displayed in C. Mean percentage from five independent experiments is shown (n = 5). A–D Untreated bacteria in THY media, acidified NaCl or Na₃PO₄ served as controls. E Human primary neutrophils were infected with GAS strain 5448 and intracellular bacteria were determined by plating serial dilution of neutrophil lysates on casein agar plates post indicated time points. The MPO activity was inhibited by indicated concentrations of MPO inhibitor 30 min prior and during the entire infection period. Dots represent the median value ± range of independent experiments with five donors (n = 5). F Assessment of SpeB positivity/negativity of 5448 strain recovered from primary human neutrophils shown in E. Mean percentages of SpeB⁺ and SpeB⁻ clones from five independent experiments are shown (n = 5). G Representative images of GAS 5448 directly plated on casein agar containing indicated concentrations of H₂O₂ and H subsequent analyses. Mean percentage from four independent experiments is shown (n = 4). I SpeB activity assay (n = 4) of bacterial supernatants. GAS were grown over-night (16 h) in THY and sterile-filtered supernatants supplemented with indicated concentrations of H₂O₂ were used for the analyses. J Western blot (upper left panel), silver staining of the loading control (upper lower panel), and image analyses (right panel) of GAS 5448 supernatants post exposure to indicated concentrations of H₂O₂. Representative images and analysis of three independent experiments are shown (n = 3). The level of significance between the groups of experiments presented in A–H was determined using Kruskal Wallis test with Dunn’s posttest. The level of significance in J was determined using Friedman test