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. 2021 May 26;12(3):e01008-21. doi: 10.1128/mBio.01008-21

FIG 4.

FIG 4

LamA-dependent fusion of specific and azurophilic granules to the L. pneumophila phagosome. (A) Representative confocal images showing colocalization of lactoferrin with the L. pneumophila phagosome. PMNs were infected with wild type or the ΔT4SS or ΔlamA mutants, formalin-killed wild type, or opsonized S. aureus for 15 min. Bacteria are stained with an anti-Legionella antibody (green), and lactoferrin is stained with anti-lactoferrin (red). (B) Quantification of lactoferrin colocalization to the L. pneumophila phagosome at 15 min postinfection. Data are shown as mean percent colocalization ± SD; n = 100. ***, Student's t test of percent colocalization of lactoferrin to phagosomes harboring the ΔT4SS or ΔlamA mutants or formalin-killed wild type versus wild type containing phagosomes; P < 0.0001. (C) Representative confocal images showing colocalization of elastase with the L. pneumophila phagosome. PMNs were infected with wild type or the ΔT4SS or ΔlamA mutants, formalin-killed wild type, or opsonized S. aureus for 15 min. Bacteria were labeled with an anti-Legionella antibody (green), and elastase is labeled with anti-elastase antibodies (red). (D) Quantification of elastase colocalization to the L. pneumophila phagosome at 15 min postinfection. Data are shown as mean percent colocalization ± SD; n = 100. ***, Student's t test of percent colocalization of elastase to phagosomes harboring the ΔT4SS or ΔlamA mutants or formalin-killed wild type versus wild type containing phagosomes; P < 0.0001. (E) Relative contribution of specific and azurophilic granules to rapid bacterial killing was determined using a pan-protease, cathepsin G, and elastase inhibitors. 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 PMNs treated with the pan-protease or elastase inhibitors at 15 min versus untreated PMNs; P < 0.0001. Unless otherwise stated, n represents technical replicates, and data shown are representative of three independent biological replicates.