Live and pnemolysin-positive pneumococci are required to fully induce Lc3-mediated response within zebrafish macrophages. (A) Representative confocal images of CMV:GFP-Lc3 control (top two panels) or cyba knockdown (bottom two panels) larvae infected systemically with approximately 1600 CFU of live (left two panels) and heat-killed (right two panels) mKate2-labeled D39 Δcps S. pneumoniae fixed at 2 hpi. Scale bars: 10 µm. (B) Quantification of Lc3 associations with intracellular live or heat-killed S. pneumoniae within infected phagocytes of fixed CMV:GFP-Lc3 control or cyba larvae at 2 hpi. Data are shown as mean ± standard deviation (SD). Data obtained from two independent experiments. (C) Anti-Ply immunoblot analysis of whole-cell lysates from the indicated S. pneumoniae strains. The position of protein markers are indicated in kiloDaltons, Pneumolysin (Ply) ≈ 53.7 KDa. Representative blot shown, n = 4. (D) Representative confocal images of control CMV:GFP-Lc3 larvae infected systemically with approximately 1600 CFU of Ply-positive (D39 Δcps, left panel) and Ply-negative (D39 Δcps Δply, right panel) mKate2-labeled S. pneumoniae fixed at 2 hpi. Scale bars: 10 µm. (E) Quantification of Lc3 associations with intracellular Ply-positive or Ply-negative S. pneumoniae within infected phagocytes of fixed CMV:GFP-Lc3 larvae at 2 hpi. Data are shown as mean ± standard deviation (SD). Data obtained from two independent experiments. (F) Representative confocal images of CMV:GFP-Lc3 control (top two panels) or cyba knockdown (bottom two panels) larvae infected systemically with approximately 1600 CFU of Ply-positive (D39 Δcps, left two panels) and Ply-negative (D39 Δcps Δply, right two panels) mKate2-labeled S. pneumoniae fixed at 2 hpi. Scale bars: 10 µm. (G) Quantification of Lc3 associations with intracellular Ply-positive or Ply-negative S. pneumoniae within infected phagocytes of fixed CMV:GFP-Lc3 control or cyba larvae at 2 hpi. Data are shown as mean ± standard deviation (SD). Data obtained from two independent experiments.
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