MK-deficient strains are sensitive to ROS in vitro but not ex vivo. (A to C) The menD::Tn and menF::Tn strains were tested for sensitivity to various concentrations of ceftriaxone (CRO), ampicillin (AMP), bacitracin (BAC), and daptomycin (DAP) (A); lysozyme and LL-37 (B); and hydrogen peroxide (C) in vitro to calculate MICs. Data represent median MICs of results from three or more independent experiments. (D) iIFNAR−/− macrophages were treated with 100 µM menadione for 1 h or infected with the wild-type strain or the ΔmenD strain at an MOI of 10 for 6 h. Cells were treated with or without 1mM N-acetylcysteine (NAC) throughout the assay and were then examined for ROS production. (E) The ΔmenD strain (MOI of 10) was examined for bacteriolysis in iIFNAR−/− macrophages 6 h postinfection. NAC (1 mM) was added to infections to scavenge ROS. Data are normalized to wild-type levels of bacteriolysis and presented as means ± SEM of results from four independent experiments. NS, not significant. Download FIG S6, EPS file, 0.7 MB.