FIG 4.

Role of L. pneumophila inositol metabolism in D. discoideum and accumulation of 2-NBDG in LCVs. (A) D. discoideum amoebae were infected (MOI, 10) with L. pneumophila JR32, the ΔiolT mutant, or the ΔiolG mutant harboring plasmid pNT28 (constitutive GFP production). Inositol at 20 mM was added 2 h postinfection, and intracellular replication was determined by fluorescence. The data represent means and SD of triplicates (Student's t test; JR32 with or without inositol, P < 0.05 at 32 to 47 h; JR32 versus the ΔiolT mutant or ΔiolG mutant, P < 0.01 at 23 to 32 h). (B) D. discoideum was infected (MOI, 10) with L. pneumophila (Lpn) JR32, ΔiolT mutant, ΔiolG mutant, ΔicmT mutant, or ΔrpoS mutant harboring plasmid pSW001 (constitutive DsRed production) for 1 h. The infected amoebae were washed and incubated with 20 μM 2-NBDG for 30 min. Subsequently, the amoebae were washed again and fixed with paraformaldehyde (PFA), stained with DAPI, and subjected to fluorescence microscopy (B and C), or homogenized using a ball homogenizer, fixed with PFA on poly-l-lysine-coated coverslips, and stained for the LCV-bound L. pneumophila effector SidC (D). Scale bars = 5 μm. (C) Mean and SD of 2-NBDG-positive LCVs were quantified from three independent experiments counting at least 50 LCVs per experiment.