FIG 3.

Generation of a robust ROS burst by PMNs in response to cytosolic hyperglucose generated by LamA-mediated glycogenolysis. (A) Determination of ROS produced by PMNs in response to L. pneumophila infection. PMNs were infected with either the wild-type bacteria or the ΔT4SS and ΔlamA mutants, the complemented lamA/C mutant, or formalin-killed wild type for 1 h. Additionally, PMNs were stimulated with zymosan or PMA as a positive control for ROS production. *, Student's t test of ROS production by PMNs infected with either ΔT4SS or the ΔlamA mutant versus wild type infected PMNs; P = 0.0277, 0.0432, respectively. (B) Representative confocal microscopy images showing spatial subcellular localization of ROS production in the pathogen containing phagosome. PMNs treated with NBT were infected with wild type or the ΔT4SS or ΔlamA mutants, the complement lamA/C mutant, or formalin-killed wild type for 1 h. Bacteria were stained using an anti-L. pneumophila antibody (red), and nuclei were stained with DAPI (blue). NBT staining is shown in differential interference contrast (DIC) images as black deposits. (C) The contribution of ROS to rapid bacterial killing was determined using the inhibitor DPI. PMNs were pretreated with DPI and infected with wild-type bacteria, and recoverable CFU were determined at 5 and 15 min postinfection. Data are shown as mean log₁₀ CFU ± SD; n = 3. ***, Student's t test of wild-type CFU in DPI-treated PMNs at 15 min versus untreated PMNs; P < 0.0001. (D) Representative confocal images showing spatial subcellular colocalization of p47phox with the L. pneumophila phagosome. PMNs were infected with wild type or the ΔT4SS or ΔlamA mutants for 15 min. Bacteria are stained with an anti-Legionella antibody (green), and p47phox is stained with anti-p47phox (red). (E) Quantification of p47phox colocalization to the pathogen-containing phagosome at 15 min postinfection. Data are shown as mean percent colocalization ± SD; n = 100. ***, *, and **, Student's t test of percent colocalization of p47phox to phagosomes harboring the ΔT4SS or ΔlamA mutants, formalin-killed wild type, or opsonized S. aureus versus wild type containing phagosomes; P = 0.0008, = 0.0288, and = 0.0043, respectively. Unless otherwise stated, n represents technical replicates, and data shown are representative of three independent biological replicates.