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Fig. S1. GFP-VipD localizes in the cytoplasm of mammalian cell. HeLa cells transfected to express GFP, GFP-0898, GFP-0898A151S, GFP-VipD or GFP-VipDS73A for 18 h were fixed and stained for the mitochondrial protein COX4I1. Samples were analysed using an Olympus IX-81 fluorescence microscope for images acquisition. Images were pseudocoloured with the IPLab software package. Bar: 20 μm.

Fig. S2. Cell rounding phenotype caused by Lpg0898 and VipD and their non-toxic mutants. 293T cells transfected to express GFP-Lpg0898 (A), GFP-Lpg0898A151S (B), GFP-VipD (C) or GFP-VipDS73A (D) for 24 h. Images were acquired with an Olympus IX-81 fluorescence microscope equipped with a CCD camera. Note the cell rounding phenotype caused by wild-type Lpg0898 and VipD. Bar: 50 μm.

Fig. S3. VipD does not cause damage in plasma membrane.

A. HeLa cells transfected to express GFP or GFP-VipD for 24 h were washed and stained with 4 μM EtBr. Retaining of the dye by the cells was analysed by microscopic analysis using an Olympus IX-81 fluorescence microscope equipped with a CCD camera. Untransfected cells treated by Triton-100 were included as a control (lower panel). Note that the nuclei of cells treated by Triton-100 were stained by the dye.

B. His6-VipD or BSA was added to culture medium at a final concentration of 8 μg ml−1. After 3 h of incubation, the integrity of the plasma membrane was assessed by the retaining of EtBr using an Olympus IX-81 fluorescence microscope equipped with a CCD camera. Bar: 60 μm.

Fig. S4. VipD-induced Cyto c release is independent of Bax and Bak.

A. Induction of caspase 3 in MEFs by transient expression. Wild-type and bax/bak−/− MEFs were transfected with plasmid DNA that allows the expression of GFP-VipD, GFP-VipDS73A and indicated controls for 24 h, and the lysates of these samples were analysed for activated caspase 3 by immunoblot (upper panel). The levels of the GFP fusions were probed by an antibody specific for GFP (lower panel) and tubulin was detected as a loading control (middle panel). Note that in wild-type MEFs, caspase 3 can be activated by the Mirus transfection reagent (Thermo Scientific, Waltham, MA), which is the only one that allows efficient transfection of these cells (lane 1).

B. Quantification of the caspase 3 in (A). Quantification was obtained by measuring the band intensity using the Odyssey imaging system. Similar results were obtained in two independent experiments.

C. Induction of Cyto c release by VipD from mitochondria isolated from MEFs. Forty micrograms of His6-VipD or His6-VipDS73A was added to 50 μg mitochondria purified from wild-type mouse embryonic fibroblast (MEF) or from MEF of bak/bax−/− mouse. After incubation at 30°C for 3 h, the release of Cyto c was evaluated by probing its presence in the soluble fraction by immunoblot.

Fig. S5. A. VipD did not detectably hydrolyse phospholipids in reactions with defined components. His6-VipD or commercial phospholipase A2 was added to liposome established with phosphoethanolamine and the reactions were allowed to proceed for 3 h at 30°C. Total lipids extracted from the reactions were separated on thin-layer chromatograph (TLC) Silica gel 60 F254 (EMD) and lipids were detected using iodine vapour.

B. Mitochondria were treated with proteinase K or buffer before incubated with VipD or its mutant. The release of cytochrome c from mitochondria was detected via immunoblot.

Fig. S6. Deletion of caspase 3 activators has little impact on the activation of this caspase by L. pneumophila. Differentiated U937 cells were infected by indicated L. pneumophila strains for 6 h, lysates of infected samples resolved by SDS-PAGE were probed for active caspase 3. Strains: Δ2 (Lp02Δ0898, Δ1625); Δ3(Lp02Δ0898, Δ1625, Δ0716); Δ4(Lp02Δ0898, Δ1625, Δ0716, Δ2831); Δ5(Lp02Δ0898, Δ1625, Δ0716, Δ2831, Δ2176). The wild-type strain Lp02 and Lp03, the mutant deficient for the Dot/Icm transporter was used as controls.

Fig. S7. An L. pneumophila deletion strain lacking more than 100 of the known Dot/Icm substrates still potently induced caspase 3 activation. Differentiated U937 cells were infected by indicated L. pneumophila strains for 6 h, lysates of infected samples resolved by SDS-PAGE were probed for active caspase 3. Similar results were obtained in multiple independent experiments.

Table S1. Bacterial and yeast strains used in this study.

Table S2. Primers used in this study.

Table S3. Plasmids used in this study.

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