Fig. 6
TBC1D5 localizes to LCVs and promotes growth of L. pneumophila. a Imaging flow cytometry (IFC) images of LCVs from homogenized D. discoideum DH1 producing in tandem GFP-TBC1D5 (pFL1304) and AmtA-mCherry as an LCV marker, infected (MOI 50, 2 h) with mPlum-producing L. pneumophila JR32 or ΔicmT (pAW14). b Quantification by IFC of the intensity of GFP-TBC1D5 on >3000 LCVs per sample at the time points post-infection indicated. The data show mean and 95% confidence intervals of one representative experiment out of three independent biological replicates (***P < 0.001; two-way ANOVA with Bonferroni post hoc test). c D. discoideum DH1 stably producing GFP-TBC1D5 was infected (MOI 20, 2 h) with DsRed-producing L. pneumophila JR32 or ΔridL harboring pCR77 (vector), ΔridL/pIF007 (RidL) or ΔridL/pKB201 (RidL-Δβ-hairpin). Intact LCVs were purified, immuno-stained for calnexin, and analyzed by fluorescence microscopy. Scale bar, 1 µm. d Quantification of c. Mean and SD of the means of three independent biological replicates (LCV isolations) are shown, each with at least 44 LCVs (average pixel GFP intensity of masked LCVs relative to the value of strain JR32; one way ANOVA; *P < 0.05, **P < 0.01). e D. discoideum Ax3 stably producing calnexin-GFP were infected (MOI 10, 1 h) with DsRed-producing L. pneumophila JR32 harboring pCR77 (vector), or ΔridL/pKB201 (RidL-Δβ-hairpin), immuno-stained for RidL, and effector translocation was analyzed by fluorescence microscopy. Scale bar 1 µm. f RAW 264.7 macrophages were infected (MOI 10, 2 h) with DsRed-producing L. pneumophila JR32 or ΔridL harboring pCR77 (vector), ΔridL/pIF007 (RidL) or ΔridL/pKB201 (RidL-Δβ-hairpin). Intact LCVs were purified, immuno-stained for TBC1D5 and SidC, and analyzed by fluorescence microscopy. Scale bar, 1 µm. g D. discoideum DH1 or an isogenic TBC1D5 knockout strain (Δtbc1d5) was infected (MOI 10, 24 h) with GFP-producing L. pneumophila JR32 (black bars) or ΔicmT mutant bacteria (white bars) harboring plasmid pNT-28, and intracellular replication was determined by fluorescence. Mean of fluorescence relative to JR32 in DH1 and SD of four independent experiments (each in technical triplicates) are shown (paired Student’s t test; *P < 0.05)
