Fig. 2.

PVL and HlgAB are required for biofilm-mediated neutrophil-killing activity. LIVE-DEAD assays measuring viability of neutrophils treated with biofilm-spent media from isogenic deletion mutants of respective leukocidins (A). LIVE-DEAD analysis of neutrophils treated with spent media from strains in a ΔlukSF background with deletion of each of three γ-hemolysin subunits (ΔlukSFΔhlgA, ΔlukSFΔhlgC, ΔlukSFΔhlgB). WT and ΔlukSFΔhlgACB-treated neutrophils are shown for comparison (B). Spent media shown in B, mixed 1:1 (volume) with those from biofilms of a ΔhlgACB strain. LIVE-DEAD measurements were made as described earlier (C). CLSM of neutrophils treated with spent media from indicated strains. Cells were stained as described for Fig. 1D (D). Percentage death was calculated in comparison with neutrophils treated with 0.1% SDS for all assays. Neutrophils in HBSS were used as a negative control. Statistical significance shown in all panels is in comparison with neutrophils treated with WT biofilm-spent media. Results represent an average of six independent experiments performed in triplicate ± SEM (**P < 0.01 and ***P < 0.001, one-way ANOVA and Tukey’s post hoc analysis; ns, not significant). (Scale bars: 10 μm.)