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

Treatment with, BAPTA-AM (A, n≥4), trans-Ned19 (B, n≥4), but not 2-APB and 8-Bromo-cADPR (B, n≥4) reduce S. aureus internalization by host cells. Genetic ablation of TPC1 (C, n=5) and SARM1 (D, n=8) reduced invasion of S. aureus 10 min p.i. HuLEC (A, B), HeLa WT (C, D), HeLa TPC1 K.O. (C) or HeLa SARM1 K.O. (D) 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. Letters indicate figure panels that supported the respective conclusion. Statistics: one sample t-test (A, B,D,), unpaired Student’s t-test (C). Bars represent means ± SD. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. Created in BioRender.

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

(A, B) Luminescence-based lysosomal exocytosis assay. HeLa cells expressing LAMP1-HiBit were treated with 1 µM ionomycin (immediately before the measurement) or 1 µM Vacuolin-1 (75 min before the measurement) in presence of LgBit and NanoLuc substrate. Lysosomal exocytosis was monitored by measuring chemiluminescence in a Tecan microplate reader. n=3 (C) S. aureus triggers lysosomal exocytosis during infection. HeLa cells expressing LAMP1-HiBit were pretreated for 75 min with 1 µM Vacuolin-1 or 200 µM trans-Ned19. Subsequently, cells were infected with S. aureus JE2 (MOI10), treated with 1 µM ionomycin or left untreated and lysosomal exocytosis was determined by measuring chemiluminescence in a Tecan microplate reader 10 min p.i. Luminescence detected in infected samples was scaled to untreated controls (0 %) and ionomycin (100%) to determine the proportion of all “releasable” lysosomes liberated during infection. n=6. (D-F) S. aureus invasion requires Syt7-dependent lysosomal exocytosis. HuLEC were treated with the lysosomal exocytosis inhibitor Vacuolin-1 or the lysosomal exocytosis inducer ionomycin and invasion efficiency of S. aureus was determined 30 min p.i. (D, E, n≥3). Invasion efficiency of S. aureus JE2 was determined in HeLa Syt7 K.O. cells 10 min and 30 min p.i. (F, n≥5; data normalized to HeLa wildtype). (G, H) 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 (G) and invasion efficiency of S. aureus was determined (n≥3). (G) 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 (H). n≥4. (I, J) Genetic ablation of ASM renders S. aureus invasion insensitive towards ARC39. HeLa ASM K.O. cells were treated with 10 µM ARC39 or were left untreated and the number of invaded bacteria was determined 30 min p.i.. Invasion efficiency was determined by normalization to wildtype HeLa cells (I) or to corresponding untreated samples (J, n≥4), (K) Genetic ablation of ASM is accompanied by massive alteration in cellular sphingolipid profiles. HeLa wildtype and ASM K.O. cells were treated with 20 µM amitriptyline (75 min) and 10 µM ARC39 (22h) or left untreated, as indicated. Whole cell sphingolipid profiles were determined by HPLC-MS/MS and the ratios of SM vs. Cer were calculated for lipid species with varying acyl side chains (chain length indicated on the x-axis). n= 4 (L) S. aureus invasion requires SM 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). (M) Experimental design for β-toxin treatment of host cells during S. aureus infection. Host cells were either pretreated with β-toxin to remove SM from the plasma membrane prior to infection (upper panel; Figure 2, L). Alternatively, β-toxin was added together with the bacteria to rescue the absence of ASM (lower panel, Figure 2, N). (N) Presence of extracellular SMase activity restores the 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. (O) Scheme of lysosomal exocytosis and ASM release as well as interfering agents. Cer: ceramide, SM: sphingomyelin, ASM: acid sphingomyelinase. xStatistics: one sample t-test (D-G, L), one-way ANOVA and Dunnett’s multiple comparisons test (C, J). Mixed-effects model (REML) and Šídák’s multiple comparisons (H). Two-way ANOVA and Tukey’s multiple comparison (K). Two-way RM ANOVA and Šídák’s multiple comparison (N) Bars represent means ± SD. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. Created in BioRender.

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 the 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. Created in BioRender.

Blocking ASM-dependent invasion affects phagosomal maturation and escape during S. aureus infection

(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 percentage of intracellular bacteria which associated 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-F) Blocking ASM-dependent internalization affects phagosomal escape. RFP-CWT and LyseninW20A -YFP expressing HeLa were treated with PCK310,ARC39 (C, F), amitriptyline (D) or Vacuolin-1 (E) and infected with S. aureus strains JE2 or Cowan I. By CLSM, proportions of bacteria that recruited RFP-CWT (phagosomal escape) were determined 3h p.i. if not indicated otherwise (n≥3). Statistics: one sample t-test (A), Mixed effects analysis (REML) and Tukey’s multiple comparison (C,), paired Student’s t-test (D, E) Graphs represent means ± SD. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. Created in BioRender.

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

(A-C) 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, B) and the percentage of phagosomal escape events that additionally were positive for LyseninW20A-YFP (C) were determined at indicated time points p.i. (n=5). (D, E) 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. (D). 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. (E). (F) Summary of experiments analyzing the influence of invasion pathway on phagosomal escape. The measured effects of different conditions on rapid ASM-dependent invasion and accompanied alterations in phagosomal escape rates are summarized. red = increased, blue=decreased. Statistics: Two-way ANOVA and Šídák’s multiple comparisons test (B-E). Graphs represent means ± SD. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. Created in BioRender.

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 were either 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 8. (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,(83)] and rapid internalization of S. aureus by host cells (7). Bacteria, which enter the host cells via the rapid pathway, experience a different intracellular fate than bacteria that employed other pathways. Statistics: Mixed effects analysis (REML) and Tukey‘s multiple comparison test. Graphs represent mean ± SD. *p≤0.05. Created in BioRender.

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) 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. (B) 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. (C, D) trans-Ned19 but not 2-APB affects invasion of S. aureus JE2 in HeLa. HeLa cells were pre-treated with trans-Ned19 (C) or 2-APB (D). 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. (E, F) 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 (E) or trans-Ned19 (F). 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. (G) 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. (H) CD38 is not involved in S. aureus internalization. HeLa cells were pretreated with the CD38 inhibitor 78c and invasion efficiency of S. aureus JE2 was determined. (I) 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. Created in BioRender.

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

(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) 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. (E) 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. (F, G) 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 (F) or ARC39 (G) in BHI medium and growth was determined by measuring OD600 in a microplate reader. (H, I) ASM activity and ASM inhibition by ARC39 and amitriptyline are 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 ASM activity assays, detecting the conversion of BODIPY-C12-SM (H), or by a flow cytometry-based ASM activity assay and conversion of visible-range FRET probe (I). (J) Microscopy-based measurement of invasion efficiency in amitriptyline- and PCK310-treated Hela cells. Hela cells were treated with 20 µM amitriptyline (75 min) or 0.5 µM PCK310 (4h) and infected with S. aureus JE2 expressing Cerulean. Number of intracellular bacteria per host cell was determined by CLSM. n=5. (K) Validation of ASM K.O. cell pools. ASM activity in HeLa wildytpe or ASM K.O.s was determined with the visible-range FRET probe and flow cytometry. The proportion of ASM-positive cells in the cell population was determined. n=4. (L) Removal of SM from the plasma membrane by β-toxin affects S. aureus host cell entry in HeLa. HeLa cells were pretreated with the indicated concentrations of β-toxin and subsequently the invasion efficiency of S. aureus JE2 was determined. (M) Treatment of HeLa with the bacterial SMases β-toxin results in generation of ceramide. HeLa cells were preloaded with 1 µM BODIPY-FL-C12-SM and treated with β-toxin for 75 min. Quantities of ceramide and SM were determined by TLC and ratio of ceramide vs. SM were calculated. Statistics: one sample t-test (D, J, L), mixed-effects model (REML) and Tukey’s multiple comparison (H, I), one-way ANOVA and Dunnett’s multiple comparison (K), unpaired Student’s t-test (M). Bars represent mean +/-SD. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. Created in BioRender.

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.). Created in BioRender.

Supporting information for phagosomal maturation and phagosomal escape measurements.

(A) amitriptyline treatment reduces the proportion of Rab7-positive bacteria. HeLa cells expressing YFP-Rab7 and mCherry-Rab5 were infected with S. aureus JE2 for the indicated time point and extracellular bacteria were removed with lysostaphin for 15 min. Then, samples were fixed and imaged by CLSM. Proportion of bacteria associated with YFP-Rab7 was determined. n=5. (B, C) 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 associated with Rab7 was determined (C). Results of treated samples were normalized to untreated controls (B, n=7). (D) 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). (E) 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) (F) 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). (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) was 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 (I) as well as the percentage of phagosomal escape events that were LyseninW20A -positive (J) were determined 3h p.i. (n=6). (K) Increasing the MOI compensates for lower numbers of invading bacteria at 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: One samples t-test (B, E), mixed effects analysis (REML) and Dunnett’s multiple comparison (D), Two-way ANOVA with Tukey’s multiple comparison (G), One-way ANOVA with Tukey’s multiple comparison (H), mixed effects analysis (REML) and uncorrected Fisher’s LSD (I), mixed effects analysis (REML) with Šídák’s multiple comparison (J).Bars represent mean +/-SD. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. Created in BioRender.

Validation of LyseninW20A-YFP and RFP-CWT reporter cell line

Hela cells expressing the SM reporter LyseninW20A-YFP(green) and the phagosomal escape reporter RFP-CWT (red) were either pretreated with 100 ng/ml bacterial SMase (A) or left untreated (B). Cells were infected with Cerulean-expressing S. aureus JE2 (cyan), extracellular bacteria were removed, and intracellular infection was monitored by time-lapse imaging at a Leica TCS SP5 microscope in 5 min intervals. White arrows indicate Lysenin-positive (A)or Lysenin-negative (B) escape events. Scale bars: 10 µm. Created in BioRender.

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 where host cells were treated with 100 ng/ml β-toxin prior to infection (G, J). Data are quantified in Figure 4, C. Created in BioRender.

Individual replicates of time-dependent phagosomal escape and replication assays

HeLa cells expressing RFP-CWT were pretreated with 20 µM amitriptyline or 10 µM ARC39, infected with S. aureus JE2 and infection was monitored by live cell imaging (for further details see Figure 6, A and B). The absolute numbers of all intracellular bacteria and bacteria that escaped from the phagosome, which were detected during live cell imaging, are depicted.

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. Created in BioRender.