S. aureus invasion is dependent on Ca2+ liberation from lysosomal stores and lysosomal exocytosis.

Treatment with Ionomycin (A, n≥3), 9-Phenantrol (B, n≥3), BAPTA-AM (C, n≥4), trans-Ned19 (D, n≥4), but not 2-APB and 8-Bromo-cADPR (D, n≥4) reduce S. aureus internalization by host cells in a TPC1 dependent fashion (E, n≥5). HuLEC (A, C, D, F), HeLa WT (B), HeLa TPC1 K.O. (E) or HeLa SARM1 K.O. (F) cells were treated with the respective substance and were subsequently infected with S. aureus JE2 for 30 min if not indicated otherwise. Extracellular bacteria were removed by lysostaphin, and the number of intracellular bacteria was determined by CFU counting. Results were normalized to untreated controls to obtain invasion efficiency (in percent of control). (F) scheme of host cell Ca2+ signaling and interfering agents. Statistics: one sample t-test (A-D, F), unpaired Student’s t-test (E). Bars represent means ± SD. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

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S. aureus invasion requires lysosomal exocytosis, ASM and plasma membrane sphingomyelin.

(A, B) S. aureus invasion requires Syt7-dependent lysosomal exocytosis. HuLEC were treated with the lysosomal exocytosis inhibitor Vacuolin-1 and invasion efficiency of S. aureus was determined 30 min p.i. (A, n=3). Invasion efficiency of S. aureus JE2 was determined in HeLa Syt7 K.O. cells 10 min and 30 min p.i. (B, n=5; data normalized to HeLa WT). (C, D) S. aureus internalization is dependent on ASM activity in endothelial and epithelial cells. Indicated cell lines were treated with the ASM inhibitors amitriptyline or ARC39 (C) and invasion efficiency of S. aureus was determined (n≥3). (D) HuLEC were treated with PCK310 or ARC39 for the indicated periods. Subsequently, cells were infected with S. aureus for 30 min and invasion efficiency was determined by CFU counting (n≥4). (E, F) S. aureus invasion requires SM and SMase activity on host cell plasma membranes. HuLEC were treated with the bacterial SMase β-toxin for 75 min and were subsequently infected with S. aureus JE2 for 30 min (n≥3). (F) Presence of extracellular SMase activity restores invasion defect in TPC1 and Syt7 K.O. cell lines. HeLa wild type as well as TPC1 or Syt7 KO cell lines were infected with S. aureus JE2 in presence of 100 ng/ml of the bacterial SMase β-toxin and invasion efficiency 10 min p.i. was determined (n=4). In each experiment the numbers of intracellular S. aureus JE2 were determined by lysostaphin protection assay and CFU counting. Results obtained for the tested condition were normalized to the wildtype cell line or the mock-treated control. (G) scheme of lysosomal exocytosis and ASM release as well as interfering agents. Cer: ceramide, SM: sphingomyelin, ASM: acid sphingomyelinase. Statistics: one sample t-test (A-C, E), one-way ANOVA and Dunnett’s multiple comparisons test (D). Two-way ANOVA and Tukey’s multiple comparison (F). Bars represent means ± SD. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

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ASM- and Ca2+-dependent uptake is rapid and predominantly mediates invasion early in infection.

(A, B) S. aureus associates with SM early during invasion. HuLEC were pretreated with BODIPY-FL-C12-SM or BODIPY-FL-C12-ceramide and infected with red-fluorescent S. aureus. Scale bar: 5 µm. Infection was monitored by live cell imaging and the proportion of bacteria that associated with the lipid analogs was quantified in each individual frame (SM: n=5, Cer: n=3). (C, D) ASM and Ca2+-dependent invasion is rapid. HuLEC were treated with ARC39, amitriptyline, BAPTA-AM, ionomycin, or the bacterial SMase β-toxin. Then, cells were infected with S. aureus and the number of invaded bacteria was determined after different time periods. The results were either normalized to 30 min time point of untreated controls (C) or to the corresponding time points of the untreated controls (D). (n ≥5). Statistics: Mixed effect analysis and Tukey‘s multiple comparison. Graphs represent means ± SD. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

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The intracellular fate of S. aureus is determined by the pathway of cell entry.

(A) Inhibition of ASM delays formation of Rab7-positive phagosomes. HeLa cells expressing mCherry-Rab5 and YFP-Rab7 were either treated with amitriptyline or left untreated. Then, cells were infected with S. aureus JE2 for indicated periods. Extracellular bacteria were removed and proportion of intracellular bacteria associating with Rab5 or Rab7 was determined by CLSM. Results obtained for amitriptyline-treated samples were normalized to untreated controls (n=5). (B) Detection of phagosomal SM and phagosomal escape by a reporter cell line expressing LyseninW20A -YFP and RFP-CWT. After internalization, S. aureus resides in a phagosome preventing the recruitment of RFP-CWT to S. aureus cell wall and LyseninW20A-YFP to luminal SM, respectively. When the bacteria lyse the phagosomal membrane, luminal SM gets exposed to the cytosol and attracts LyseninW20A -YFP, while RFP-CWT is recruited to the staphylococcal surface. (C) Blocking ASM-dependent internalization affects phagosomal escape. RFP-CWT and LyseninW20A -YFP expressing HeLa were treated with PCK310, or ARC39 and infected with S. aureus strains JE2 or Cowan I. Thereby, β-toxin was either removed prior to infection (pretreatment only) or was present during the whole experiment (pretreatment + during infection). By CLSM, proportions of bacteria that recruited RFP-CWT (phagosomal escape) were determined (n≥3). (D-F) Phagosomal escape depends on presence of plasma membrane SM during invasion, but not presence of SM within phagosomal membranes HeLa RFP-CWT LyseninW20A -YFP were pretreated with β-toxin to remove surface SM (2a, 2b) or left untreated (2c). Then, cells were infected with S. aureus JE2 in presence (3a) or absence (3b) of β-toxin. Untreated samples (3c) were infected with S. aureus JE2 harboring a plasmid either encoding β-toxin and the fluorescence protein Cerulean (pCer+hlb) or solely Cerulean (pCer). Proportion of bacteria that recruited RFP-CWT (phagosomal escape, E) and the percentage of phagosomal escape events that additionally were positive for LyseninW20A-YFP (F) were determined at indicated time points p.i. (n=5). (G, H) Early ASM-dependent invaders possess lower escape rates than late invaders. HeLa cells expressing RFP-CWT were infected with indicated MOIs of S. aureus JE2 either for 10 min (early invaders) or 30 min (early+late invaders). Phagosomal escape rates were determined 3h p.i. (G). HeLa reporter cells expressing YFP-CWT were infected with an MOI=5 of S. aureus JE2 expressing a fluorescent protein (e.g. RFP) for 30 min (early+late invaders). After 20 min, the same samples were infected with S. aureus JE2 expressing another fluorophore (e.g. Cerulean) for 10 min (early invaders) and phagosomal escape was determined 3 h p.i. (H). Statistics: one sample t-test (A), Mixed effects analysis (REML) and Tukey’s multiple comparison (C,G, H), Two-way ANOVA and Šídák’s multiple comparisons test (E, F). Graphs represent means ± SD. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

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Blocking ASM-dependent internalization affects intracellular replication and host cell survival.

(A, B) HeLa cells expressing RFP-CWT and LyseninW20A-YFP were treated with amitriptyline or ARC39 or were left untreated (Ctrl). The cells were subsequently infected with S. aureus and infection was monitored for 10 h by CLSM. Proportion of escaped bacteria (A) or the intracellular replication (B) was determined (n= 5). (C) HuLEC were stained with BODIPY-FL-C12-SM (green), and either were pretreated with 100 ng/ml of β-toxin or were left untreated. Then, cells were infected with S. aureus (red) and infection was monitored by CLSM for 19 h. (D, E) HuLEC were treated with amitriptyline or β-toxin and subsequently infected with S. aureus. After 21 h, plasma membrane integrity was measured by 7-AAD staining (D), and proportion of apoptotic cells was determined by annexin V staining (E) (n=5). See also Supp. Figure 6. (F) Model: S. aureus interacts with an unknown primary receptor (1) that triggers production of NAADP, which in turn activates TPC1 to mediate lysosomal Ca2+ release and activation of Syt7 (2). Syt7-dependent lysosomal exocytosis (3) leads to release of ASM (4) and production of Cer on the plasma membrane (5) thereby resulting in the recruitment of co-receptors (6) and rapid internalization of S. aureus by host cells (7). Bacteria that enter the host cells via the rapid pathway experience a different intracellular fate than bacteria that entered via other pathways. Statistics: Mixed effects analysis (REML) and Tukey‘s multiple comparison test. Graphs represent mean ± SD. *p≤0.05.

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Oligonucleotides used in this study

Bacterial Strains used in this study

Host cell treatment with various compounds

Supporting information for Ca2+-dependent internalization of S. aureus.

(A) High ionomycin concentrations interfere with growth of S. aureus JE2. S. aureus JE2 was grown in in presence of varying concentrations of ionomycin. Growth (OD600) was determined in a microplate reader. (B) Ionomycin does not affect survival of S. aureus JE2 during infection of host cells. HeLa cells were pretreated with varying concentrations of ionomycin and subsequently, infected with S. aureus JE2. After 30 min, cells were lysed and surviving extra- and intracellular bacteria were recovered by CFU count. (C) Ionomycin does not affect host cell survival. HeLa cells were treated with the indicated concentrations of ionomycin, and cytotoxicity was determined by measuring in lactate dehydrogenase (LDH) release. (D) 9-Phenantrol does not affect survival of S. aureus JE2. HeLa cells were pretreated with varying concentrations of 9-Phenantrol and infected with S. aureus JE2. After 30 min, cells were lysed and surviving extra- and intracellular bacteria were recovered by CFU plating. (E) Chelation of intracellular but not presence of extracellular Ca2+ affects S. aureus internalization by HeLa cells. HeLa cells were preloaded with varying concentrations of the cell-permeable Ca2+ chelator BAPTA-AM and then infected with S. aureus JE2 in presence (+1.8 mM CaCl2) or absence (Ca2+-free) of Ca2+ in the cell culture medium. Invasion efficiency was determined by CFU plating. (F) α-toxin plays only a subordinate role during S. aureus uptake by host cells. HeLa, HuLEC or HuVEC were infected with S. aureus JE2 wildtype or a strain deficient in α-toxin production (Δhla). Invasion efficiency was determined by CFU plating. The number of intracellular bacteria was normalized to samples infected with the wildtype. (G, H) trans-Ned19 but not 2-APB affects invasion of S. aureus JE2 in HeLa. HeLa cells were pre-treated with trans-Ned19 (G) or 2-APB (H). 2-APB was removed from the cells shortly before infection to avoid direct contact of inhibitor and bacteria (due to a bactericidal effect of 2-APB; data not shown). Then, cells were infected with S. aureus JE2 and invasion efficiency was determined. (I, J) trans-Ned19 and 2-APB have no effect on S. aureus JE2 in our infection protocol. HeLa cells were pre-treated with or 2-APB (I) or trans-Ned19 (J). 2-APB was removed from the cells shortly before infection. Then, cells were infected with S. aureus JE2 and, after 30 min, total bacteria (extra- and intracellular) were recovered by CFU plating. (K) Reduction of TPC1 expression in a Cas9-treated cell pool (HeLa TPC1 K.O.). TPC1 as well as GAPDH (loading control) was detected in lysates of wildtype and TPC1 K.O. cells via Western blot. (L) CD38 is not enrolled in S. aureus internalization. HeLa cells were pretreated with the CD38 inhibitor 78c and invasion efficiency of S. aureus JE2 was determined. (M) Reduction of SARM1 expression in a Cas9-treated cell pool (HeLa SARM1 K.O.). TPC1 as well as GAPDH (loading control) was detected in lysates of wildtype and SARM1 K.O. cells via Western blot. Statistics: one sample t-test, Bars represent mean +/- SD. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

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Supporting information for the involvement of lysosomal exocytosis and ASM in S. aureus invasion

(A) Blocking lysosomal exocytosis by Vacuolin-1 reduces S. aureus invasion in HeLa. HeLa cells were treated with increasing concentrations of the lysosomal exocytosis inhibitor Vacuolin-1. The invasion efficiency of S. aureus JE2 was determined 30 min p.i. (B) Vacuolin-1 has no bactericidal effect. HeLa cells were treated with Vacuolin-1 and infected with S. aureus JE2. After 30 min, total bacteria (extra- and intracellular) were recovered by CFU plating and normalized to untreated controls. (C, D) The ASM inhibitors amitriptyline and ARC39 have no effect on growth of S. aureus. S. aureus JE2 was grown in presence of varying concentrations of amitriptyline (C) or ARC39 (D) in BHI medium and growth was determined by measuring OD600 in a microplate reader. (E, F) ASM activity and ASM inhibition by ARC39 and amitriptyline is similar among human cell lines. Several cell lines were treated with amitriptyline or ARC39 and ASM activity within cell lysates was assessed by either thin layer chromatography-based TLC-based ASM activity assays, the conversion of BODIPY-C12-SM (E), or by a flow cytometry-based ASM activity assay and conversion of visible-range FRET probe (F). (G) Removal of SM from the plasma membrane by β-toxin affects S. aureus host cell. HeLa cells were pretreated with the indicated concentrations of β-toxin and subsequently the invasion efficiency of S. aureus JE2 was determined. Statistics: one sample t-test (A, G), mixed-effects model (REML) and Tukey’s multiple comparison (E, F), Bars represent mean +/- SD. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

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S. aureus associates with a visible-range SM probe during host cell invasion.

(A) Live cell imaging of S. aureus infection reveals association of the bacteria with a SM analog during cell entry. HuLEC were treated with 10 µM of a visible range FRET probe in presence of 1% FBS. Then, cells were infected with S. aureus JE2 in presence of the probe and infection was monitored by live cell confocal imaging. After 40 min, lysostaphin was added to remove extracellular bacteria. The BODIPY-TR signal demonstrates the localization of the probe, the FRET signal indicates an intact, non-metabolized probe, whereas FITC fluorescence indicates probe cleavage by ASM. Bacteria of interest (white arrows) adhere to host cell between 29 and 31 min p.i. (B) Hypothetical model of S. aureus invasion. (1). The interaction of bacteria with the host cell surface triggers lysosomal exocytosis, release of ASM (2) and the uptake of the bacteria (cmp. to A: at 37-39 min p.i.) by ASM-dependent membrane remodeling (3). ASM is co-internalized together with bacteria (4) and subsequently, cleaves the probe within the S. aureus-containing phagosome (5) (cmp: to A: increasing FITC signal starting at 47 min p.i.).

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Supporting information for phagosomal maturation and phagosomal escape measurements.

(A) Phagosomal escape assay in early infection. A HeLa reporter cell line expressing YFP-Rab7 and RFP-CWT was treated with amitriptyline, β-toxin and PCK310 and subsequently infected with S. aureus JE2 for 30 min. Extracellular bacteria were removed by lysostaphin and the proportion of bacteria that acquired the phagosomal escape marker RFP-CWT was determined 45 and 60 min p.i. (n=8). (B) Reduced association of S. aureus with Rab7 is not due to changes in phagosomal escape. HeLa cells expressing YFP-Rab7 and the phagosomal escape marker RFP-CWT were treated with amitriptyline, PCK310 or β-toxin and subsequently, infected with S. aureus JE2 for 45 min and analyzed by CLSMs. Bacteria that escaped from phagosomes were subtracted from the data set and the proportion of residual bacteria associating with Rab7 was determined. Results of treated samples were normalized to untreated controls (n=7). (C) β-toxin treatment and ASM inhibition reduced invasion efficiency of S. aureus JE2 in a dual reporter HeLa cell line. A HeLa reporter cell line expressing RFP-CWT and LyseninW20A-YFP was treated with the indicated concentrations of β-toxin and amitriptyline for 75 min, the indicated concentrations of ARC39 for 22 h, 0.5 µM PCK310 for 4h or 50/100 nM PCK310 for 22h (o.n.) and invasion efficiency of S. aureus JE2 was determined. (n≥5) (D) Inhibition of ASM has no influence on the proportion of LyseninW20A-positive escape events. The dual reporter cell line was infected with S. aureus JE2. Extracellular bacteria were removed and the proportion of bacteria that acquired the phagosomal escape marker RFP-CWT as well as the SM reporter LyseninW20A-YFP was determined by CLSM. The proportion of escaped (RFP-CWT-positive) bacteria that additionally acquired LyseninW20A-YFP was calculated. (n=9). (E) ASM inhibition by amitriptyline increases phagosomal escape of S. aureus JE2. The dual reporter cell line was infected with S. aureus JE2 and phagosomal escape rates were determined 3h p.i. (n=8). (F) Blocking lysosomal exocytosis by Vacuolin-1 enhanced phagosomal escape. HeLa RFP-CWT/LyseninW20A-YFP was pretreated with 1 µM Vacuolin-1 and infected with S. aureus JE2 for 10 min. Phagosomal escape rates were determined by CLSM 3 h p.i. (n=4). (G) β-toxin pretreatment increases phagosomal escape of S. aureus JE2 but not S. aureus Cowan I. RFP-CWT and LyseninW20A -YFP expressing HeLa were treated with 100 ng/ml β-toxin infected with S. aureus strains JE2 or Cowan I. Thereby, β-toxin was either removed prior to infection (pretreatment only) or was present during the whole experiment (pretreatment + during infection). By CLSM, proportions of bacteria that recruited RFP-CWT (phagosomal escape) were determined (n≥3). (H) S. aureus strains vary in β-toxin expression. The neutral SMase activity in culture supernatants of the indicated S. aureus strains or isogenic β-toxin mutants (Δhlb) were determined by a TLC-based approach (n=3). (I, J) β-toxin pretreatment of host cells but not β-toxin expression of endocytosed S. aureus affects phagosomal escape. HeLa RFP-CWT/LyseninW20A-YFP were infected with S. aureus 6850 or an isogenic strain deficient in β-toxin production (Δhlb). The proportion of bacteria that escaped from the phagosome as well as the percentage of phagosomal escape events that were LyseninW20A -positive were determined 3h p.i. (n=6). (K) Increasing the MOI compensates for shorter infection times. HeLa cells were either infected for 10 min or 30 min with varying MOIs. Then, the number of intracellular bacteria per host cell was determined 3h p.i. by CLSM. (n=7). Statistics: Mixed effects analysis (REML) with Šídák’s multiple comparison (A, G), Mixed effects analysis (REML) and Tukey’s multiple comparison (I, J), One-way ANOVA with Tukey’s multiple comparison (H), one samples t-test (B, C), unpaired Student’s t-test (E, F). Bars represent mean +/- SD. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

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Evaluation of phagosomal escape and LyseninW20A recruitment during S. aureus infection.

HeLa reporter cells expressing RFP-CWT and LyseninW20A-YFP were infected with S. aureus JE2, fixed and imaged by CLSM (A). Individual bacteria within the images were identified as regions of interest (ROIs) and extracted in each color channel. All extracted ROIs are depicted in a montage (B). If bacteria signals are omitted, the montage of extracted ROIs shows the proportion of reporter recruitment. The average fluorescence intensity of both reporter fluorophores is measured within each individual ROIs is plotted as dot plots (D). From these results, the proportion of bacteria that recruited the markers (CWT-RFP = phagosomal escape and LyseninW20A = SM-rich-phagosome) is calculated. (E-J) Exemplary escape assay. Montages of extracted ROIs (without bacterial fluorescence) as well as the resulting intensity measurement in individual ROIs 3 h p.i. are depicted for infections of S. aureus Cowan I (E, H) and JE2 (F, I) as well as an infection with S. aureus JE2 in which host cells were treated with 100 ng/ml β-toxin prior to infection (G, J). Data are quantified in Figure 4, C.

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Inhibition of ASM and β-toxin treatment increase number of host cells remaining attached to the substratum and reduce host cell lysis during infection.

HuLEC were treated with amitriptyline or β-toxin (A) or PCK310 and β-toxin (B) and were then infected with S. aureus JE2. After 30 min, extracellular bacteria were removed and the number of cells remaining attached to the substratum was determined (A) or the proportion of host cells that were lysed during the infection was measured by lactate dehydrogenase (LDH) release (B). Statistics: Mixed-effects analysis and Dunnett’s multiple comparison (A, B). Bars represent mean +/- SD. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

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