(A) Immunoblot analysis of LC3-I and LC3-II levels in uninfected cells (control) and cells infected for 2-hours with either wild type Legionella (WT) or a dotA mutant. Treatment of cells with Bafilomycin A1 is indicated. (B) Images show LC3 (green) distribution in HEK293 cells infected with the indicated Legionella strains (red) for 2-hours. Scale bar, 1 μm. (C) Graph shows percent of cells containing LC3-puncta calculated from three independent assays where a total of 100 cells were scored in each assay. Data represent the average ± s.d., *p<0.0001 compared to the uninfected control. (D) Mouse bone marrow-derived macrophages were infected for 2-hours with a Legionella ΔflaA strain producing a BlaM∷RalF fusion protein. Immunoblot analysis of was used to detect LC3-I and LC3-II levels in injected and uninjected cells (). (E) Representative images of bone marrow-derived macrophages from GFP-LC3 transgenic mice infected with the indicated Legionella strains (red) for 2-hours. Cells were stained using an antibody specific for GFP (green). Scale bar, 1 μm. (F) Graph shows data on GFP-LC3 puncta staining for uninfected and infected mouse bone marrow-derived macrophages. The percent of cells containing GFP-LC3 puncta was calculated from three independent assays where a total of 100 cells were scored in each assay. Data represent the average ± s.d., *p<0.0001 compared to the uninfected control.

(A) Immunoblot analysis of LC3-I and LC3-II levels in HEK293 cells infected for 2-hours with either wild type Legionella (WT), a dotA mutant, the pentuple mutant, and mutants with single chromosomal deletions (pentuple, Δ2ab, Δ3, Δ4a, Δ6a, and Δ7a). (B) Immunoblot analysis of LC3-I and LC3-II levels in HEK293 cells producing the indicated Legionella effector proteins fused to GFP. (C) Long-term protein degradation was measured in cells producing GFP alone, GFP-RavZ and GFP-RavZ_C258A. No significant differences were observed for cells grown in complete medium (black bars). An increase in protein degradation resulting from starvation-induced autophagy was observed in cells producing GFP alone or GFP-RavZ_C258A but cells producing GFP-RavZ displayed a significant defect in autophagy-dependent protein degradation. Data represent average ± s.d. from 3-independent triplicates. *p<0.001. (D) Immunoblot analysis of LC3-I and LC3-II levels in HEK293 cells infected for 2-hours with wild type Legionella (WT), a dotA mutant, a ΔravZ mutant, or the complemented RavZ-deficient strain (ΔravZ + pRavZ). (E) HEK293 cells infected for 2-hours with the indicated strains of Legionella were fixed and stained for LC3. Infected cells were scored for the presence of LC3-positive puncta. The graph shows percent of cells containing LC3-puncta calculated from three independent assays where a total of 100 cells were scored in each assay. Data represent the average ± s.d., *p<0.0001 compared to the pentuple mutant control.

(A) Coomassie-stained SDS-PAGE gel from an in vitro GR conjugation assay containing the conjugating enzymes Atg7 and Atg3, the RavZ protein, the unconjugated GR protein and the lipid-conjugated GR protein (GR-PE). Lanes from the whole reaction (rxn) and reactions missing individual components are marked. The RavZ lane contained all the components of the whole rxn plus purified RavZ protein. (B) Indicated above each lane are the concentrations of purified RavZ added to a conjugation assay containing GR at a concentration of 12 μM. (C) Liposomes containing GR conjugated to PE were isolated on a flotation gradient (Float-Up lane) and treated with RavZ (+RavZ lane). (D) Liposomes containing GR-PE were treated with RavZ in the presence of the indicated concentrations of NEM or were treated with the RavZ_C258A protein. (E) Fluorescence anisotropy was used to analyze the release kinetics of Texas-Red-labeled GR from liposomes in vitro upon treatment with RavZ (red line), Atg4B (blue line), and RavZ_C258A (green line). (F) Stained SDS-PAGE gels show that GR-YFP was rapidly cleaved by Atg4B to generate the products GR and YFP. No cleavage of GR-YFP was observed by RavZ after a 120 min reaction. (G) LC-MS/MS chromatographs from samples containing purified GR (top graph) and GR that was isolated after RavZ-mediated deconjugation from PE-containing liposomes (bottom graph). Arrow indicates that the native C-terminal peptide in GR (DESVYG) was abundant in the untreated sample and that RavZ-mediated deconjugation resulted in a loss of the C-terminal glycine (DESVY).