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. 2023 Dec 12;12(1):e02912-23. doi: 10.1128/spectrum.02912-23

FIG 4.

FIG 4

A H2O2-dependent molecule is formed in cultures of Spn with Hb-Fe3+. (A, C, and E) THY containing Hb-Fe3+ (Hb-Fe3+) was infected with TIGR4 (A), EF3030 (C), D39 (E), and ATCC 33400 (E) and incubated for 2, 4, or 6 h. As a control, Hb-Fe3+ was left uninfected but incubated under the same conditions. Supernatants were collected, and the spectra between 250 and 500 nm were obtained using a spectrophotometer (BMG LabTech Omega). (B) TIGR4, its mutant derivative, or complemented strains were cultured under similar conditions as above for 4 h. Uninfected THY-Hb-Fe3+ (Hb-Fe3+) served as a control. The supernatants were purified and analyzed as above. The absorbance at 305 nm obtained for each assessed strain was used to construct the graph. (D) Supernatants from C were also collected for quantification of H2O2 using the Amplex Red Hydrogen Peroxide Assay. (F-H) THY-Hb-Fe3+ (Hb-Fe3+) was inoculated with TIGR4 and treated with (F) DNase I (50 U), (G) a cocktail of serine and cysteine protease inhibitor (PI), or (H) catalase (200, 400, 600, 800, or 1,000 U). As a control, Hb-Fe3+ was left uninfected (Hb-Fe3+) in F-H or treated with DNase I (50 U) in F. The absorbance at 305 nm obtained for each assessed treatment was used to construct the graph. Error bars represent the standard errors of the means calculated using data from at least two independent experiments. The level of significance was determined using a t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant.