GBS lacking the GBC are resistant to hGIIA.

(A) HGIIA kills GBS strain NEM316 WT but not ΔgbcO in a concentration-dependent manner and phenotype is restored in complemented strain ΔgbcO/pTCV. The killing is represented as the percentage of CFU surviving after hGIIA exposure compared to the inoculum. GBS killing is prevented when (B) exposed to catalytically inactive hGIIA H48Q and (C) by the hGIIA-specific inhibitor LY311727. (D) Treatment of NEM316 WT with the gbcO-type inhibitor tunicamycin reproduces the ΔgbcO phenotype with regard to hGIIA-mediated killing GBS more resistant to hGIIA-mediated killing. (E) Visualization of bacteria-bound hGIIA H48Q to GBS NEM316 by fluorescence microscopy (+). As control, H48Q hGIIA mutant protein was omitted (-). hGIIA was detected with a mouse anti-human hGIIA monoclonal antibody. An irrelevant IgG1 isotype antibody served as negative control. The cell wall was labeled with Ester-350, and newly formed septa were visualized with fluorescently labeled vancomycin (Vanc-FL), which stains sites of peptidoglycan insertion. Shown are representative cells. (F) Quantification of hGIIA binding to polar or non-polar regions of GBS are based on analysis of 12 fields including 578 stained cells from two separate experiments. For all other panels, data represent mean +/- SD of three independent experiments. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.