FIG 2.

Increased ROS in cells infected with Spn-H₂O₂. (A) Schematic representation of an increase in ROS leading to toxicity in human alveolar cells. Extracellular and intracellular pneumococci produce H₂O₂, which increases the pool of ROS but also oxidizes hemoproteins, further increasing other ROS molecules, leading to cell toxicity. (B-C) Human alveolar A549 cells were infected with TIGR4, TIGR4∆spxB∆lctO, EF3030, EF3030∆spxB∆lctO, treated with 100 µM menadione or 750 µM H₂O₂, or left uninfected and incubated for (B) 8, (C) 6, or 16 h. Post-incubation, cells were treated with CellROX (5 µM), fixed, and stained with DAPI (DNA); the membranes of some cells were also stained with WGA-Alexa 555. Confocal optical XY middle sections are shown except the far-right panel, which also shows XZ and YZ optical sections. (D) Intracellular ROS was quantified from confocal micrographs as detailed in Materials and Methods and reported as a ratio of GFP:DAPI channel intensity within the same samples. 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 one-way analysis of variance with Dunnett’s post hoc t-test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant.