Fig. 5
From: Quorum sensing modulates the formation of virulent Legionella persisters within infected cells
Intravacuolar nongrowers express hallmark genes of motility and virulence. A. castellanii was infected (MOI 1, 5 h or 24 h) with L. pneumophila harboring the fluorescent reporters a Ptac-timer−PflaA-mCerulean or b Ptac-timer−PsidC-mCerulean, fixed and analyzed by confocal microscopy. Micrographs show the fluorescence for Timer at 500 and 600 nm, for mCerulean at 479 nm and the bright field. The white arrows indicate intracellular red/orange nonreplicating and mCerulean-producing, transmissive bacteria. Scale bar 20 μm. c A. castellanii was infected (MOI 1, 24 h) with L. pneumophila harboring the fluorescent reporters PflaA-gfp or PsidC-gfp, fixed and analyzed by confocal microscopy. Micrographs show the overlay of bright field and fluorescence for PflaA-gfp (500 nm, GFP; 600 nm, mCherry, all bacteria) and for PsidC-gfp (500 nm, GFP; 405 nm, DAPI (false red color), all bacteria). The white arrows indicate intracellular nonreplicating and GFP-producing bacteria. Scale bar 10 μm. d The subpopulation fraction and e the absolute number of intracellular GFP producers in the infected cell lysates were determined by flow cytometry. f D. discoideum amoebae producing the LCV marker and PtdIns(4)P probe P4C-GFP were infected (MOI 1, 24 h) with L. pneumophila (Ptac-timer−PflaA/sidC-mCerulean), fixed and analyzed by confocal microscopy. Micrographs show overlays of the fluorescence at both 500 and 600 nm (to detect Timer and P4C-GFP) and 479 nm (mCerulean). White arrows show GFP-positive membrane surrounding both intracellular growers and nongrowers. Only the nongrowers express hallmark genes for motility and virulence. Scale bars 20 μm. Data represent the mean ± SEM of three biological replicates (n = 3; light gray filled circles). Source data are provided as a Source Data file
