Fig. 2
From: Quorum sensing modulates the formation of virulent Legionella persisters within infected cells
L. pneumophila intracellular nongrowers are viable and persisters. a, b FACS-sorting of intracellular L. pneumophila subpopulations. A. castellanii were infected with L. pneumophila/Timer (MOI 1, 24 h) and lysed. Released intracellular bacteria were FACS-sorted according to their Timer green/red color ratio, sorted subpopulations were reanalyzed by flow cytometry and compared with the initial infected cell lysate (pre-sort) to evaluate the separation efficiency. a Graph, (representative for four biological replicates) and b the quantification is shown. Pre-sort, black; growers, yellow; nongrowers, red. c Intracellular growers and nongrowers resume growth on CYE plates. To determine re-growth, FACS-sorted intracellular growing and nongrowing L. pneumophila/Timer subpopulations were plated on CYE or CYE/Cam plates and CFU were determined (plating efficiency: CFU*100/sorted fluorescent particles). d Growth rate heterogeneity is a reversible phenotypic trait. FACS-sorted intracellular growing and nongrowing L. pneumophila/Timer subpopulations were plated on CYE. Three colonies were harvested, grown to stationary phase in AYE broth and used to re-infect A. castellanii (MOI 1, 24 h). The size of the nongrowing population was determined by flow cytometry on infected cell lysates. e Ex vivo, nongrowers are more tolerant to antibiotic treatment. FACS-sorted growing and nongrowing L. pneumophila were treated (1 h) or not with the antibiotics ofloxacin (3 μg mL−1), erythromycin (3 μg mL−1) or gentamycin (20 μg mL−1), and the percentage of survivors was calculated as CFUtreated/CFUnontreated × 100. f Formation of L. pneumophila persisters in infected amoebae. After host cell lysis, bacteria were resuspended in AYE supplemented with erythromycin (60 μg mL−1; gray), ofloxacin (30 μg mL−1; black), or without antibiotic (red dots). Bacteria were incubated at 25 °C and plated at defined time points. The antibiotics caused bi-phasic kill curves, indicating the existence of persisters. Survivors of 50 h of antibiotic treatment were harvested from plates and showed similar kill curves in follow-up experiments (dashed-lines). Data represent the mean ± SEM of at least three biological replicates (n ≥ 3; light gray filled circles). Student’s t test two-tailed. ***P < 0.001, **P < 0.01. Source data are provided as a Source Data file
