RB50Δ sigE is more efficiently phagocytosed and killed by PMNs than RB50. (A) GFP-expressing RB50 (white bars) and RB50ΔsigE (grey bars) were incubated with freshly isolated human peripheral blood PMNs for 20 min at an MOI of 50. Attachment levels were measured as mean intensities ± SE of green fluorescence associated with PMNs. (B) Cell surface-bound bacteria were detected by incubation with RPE-labeled goat F(ab’)2 fragments of anti-mouse IgG, after incubation with immune serum. Mean phagocytosis levels ± SE were calculated from the decrease in red fluorescence of GFP-positive cells incubated for an additional 30 min at 37°C allowing for internalization (RPE2, 50 min total incubation time) compared to that of cells incubated for only 20 min (RPE1). Percent phagocytosis is (1-RPE2/RPE1) × 100%. (C) To determine killing of bacteria by PMNs, cells incubated with bacteria for 50 min were treated with antibiotics to kill extracellular bacteria. Viable bacteria per PMN (left) and percent killing of internalized bacteria (right) were expressed as mean ± SE. AU indicates arbitrary units; * indicates a P-value of < 0.05.