Fig. 4
From: Quorum sensing modulates the formation of virulent Legionella persisters within infected cells
Intracellular L. pneumophila nongrowers are highly virulent. a Intracellular nongrowers are virulent. A. castellanii were infected (MOI 1, 24 h) with L. pneumophila/Timer, lysed and FACS-sorted according to the Timer green/red color ratio. Sorted growers and nongrowers were used to re-infect A. castellanii, and intracellular replication was determined by CFU (fold change: CFU30 min p.i./CFU 96 h p.i.). b T4SS-dependent survival of intracellular nongrowers. A. castellanii were infected (MOI 1) with L. pneumophila wild-type (black), the isogenic avirulent ΔicmT strain (yellow) and the complemented strain ΔicmT(icmTS) (green) expressing timer. At given time points, the nongrowers from infected amoebae lysates were quantified by flow cytometry and analyzed by comparison with WT. c, d Intracellular nongrowers reside in individual LCVs. c D. discoideum amoebae producing the ER/LCV marker calnexin-GFP or the PtdIns(4)P probe P4C-GFP were infected (MOI 1, 24 h) with L. pneumophila/Timer WT or the ΔicmT/Timer strain, fixed and analyzed by confocal microscopy. Micrographs show overlays of the fluorescence at 500 nm (GFP, Timer) and 600 nm (Timer). White arrows show GFP-positive membrane only around intracellular WT growers and nongrowers. Scale bars 10 μm. d D. discoideum/P4C-GFP was infected (MOI 1, 24 h) with L. pneumophila/Timer WT or ΔicmT/Timer and P4C-GFP enrichment measured by imaging flow cytometry. Data represent the mean ± SEM of three biological replicates (n = 3; light gray filled circles). Student’s t test two-tailed; ***P < 0.001, *P < 0.05. Source data are provided as a Source Data file
