FIG 6.

OprF promotes complement-mediated internalization by human neutrophils. (A) Neutrophils were infected with normal human serum (NHS) or heat-inactivated serum (HIS) opsonized wild-type and oprF-deficient P. aeruginosa strains as described in Materials and Methods. A representative image shows internalization of NHS-opsonized P. aeruginosa by neutrophils. External bacteria exposed to the antibodies before permeabilization stained green. Bacteria exposed to the antibodies after permeabilization stained red (arrows indicate internalized bacteria). DAPI was used to stain the phagocyte nucleus (blue; magnification, ×60). Quantitation of internalized bacteria is denoted in parentheses below each image and represents an average of 100 infected neutrophils that were examined from triplicate coverslips in each test group. (B) C5-depleted-serum-opsonized OprF-expressing E. coli and vector control were incubated with neutrophils and stained with C3 and OprF antibody. Vector control E. coli bound only with C3 antibody and stained green (i), while OprF-expressing E. coli bound with both C3 and OprF antibodies and stained yellow (ii); blue DAPI fluorescence is used to stain the phagocyte nucleus (as above). Quantitation of bacterial internalization with neutrophils is denoted in parentheses below each image and represents an average for 100 infected neutrophils examined from triplicate coverslips in each test group. Shown are results representative of three independent experiments.