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cmi12233-sup-0002-si.eps2294K

Fig. S1. 293 cells expressing LAMP1-YFP demonstrate that strain 6850 localizes to LAMP1-YFP-decorated vesicles (green) at 2 h p.i. but is not associated with fluorescent membranes at 6 h p.i. By contrast, strain Cowan I remains enclosed by LAMP1-positive membranes.

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Fig. S2. PIPLC does not synergize with PSMα in transgenic S. aureus SA113.

Escape marker recruitment assays at 3 h p.i. with recombinant S. aureus SA113 show low escape rates from HeLa phagosomes regardless of expression of either PSMα alone or in combination with PIPLC (PSMα-PIPLC) when compared with a positive control (pHLD-HLB; Giese et al., 2011).

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Fig. S3. Phagosomal escape of S. aureus strains in HeLa and EA.hy926. Phagosomal escape of the S. aureus strains 6850 (A, D), LAC (B, E) and MW2 (C, F) in HeLa epithelial cells (A, B, C) and EA.hy926 endothelial cells (D, E, F) was determined by flow-cytometric pH assessment in the bacterial microenvironment. Strongly negative ΔAFU values correlate with acidification of S. aureus containing phagosomes. In turn, less negative values at 6 h p.i. indicate translocation to the neutral cytoplasm. Data are displayed as means from at least 3 independent experiments performed in duplicate ± SEM. *P < 0.05, **P < 0.01. (G) Microscopic escape analyses with S. aureus LAC Δpsmα, Δpsmβ, as well as Δpsmαβ, a psmα and β double knock-out, corroborates the data as it shows drastically decreased escape efficiencies of S. aureus LAC only when PSMα was deleted. A knock-out of PSMβ or δ-toxin (Δhld) alone had no effect.

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Fig. S4. Differences in psm gene expression of strains 6850 and LAC. Relative expression levels (2ΔCT) as measured by quantitative real-time PCR from in vitro cultures demonstrate that in S. aureus 6850 (A) psmβ is expressed at higher levels than psmα, whereas in LAC (B) psmα expression is more prominent. Both strains display a distinct expression dynamics: whereas 6850 expresses PSMs at a high rate already early during bacterial growth (2 h), PSM transcription of strain LAC peaked at 5 h. Values were normalized to gyrB. Data shown are results of 3 independent experiments performed in triplicates ± SEM. *P < 0.05.

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Fig. S5. In escape-deficient S. aureus psmβ and agrA remain transcriptionally active. Relative expression levels (2ΔCT) of psmα, psmβ and agrA at 2 or 6 h p.i and of the inoculum as measured by quantitative real-time PCR in 293 cells infected with (A) S. aureus strains LAC (wt), LAC Δpsmα and LAC Δpsmβ or (B) S. aureus 6850 (wt) and 6850 Δpsmα. Data shown are results of 3 independent experiments performed in triplicates ± SEM. Expression was normalized to the housekeeping gene gyrase (gyrB). *P < 0.05, **P < 0.01.

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Fig. S6. Flow cytometric invasion assays demonstrate that invasiveness of LAC mutants does not differ. LAC as well as Δpsmα, Δpsmβ, Δpsmαβ and Δhld mutants were transformed with pmRFP resulting in red-fluorescent bacteria. Infections of HeLa were performed at an moi of 10 for 1 h. By comparison, all mutants invaded HeLa with similar levels of efficiency.

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

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Table S2. Plasmids used in this study.

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Table S3. List of oligonucleotides.

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