Fig. 1
From: Quorum sensing modulates the formation of virulent Legionella persisters within infected cells
L. pneumophila shows growth rate heterogeneity in infected amoebae. a Timer color ratio reflects the division rate at a single cell level. Stationary phase grown L. pneumophila/Timer was diluted in AYE broth and let grow for 24 h. At given time points, bacteria were harvested and analyzed by flow cytometry. The Timer color ratio - Log10[500 nm (green)/600 nm (red)] - was calculated for individual bacteria. b–d L. pneumophila intracellular growth rate heterogeneity. b Confocal microscopy of A. castellanii infected (MOI 1; 5, 24 h) with L. pneumophila/Timer wild-type (WT) or the isogenic avirulent ΔicmT/Timer strain. Micrographs show overlays of bright field and the Timer fluorescence (500 nm and 600 nm); growing and nongrowing bacteria appear green or red/orange, respectively. Magnifications: growth rate heterogeneity of L. pneumophila subpopulations (24 h p.i.) with different color ratios (R: Log10[green/red] color ratio) and the corresponding division rate (μ). Scale bars 10 μm. c Flow cytometry or d imaging flow cytometry of lysed infected A. castellanii shows growth rate heterogeneity of released intracellular bacteria. Black, whole population; red, nongrowers (NG); orange, slow-growers (GS); green, fast-growers (GF). gray, ΔicmT. BF, Bright field. e L. pneumophila forms a high percentage of nongrowers in infected Acanthamoebae. Quantification by flow cytometry of nongrowing L. pneumophila/Timer wild-type (WT) or ΔicmT/Timer in infected cell lysates (MOI 1; 5, 24 h). f Intracellular nongrowers produce Timer protein de novo. A. castellanii was infected (MOI 1, 24 h), L. pneumophila/Timer and immobilized in PYG/0.1% agarose. Photobleaching (PB) was set up using the FRAP-wizard algorithm of the Leica SP8 microscope. Regions of interest (ROI; dashed squares) were photobleached (100% 488 nm laser intensity, iteration 30×), and fluorescence recovery at 500 nm was recorded (every 10 min). Micrographs illustrate FRAP kinetics in inverted color for clarity. Scale bar 20 μm. Fluorescence recovery measurements shown for three nongrowers (yellow) and three growing bacterial clusters (green). Data represent the mean ± SEM of three biological replicates (n = 3; light gray filled circles). Student’s t test two-tailed, ***P < 0.001. Source data are provided as a Source Data file
