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

FIG 3.

FIG 3

Cytotoxic role and peroxidase activity of Hb-Fe3+ with Spn-H2O2 and human cells. (A) Human alveolar A549 cells were incubated for 14 h with sterilized TIGR4 supernatants grown for 6 h in THY containing Hb-Fe3+. (B) Proposed mechanisms for radical production by Spn-H2O2 through the Fenton reaction. Spn scavenges free iron through Hb oxidation, and Spn-H2O2 reacts with the free iron to produce •OH or Hb-Fe4+. (C) Cell culture medium (DMEM), DMEM supplemented with 10 µM Hb-Fe3+ (DMEM + Hb-Fe3+), or A549 cells in DMEM (A549) were infected with TIGR4 and incubated for 2, 4, or 6 h. H2O2 concentration is per mL of media. (D-E) THY or THY containing Hb-Fe3+ (Hb-Fe3+) was infected with TIGR4 and incubated for 2, 4, or 6 h. As a control for D, b-Fe3+ was left uninfected but incubated under the same conditions. Supernatants from C and E were collected, and H2O2 was quantified using Amplex Red. Supernatants from D were also collected to observe spectra between 350 and 500 nm via a spectrophotometer (BMG LabTech Omega). Error bars represent the standard errors of the means calculated using data from at least two independent experiments. Statistical significance was determined using a one-way analysis of variance with Tukey’s post hoc test for multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.