Figure 4. MA-induced cytCa²⁺ increase spreads from infected to uninfected alveoli.

Images and quantifications are of alveolar fluorescence after epithelial transfection with the Ca²⁺-sensing YC2.3 FRET probe. CytCa²⁺ was measured in terms of the FRET-induced YFP/CFP fluorescence ratio. (A–D) Confocal images in A show epithelial (yellow) FRET-induced YFP (FRET) fluorescence of 2 representative alveoli (alv) containing MAs (red) of WT or Hla-deficient (hla^–) USA300 as indicated. Bacteria are MitoTracker (MT) dye loaded. High-magnification images in B and C show MA-associated epithelial sites (1 and 3 in A), before (B) and 1 hour after (C) bacterial microinstillation. Images in D show high-magnification views of MA-free sites (2 and 4 in A). Note, fluorescence increased at sites 1 and 2, but not 3 and 4. Bacterial fluorescence was digitally removed in C. Scale bars: 20 (A) and 5 (B and D) μm. (E) Time course of FRET ratio increase at 2 representative MA-associated alveolar sites, after microinstillation of the indicated bacteria. Arrow indicates the time point of instillation. (F) Group data 1 hour after the indicated microinstillations. Right bar shows alveoli that were pretreated with BAPTA before WT instillation. Bars: mean ± SEM; n = 4 lungs (4 sites quantified per lung); *P < 0.05 using ANOVA with Bonferroni correction. (G) Time-dependent effects of the indicated microinstillations. Arrow indicates the time point of instillation. HCl, hydrochloric acid (pH 1.0–1.2). Points: mean ± SEM; n = 3 lungs (3 sites quantified per lung); *P < 0.05 vs. HCl using 2-tailed t test. (H) Group data 1 hour after alveolar WT microinstillation. Instilled alveoli were pretreated with vehicle (Veh) or BAPTA before instillation. Quantified alveoli did not contain bacteria. Bars: mean ± SEM; n = 4 lungs (4 sites quantified per lung); *P < 0.05 using 2-tailed t test.