Introduction

The most abundant sphingolipid, sphingomyelin (SM), resides in the extracellular leaflet of the plasma membrane and hence is exposed to the environment (1, 2). SM is a substrate for sphingomyelinases (SMases) that cleave off the phosphocholine head group to produce the non-polar ceramide. Acid sphingomyelinase (ASM) is a lysosomal enzyme involved in recycling of SM (3, 4) and is linked to infectious diseases, where it was shown to be involved in cellular uptake of several human pathogens (5–11).

Staphylococcus aureus is a Gram-positive human commensal that asymptomatically colonizes about one third of the human population (12). It is an opportunistic pathogen causing diseases ranging from soft tissue and skin infections (13) to lethal diseases (14, 15). S. aureus is notorious for acquiring antibiotic resistances thereby causing >100,000 annual deaths (16). S. aureus possesses intracellular virulence strategies (17, 18) that contribute to antibiotic resistance (19, 20) and immune evasion (21). Internalization of S. aureus by host cells is mediated by several adhesins on the staphylococcal surface that bind a plethora of receptors on the host cell plasma membrane (5, 22–32). For instance, staphylococcal fibronectin-binding proteins that form fibronectin bridges to α₅β₁ integrins on host cells to initiate internalization (22) or clumping factor B which can interact with the host receptor annexin A2 (25).

After internalization, S. aureus resides within a phagosome-like compartment that matures by acquiring proteins associated with early and then late endosomes as well as lysosomes (33–35). In epithelial and endothelial cells, the bacteria translocate to the host cytosol (“phagosomal escape”) where they replicate and cause host cell death (33, 36–38).

The mechanism by which pathogens enter their host cells has been suggested to dictate the outcome of infections (39–42). For instance, phagosomes formed during natural internalization of Brucella arbortus by macrophages possessed different characteristics compared to phagosomes that were artificially generated by phorbol myristate acetate (42). The protozoic parasite Toxoplasma gondii invades its host cells by a proactive mechanism. Phagosomes generated during active invasion differed from those that were generated during Fc receptor-mediated uptake of antibody-coated parasites (41). Since previous observation connecting host cell entry and intracellular fate of pathogens were based on artificial induction of internalization, it is unclear whether pathogens can enter host cells within the same population via different naturally occurring mechanisms and if the entry route has a direct influence on the outcome of an intracellular infection.

Here, we describe the internalization of S. aureus by tissue cells via a rapid pathway taking place within minutes after contact between bacteria and host cell surface. This pathway requires nicotinic acid adenosine dinucleotide phosphate (NAADP)-dependent mobilization of lysosomal Ca²⁺, followed by lysosomal exocytosis and thereby release of ASM. Since the rapid uptake is concurrent to previously known internalization pathways, it probably has been missed due to long infection times used in traditional infection protocols (e.g., (22, 25, 29, 30)).

S. aureus bacteria, which enter host cells of the same cell population via the rapid pathway, cause a distinct infection outcome with altered phagosome maturation, bacterial translocation to host cytosol and eventually host cell death when compared to bacteria that enter host cells at later time points during infection. Thus, the outcome of an infection is decided at the single cell level during bacterial uptake at the host plasma membrane. Bacteria-containing phagosomes that were formed by the ASM-dependent rapid uptake pathway are delayed in their maturation and the bacteria do not escape these organelles efficiently, hence demonstrating a direct link between concurrently active modes of bacterial uptake and the resulting outcomes of intracellular S. aureus infection.

Results

S. aureus triggers lysosomal Ca²⁺ mobilization for host cell invasion

We previously identified that internalization of S. aureus by host cells is associated with cellular Ca²⁺ signaling (43). To delineate the role of Ca²⁺ in this process mechanistically, we pretreated human microvascular endothelial cells (HuLEC) with the calcium ionophore ionomycin, which reduced bacterial invasion efficiency dose-dependently (Figure 1, A), at concentrations neither affecting bacterial (Supp. Figure 1, A, B) nor host cell viability (Supp. Figure 1, C).

Figure 1

Since focal adhesions constitute major cellular entry sites for S. aureus and the Ca²⁺-activated transient receptor potential ion channel 4 (TRPM4) is located therein, we inhibited TRPM4 with 9-Phenantrol. 9-Phenantrol reduced invasion efficiency (Figure 1, B), but was not bactericidal (Supp. Figure 1, D). Further, treatment of HuLEC (Figure 1, C) or HeLa (Supp. Figure 1, E) with the Ca²⁺ chelator BAPTA-AM (44) led to reduction of S. aureus invasion in a concentration-dependent manner.

The pore-forming staphylococcal α-toxin has been shown to mediate Ca²⁺ influx into host cells (45). However, S. aureus invasion was independent of toxin production in HeLa and only slightly reduced in endothelial cells (Supp. Figure 1, F). Consequently, omission of Ca²⁺ from the cell culture medium did not affect internalization of bacteria, indicating that invasion is not dependent on Ca²⁺ influx (Figure 1, C).

Since intracellular Ca²⁺ stores can serve as alternative source of Ca²⁺, we next blocked Ca²⁺ liberation from the ER using 2-APB (46) or 8-Bromo-cyclic ADP ribose (8-Bromo-cADPR; (47)) and from lysosomes using trans-Ned19 (48) in HuLEC (Figure 1, D) or HeLa (Supp. Figure 1, G, H) at concentrations not affecting bacterial viability (Supp. Figure 1, I and J). Interference with lysosomal, but not ER Ca²⁺ release reduced internalization of S. aureus.

Ned19 antagonizes NAADP, a second messenger that mediates opening of two pore channels (TPCs) which transport Ca²⁺ from the endo-lysosomal compartment into the cytosol (48). Hence, a HeLa cell pool depleted of TPC1 (Supp. Figure 1, K) substantially reduced invasion when compared to unmodified HeLa cells. This was particularly pronounced after a 10 min infection pulse, suggesting that Ca²⁺-dependent invasion is important early in infection (Figure 1, E).

CD38 is a well-known producer of NAADP in immune cells (49–51). Thus, we tested S. aureus invasion in HeLa cells treated with the specific CD38 inhibitor 78c, even though expression of CD38 in these cells is predicted to be very low (proteinatlas.org). We observed no effect on S. aureus invasion by CD38 inhibition (Supp. Figure 1, L). An alternative NAADP producer, with higher expression in our infection model, is Sterile Alpha and TIR Motif Containing 1 [SARM1, (52)]. We measured a slightly decreased invasion efficiency 10 min p.i. in a cell pool lacking SARM1 when compared to WT cells (Figure 1, F; Supp. Figure 1, M), suggesting that SARM1 might be enrolled in NAADP production during internalization of S. aureus by host cells.

Altogether, our findings support the important role of lysosomal Ca²⁺ mobilization in S. aureus invasion as particularly required early in infection.

Rapid S. aureus invasion requires lysosomal exocytosis, ASM and its substrate sphingomyelin on the host cell surface

Ionomycin treatment results in lysosomal exocytosis, a Ca²⁺-dependent process in which lysosomes release their content to the extracellular space (45, 53). Hence, we treated host cells with Vacuolin-1, an inhibitor blocking this process (54), which reduced invasion efficiency in HuLEC (Figure 2, A) and HeLa cells (Supp. Figure 2, A), while not affecting survival of the bacteria (Supp. Figure 2, B).

Figure 2

Synaptotagmin 7 (Syt7) is known to support fusion of lysosomes with the plasma membrane in a Ca²⁺-dependent manner (55). Accordingly, Syt7-depleted HeLa cells demonstrated lower bacterial invasion (Figure 2, B). As already observed for TPC1 (Figure 1, E), this was most pronounced early in infection further supporting our hypothesis that lysosomal exocytosis is important for rapid invasion events.

Lysosomal exocytosis results in release of ASM (45, 56). Therefore, prior to infection, the cells were exposed to amitriptyline, a functional inhibitor of ASM (FIASMA), or a competitive inhibitor [ARC39, (57)] at concentrations not affecting bacterial survival (Supp. Figure 2, C, D). While invasion in HuLEC, human umbilical vein endothelial cells (HuVEC) and HeLa was sensitive to both inhibitors, invasion in 16HBE14o^- and EA.hy926 was not or only marginally affected (Figure 2, C). We excluded that differences among cell lines arose from differential ASM activity or sensitivity to the inhibitors by monitoring enzymatic activity [Supp. Figure 2, E; (58) and Supp. Figure 2, F; (59)].

Next, we treated HuLEC with 10 µM ARC39 or 0.5 µM PCK310, a fast-acting ASM inhibitor. Already after 1h preincubation, PCK310 reduced invasion of S. aureus, whereas ARC39 required overnight treatment for a similar reduction (Figure 2, D). Taken together, these results show that ASM is involved in S. aureus invasion of host cells.

We removed SM, the substrate of ASM, from plasma membranes of HuLEC (Figure 2, E) and HeLa (Supp. Figure 2, G) by pretreatment with the bacterial SMase β-toxin. Most human pathogenic S. aureus strains including JE2, the strain we use in the present study, do not produce β-toxin, due to a genomic integration of a prophage (60). SMase pretreatment reduced invasion efficiency in a concentration-dependent manner in both cell lines. 10 ng/ml β-toxin were sufficient to decrease invasion to 34.37 ± 6.24% of that measured in untreated control cells. Interestingly, a 250-fold higher concentration did not lead to further reduction. In line with our previous experiments, this suggests that multiple S. aureus invasion pathways exist within the same host cell population, of which at least one requires SM and its enzymatic breakdown on the plasma membrane.

Next, we infected wild-type as well as the TPC1 or Syt7 K.O. cells with S. aureus in presence (but without pretreatment) of 100 ng/ml β-toxin and determined the number of intracellular bacteria after 10 min (Figure 2, F). Presence of β-toxin in the culture medium completely rescued the invasion defect of the K.O. cells. This indicates that reduced bacterial internalization in absence of TPC1 and Syt7 results from a lack in ASM delivery to the cell surface by Ca²⁺-dependent lysosomal exocytosis.

ASM- and Ca²⁺-mediated invasion is rapid

To study the role of SM in S. aureus uptake kinetics, we incubated HuLEC with fluorescent sphingolipid analogs and recorded S. aureus infection by time lapse imaging [Figure 3, A; Supp. Video 1; Supp. Figure 3; Supp. Video 2; (59)]. We observed that the bacteria were rapidly engulfed by SM-containing membrane compartments. While association of bacteria with BODIPY-FL-C12-SM increased over time during the first 30 min of infection, this was not observed for BODIPY-FL-C12-Ceramide, suggesting that S. aureus invasion specifically relies on interaction with SM in plasma membranes (Figure 3, B). Next, we determined the invasion efficiency of S. aureus in a time-dependent manner and blocked invasion by either ASM inhibitors, ionomycin, BAPTA-AM or β-toxin pretreatment. We found a time-dependent increase in intracellular bacteria, whereby all treatment conditions reduced bacterial invasion at every time point with the untreated control at 30 min set to 100% (Figure 3, C). By normalizing the data to the untreated control at the corresponding time points, we found that -with exception of β-toxin-the effect of the treatments was most pronounced early (5-10 min) after infection (Figure 3, D). Thus, early during infection bacteria enter the host cell predominantly in an ASM- and Ca²⁺-dependent manner.

Figure 3

The mode of S. aureus invasion affects infection outcome

After invasion, S. aureus resides within Rab5-positive early and subsequently, in Rab7-positive late phagosomes (33). We pretreated a HeLa cell line stably expressing mCherry-Rab5 as well as YFP-Rab7 with amitriptyline and infected with S. aureus for different time periods and determined the proportion of bacteria that associated with Rab5 and/or Rab7 (Figure 4, A).

Figure 4

While ASM inhibition marginally affected S. aureus association with Rab5-positive phagosomes, that with the Rab7-positive compartment was significantly reduced compared to the untreated control. This was restricted to shorter infection pulses (5-45 min) and was not observed for long infection periods (60 min). We also observed a reduced proportion of bacteria associating with Rab7-positive membranes after treatment with PCK310 or bacterial SMase. However, this was not caused by translocation of S. aureus to the host cytosol (Supp. Figure 4, A, B). Hence, phagosomes generated ASM-dependently possess different maturation dynamics when compared to phagosomes formed in an ASM-independent fashion.

Next, we measured phagosomal escape while simultaneously detecting SM content of disrupted phagosomal membranes. Therefore, we used a HeLa reporter cell line that cytosolically expresses the phagosomal escape reporter RFP-CWT (36) as well as the SM reporter Lysenin^W20A-YFP (61), respectively (Figure 4, B). Reporter gene expression in the cell line did not alter S. aureus invasion dynamics under different treatment conditions (Supp. Figure 4, C). The reporter cells were left untreated or were pre-exposed to ASM inhibitors and were infected with S. aureus strain JE2. In addition, S. aureus Cowan I, a strain known to have low escape rates, was used for infection. The proportion of bacteria that escaped from the phagosome (Figure 4, C) as well as the proportion of escape events that additionally were positive for cytosolic SM (Supp. Figure 4, D) were determined 1.5 h and 3 h p.i. (see also Supp. Figure 5, A-D).

Whereas no differences for Lysenin-positive escape events were detected (Supp. Figure 4, D), a higher proportion of bacteria escaped from phagosomes on abrogation of ASM activity regardless of the inhibitor used (Figure 4, C; Supp. Figure 4, E). A similar increase was observed when we blocked lysosomal exocytosis by Vacuolin-1 (Supp. Figure 4, F).

Since blocking of ASM-dependent uptake reduced the number of intracellular bacteria but increased their phagosomal escape rates, we hypothesized that the presence of SM on the plasma membrane affects downstream events during S. aureus infection. To address this question, we pretreated host cells with the bacterial SMase β-toxin to block SM-dependent invasion (Figure 4, D; 2a, 2b) and either infected the cells in presence (3a) or after removal of the toxin (3b). β-toxin pretreatment, resulted in enhanced phagosomal escape rates of S. aureus JE2, independent of the presence of β-toxin during infection (Figure 4, E; 4a, 4b). This was not observed for infection with the non-cytotoxic Cowan I strain, indicating that removal of SM by β-toxin did not affect general phagosomal membrane stability (Supp. Figure 4, G). Moreover, β-toxin treatment strongly reduced recruitment of Lysenin^W20A, demonstrating effective removal of SM from membranes by β-toxin (Figure 4, F and Supp. Figure 5, E-J).

To investigate if removal of SM was important at the cell surface or within the phagosome, we infected the cells with an S. aureus JE2 strain recombinantly overexpressing β-toxin and Cerulean, or a strain solely expressing Cerulean. We did not observe increased phagosomal escape upon overexpression of β-toxin (Figure 5, E; 4c), although SM levels of phagosomal membranes were reduced (Figure 5, F) comparably to samples pretreated with β-toxin (see above). Moreover, we did not detect substantial differences in escape rates of S. aureus 6850, a strain naturally producing β-toxin (Supp. Figure 4, H), and an isogenic β-toxin mutant. By contrast, escape of S. aureus 6850 again was enhanced upon pretreatment with β-toxin (Supp. Figure 4, I) accompanied by absence of SM from phagosomal membranes (Supp. Figure 4, J).

Figure 5

Phagosomal escape of S. aureus thus is independent of SM levels within the infected phagosomes, but rather reflects the presence of SM and ASM at the plasma membrane during host cell entry.

To demonstrate that phagosomal escape is dependent on the pathway of cell entry, we infected reporter cells with S. aureus for either a 10 min (“early invaders”) or a 30 min infection pulse (“early and late invaders”). Subsequently, we determined phagosomal escape rates (Figure 4, H) as well as the number of invaded bacteria (Supp. Figure 4, K). Independent of the multiplicity of infection (MOI) used, bacteria taken up within the first 10 min demonstrated significantly lower escape rates when compared to bacteria that were cocultured with host cells for 30 min.

This was corroborated by HeLa YFP-CWT infected with S. aureus expressing a fluorescent protein (e.g. mRFP) for 30 min (“early and late invaders”). After 20 min, we initiated a second infection pulse for 10 min (“early invaders”) with a S. aureus strain expressing another fluorescence protein (e.g. Cerulean) and determined phagosomal escape rates of both bacterial recombinants (Figure 4, I). Again, early invaders demonstrated lower escape rates when compared to the 30 min control. This was independent of the fluorescence expressed by the strain that was used for each infection pulse.

We next monitored phagosomal escape over 10 h in cells pretreated with ASM inhibitors (Figure 5, A). The compounds enhanced escape rates 3 h p.i. when compared to untreated controls. In contrast, 6 h p.i. escape rates were found decreased upon ASM inhibition.

Taken together, the efficiency of phagosomal escape of S. aureus is directly affected by the mode of cell entry, whereby the rapid SM/ASM-dependent pathway results in delayed phagosomal escape, while bacteria that entered host cells ASM-independently escape earlier during infection.

Since translocation to the host cytosol is a prerequisite for S. aureus replication in non-professional phagocytes, we recorded intracellular growth of S. aureus. In untreated controls replication started about 5 h p.i., and thus shortly after phagosomal escape (3-4 h p.i., Figure 5, B). By contrast, we did not observe significant bacterial replication in cells treated with ASM inhibitors (Figure 5, B).

We next treated HuLEC with β-toxin, stained the cells with BODIPY-FL-C12-SM and infected the cells with fluorescent S. aureus. Live cell imaging demonstrated that most untreated host cells died after 11 h p.i., whereas β-toxin-treated samples showed higher survival rates (Figure 5, C). This was also observed by cell death assays measuring i) membrane integrity (7-AAD staining, Figure 5, D), ii) apoptosis (Annexin V staining, Figure 5, E), iii) number of host cells that remained attached to the substratum (Supp. Figure 6, A) and iv) host cell lysis by LDH release (Supp. Figure 6, B).

Discussion

ASM previously has been implicated in the invasion of several viral, bacterial, and eukaryotic pathogens (5–11). However, the dynamics of ASM-mediated host cell entry, the underlying receptors as well as effects on post-invasion events are only poorly understood.

We here show that ASM is involved within minutes after the pathogen contacts host cells and is concurrent with ASM-independent uptake pathways. We demonstrate that the infection outcome of bacteria that enter the host cells via this rapid pathway is distinct from other entry mechanisms.

We observed a reduction of bacterial invasiveness upon treatment with different ASM inhibitors (Figure 2, C, D) or removal of the enzyme substrate SM from the plasma membrane (Figure 2, E) confirming that ASM activity on the host cell surface is crucial for pathogen internalization.

Release of ASM is caused by lysosomal exocytosis (56). Consequently, the inhibitor Vacuolin-1 as well as genetic ablation of Syt7, a key protein in lysosomal exocytosis (55), resulted in drastically reduced internalization of bacteria (Figure 2, A, B).

Elevation of cytosolic Ca²⁺ levels lead to recruitment of lysosomes to the cell surface (53). Whereas most studies implicated Ca²⁺ influx from the extracellular milieu (8, 10), we here demonstrate that S. aureus mainly triggers lysosomal Ca²⁺ mobilization as was shown by treatment with the inhibitor trans-Ned19 as well as a genetic K.O. of the endo-lysosomal Ca²⁺ channel TPC1 (Figure 1, D and E; Supp. Figure 1, G). TPCs have previously been associated with regulation of phagocytic processes (62) and host cell entry of several viruses such as Ebola virus (63), Middle East respiratory syndrome coronavirus [MERS-CoV, (64)] or SARS-CoV-II (65).

TPC-dependent Ca²⁺ liberation was shown to induce exocytosis, for instance, during release of cytolytic granules from T cells (66) or in interplay with TRPM4 during secretion of insulin (67). Consistently, the TRPM4 channel blocker 9-Phenantrol reduced internalization of S. aureus by host cells (Figure 1, B). Interestingly, exogenous addition of a bacterial SMase immediately before infection rescued the invasion defect in TPC1 and Syt7 K.O. cells (Figure 2, F) indicating that reduced invasion results from a decreased delivery of ASM to the host cell surface. Thus, NAADP-dependent Ca²⁺ liberation from lysosomal stores mediates Syt7-dependent fusion of lysosomes with the plasma membrane resulting in the release of ASM (Figure 5, F).

Inhibition of CD38, a known NAADP producer (49, 50), did not influence uptake of staphylococci (Supp. Figure 1, L). By contrast, we detected a decreased invasion in cells deficient for the Toll-like receptor adaptor protein SARM1, an alternative NAADP producer (52). However, the role of SARM1 in lysosomal exocytosis and ASM release requires further investigation. The impact of SARM1 on internalization of S. aureus was rather moderate, suggesting the existence of other NAADP-generating enzymes.

One important characteristic of the ASM-dependent internalization pathway is its velocity - taking place within the initial ten minutes of an infection with S. aureus, during which treatment with small molecules (Figure 3, D), as well as genetic ablation of TPC1 (Figure 1, E) and Syt7 (Figure 2, B) resulted in reduction of intracellular bacteria. This was accompanied by the quick formation of S. aureus-containing phagosomes decorated with fluorescent SM analogs (Figure 3, A; Supp. Figure 3, Supp. Video 1, Supp. Video 2).

When we blocked the rapid internalization pathway by ASM inhibitors (Figure 4, A, C; Supp. Figure 4, B), Vacuolin-1 (Supp. Figure 4, F) or by removal of surface SM (Figure 4, A, E; Supp. Figure 4, B, H), we observed changes in both, maturation of bacteria-containing vesicles and phagosomal escape. A delayed maturation of phagosomes may affect acidification of the vesicles, a property that is sensed by S. aureus leading to expression of a different subset of virulence factors (68) and hence may alter intracellular pathogenicity. Phagosomal escape of S. aureus only was dependent on SM on the plasma membrane during invasion but not on the presence of SM within the phago-endosome (Figure 4, D-G; Supp. Figure 4, H, I). This not only corroborated that phagosomal escape of clinically relevant S. aureus strains is independent of the bacterial SMase β-toxin (36), but also demonstrated that the outcome of phagosomal escape is influenced by processes at the plasma membrane during cell entry.

To exclude that inhibitor or bSMase treatments caused changes in phagosomal escape by affecting cellular processes other than bacterial uptake, we infected untreated WT cells with S. aureus for 10 or 30 min, respectively, and monitored phagosomal escape. Bacteria that invaded within the first 10 minutes after contacting host cells (“early invaders”) are predominantly ASM-dependent (Figure 3) and demonstrated significantly less phagosomal escape (Figure 4, G, H). Consistently, blocking the ASM-dependent pathway led to increased proportions of ASM-independent invaders, which in turn resulted in higher escape rates (Figure 4, C, E; Supp. Figure 4, F). Intracellular S. aureus infections thus are directly influenced by the mode of internalization. This ultimately resulted in lower host cytotoxicity (Figure 5, C-E; Supp. Figure 6) and bacterial replication (Figure 5, B) when we blocked the ASM-dependent invasion.

An uptake-related intracellular fate has been suggested for Mycobacterium bovis (39), Toxoplasma gondii (41) or Brucella abortus (42). However, in these studies the host cells were either depleted from cholesterol, which causes a variety of side effects (40), or internalization was artificially induced by chemicals or ectopic expression of receptors, respectively.

Taken together, we here describe a rapid internalization pathway for S. aureus into human epithelial and endothelial cells. Bacteria that enter the host cells via this infection route face a distinct intracellular fate. Here we show, to our knowledge, for the first time that host cells within the same population can be invaded by a pathogen via distinct routes and that this results in a differential infection outcome. Rapid internalization may prove beneficial for pathogens in an in vivo setting since internalization by host cells shortens the exposure to the innate immune system within a host. Interestingly, the absence of ASM in Smpd1^-/- mice enhanced the potency of antibiotics to clear S. aureus sepsis (69). It is tempting to speculate that reduced host cell invasion in absence of ASM caused a prolonged exposure of the pathogen to antibiotics. Several FIASMAs are already approved drugs (70), and may have future use in infection treatment.

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (DFG; https://www.dfg.de) within the research training group RTG2581 (M.R., F.S.). C.K. and C.A. are grateful to the DFG AR 376/22-1. M.R. was supported by funds of the Bavarian State Ministry of Science and the Arts and the University of Würzburg to the Graduate School of Life Sciences (GSLS), University of Würzburg. The DFG funded the Leica TCS SP5 CLSM under project code 116162193 and the BD FACSAriaIII cell sorter under project code 206080318.

We thank Sibylle Schneider-Schaulies and Thomas Rudel for valuable discussions and critically reading the manuscript, and Nadine Vollmuth as well as Christian Stigloher for scientific advice and intense discussions. We are indebted to Kathrin Stelzner for conducting FACS of cell lines and generation of S. aureus strains. pmRFP-LC3 was a gift from Tamotsu Yoshimori (Addgene plasmid # 21075; http://n2t.net/addgene:21075; RRID:Addgene_21075, (71)). pLVTHM (72), pMD2.G and psPAX2 (unpublished) were a gift from Didier Trono (Addgene plasmids # 12247, 12259 and 12260, respectively). pLX304-Flag-APEX2-NES was a gift from Alice Ting (Addgene plasmid # 92158, (73)) and pSpCas9(BB)-p2A-GFP (PX458) (74) was a gift from Feng Zhang (Addgene plasmid # 48138; http://n2t.net/addgene:48138). Figures were created in BioRender.

Additional information

Author Contributions

M.R., M.F. conceptualized the work, designed the methodology and wrote the original draft manuscript. M.F. acquired funding. M.R. F.S., K.U., M.P., A.M., N.K., J.W., A.I., C.K., K.P. performed the experiments; M.R., M.F. supervised the experiments. M.R., C.A., M.F. analyzed the data; all authors edited and revised the manuscript.

Declaration of interests

Patentability of PCK310 is currently evaluated by C.K. and C.A.

Materials and methods

Cell culture

HeLa (ATCC CCL-2^TM), 16HBE14o- (kindly provided by Prof. Jan-Peter Hildebrandt, University of Greifswald) and EA.hy 926 (75) were cultivated in RPMI+GlutaMAX^TM medium (Gibco^TM, Cat. No. 72400054) supplemented with 10% (v/v) heat-inactivated (56°C at 30 min) fetal bovine serum (FBS, Sigma Aldrich, Cat. No. F7524) and 1 mM sodium pyruvate (Gibco^TM, Cat.No. 11360088).

HuLEC-5a (ATCC CRL-3244^TM) and HuVEC (Gibco^TM, Cat. No.C01510C) were cultured in MCDB131 medium (Gibco^TM, Cat. No. 10372019) complemented with microvascular growth supplement (Gibco^TM, Cat. No. S00525), 2 mM GlutaMAX^TM (Gibco^TM, 35050061), 5 % (v/v) heat-inactivated (56°C at 30 min) FBS, 2.76 µM hydrocortisone (Sigma Aldrich, Cat. No. H0888), 0.01 ng/ml human epidermal growth factor (Pep Rotech, AF-100-15) and 1x Penicillin-Streptomycin (Gibco^TM, Cat. No. 15140122).

HeLa

HuLEC and HuVEC were detached by StemPro^TM Accutase^TM (Gibco^TM, Cat. No. A1110501) and seeded at the indicated density two days prior to the experiment, whereas HeLa, 16HBE14o-and EA.hy 926 were detached with TrypLE^TM (Gibco^TM, Cat. No. 12604013) and seeded one day prior to the experiment.

Generation of HeLa KO cell lines

Generation of K.O. cell lines is based on a previous protocol (74). sgRNAs (Table 1) were designed with the CHOPCHOP online tool (chopchop.cbu.uib.no) and synthesized as primer pairs (Sigma Aldrich). After phosphorylation with T4 nucleotide kinase (ThermoFisher, Cat. No. ER0031), the sgRNAs were cloned into the BbsI sites of pSpCas9(BB)-p2A-GFP (AddGene #48138, (74)) via forced ligation by BbsI (ThermoFisher, Cat. No. ER1012) and T4 Nulceotide ligase (ThermoFisher, Cat. No. EL0011). The constructs were then transformed in E. coli DH5α (ThermoFisher, Cat. No EC0112) and subsequently sequence verified.

Table 1

HeLa cells were transfected with 1 μg each of two distinct sgRNA constructs using JetPrime (Polyplus) following the manufacturer’s instructions. After 36-48h, cells were sorted for strong GFP expression in a FACS Aria III (BD Bioscience). The resulting cell pools were cultivated and tested for loss of expression of the respective sgRNA target by Western Blotting.

Following antibodies were used: mouse anti-PTK7 monoclonal IgG1 (bio-techne, Cat. No. MAB4499) with a horse radish peroxidase (HRP)-conjugated anti-mouse antibody (SantaCruz, Cat. No. sc-516102) and rabbit anti-TPC1 polyclonal (Thermo Fisher, Cat. No. PA5-41048) with an HRP-conjugated anti-rabbit antibody (Biozol, Cat. No. 111-035-144).

Generation of reporter constructs

pLV-mRFP-CWT. mRFP and the cell wall-targeting domain of lysostaphin CWT were amplified using primers MP-mRFP-f and euk_mRFP-r, as well as infus-CWT-f and infus-CWT-r, respectively. As PCR templates we used pmRFP-LC3 (PMID 17534139) and pLV-YFP-CWT (76). The resulting plasmid was termed pLV-mRFP-CWT. The PCR products were subsequently cloned into pLVTHM (72) restricted with PmeI and SpeI by InFusion cloning following the manufacturer’s recommendations.

pLV-YFP-Lysenin. The sequence of the sphingomyelin-binding but not pore-forming earthworm toxin mutant Lysenin^W20A (61) was synthesized by GeneArt (ThermoFisher) using a human codon usage and 5’ flanking region encoding a glycine-rich linker sequence. The synthesized template served as template for subsequent PCRs.

Lysenin^W20 was amplified using oligonucleotides InfReamp-f and hLysenin-infus-r (Table 1). YFP was amplified with Pm-eYFP-inf and eYFP-rev from template pLVTHM-YFP-CWT (76). Both PCR products were joined with pLVTHM vector restricted with PmeI and SpeI by InFusion cloning following the manuacturer’s recommendations, thereby creating pLV-YFP-Lysenin.

Constructs were transformed into E. coli DH5α and sequences were validated. Plasmid preparations were performed using standard laboratory procedures. VSV-G-pseudotyped Lentiviral particles were generated by transfecting with each lentiviral vector as well as the plasmids pMD2.G (VSV G) and psPAX2 following the protocol of (72) and target cells were transduced as described previously (76).

S. aureus culture

S. aureus liquid cultures were either grown in 37 g/L brain heart infusion (BHI) medium (Thermo Fisher, Cat. No. 237300) or in 30 g/l TSB medium (Sigma Aldrich, Cat. No. T8907). E. coli liquid cultures were grown in LB medium [10 g/l tryptone/peptone (Roth, Cat. No. 8952.4,) 5g/l yeast extract (Roth, Cat. No 2363.2), 10 g/l NaCl (Sigma Aldrich, Cat. No. S5886)].

For agar plates, 15 g/L agar (Otto Norwald, Cat. No. 257353), was added to either TSB (S. aureus) or LB (E. coli).

Media and plates were supplemented with appropriate antibiotics [5 µg/ml erythromycin (Sigma Aldrich, Cat. No. E5389), 10 µg/ml chloramphenicol (Roth, Cat. No. 3886.2), 100 ng/ml Carbenicillin (Roth, Cat. No. 6344.2)].

Strains used in this study are listed in Table 2.

Table 2

S. aureus growth curves

S. aureus overnight cultures were grown in BHI medium, and 1 ml was centrifuged at 14,000 for 1 min. Bacteria were washed thrice with DPBS and either resuspended in BHI (for amitriptyline and ARC39 treatment) or infection medium [MCDB131 medium (Gibco^TM, Cat. No. 10372019) complemented with 2 mM GlutaMAX^TM (Gibco^TM, 35050061) and 10 % (v/v) heat-inactivated (56°C at 30 min) FBS, for ionomycin treatment). Bacteria suspensions were diluted to OD₆₀₀=0.1 in the respective medium. Then, 400 µL per well of the suspension as well as respective blanks were transferred into a 48 well plate and OD₆₀₀ was determined every 16 min in Tecan mPlex200 microplate reader.

S. aureus infections

Host cells were seeded in 6 well plates (2x10⁵ cells per well), 12 well plates (1x10⁵ cells per well) or 24 well plates (0.5x10⁵ cell per well) either one day (HeLa, EA.hy 926, 16HBE14o^-) or two days (HuLEC, HuVEC) prior to the experiment. Host cells were washed thrice with DPBS and infection medium or Ca²⁺-free infection medium [DMEM w/o calcium (Gibco^TM, Cat. No. 21068028) complemented with 10 % (v/v) heat-inactivated (56°C at 30 min) FBS and 200 µM BAPTA (Merck Millipore, 196418)] with or without 1.8 mM CaCl₂ (Roth, Cat. No. 5239.1) was added. If indicated, cells were pretreated with compounds prior to infection (for details about treatments see Table 1). For experiments involving blocking of receptors by antibodies, respective solvent controls were implemented (final concentrations in infection medium: 0.002% (w/v) NaN₃ (Sigma Aldrich, Cat. No. S2002) 5% glycerol (Roth, Cat. No 3783.2) in 10% DPBS for 30 ng/ml anti-NRCAM antibody as well as well as 0.01 % (w/v) NaN₃ for 50 ng/ml anti-CD73 and 50 ng/ml anti-MELTF antibodies.

Table 3

An S. aureus overnight culture grown in BHI containing the appropriate antibiotics was diluted to an OD₆₀₀=0.4 in the same medium. When inducible expression of genes was required for the strains S. aureus JE2 pCer and S. aureus JE2 pCer+hlb, 200 ng/ml anhydrous tetracycline (AHT, AcrosOrganics, 233131000) was added. The culture was grown to an OD₆₀₀= 0.6-1.0 and 1 ml bacterial suspension was harvested by centrifugation and washed twice with Dulbecco’s Phosphate Buffered Saline (DPBS, Gibco^TM, Cat. No 14190169). The bacteria were resuspended in infection medium (or Ca²⁺-free infection medium when infection was performed in absence of extracellular Ca²⁺). The number of bacteria per ml in the suspension was determined with a Thoma counting chamber and the MOI was determined. If not indicated otherwise, an MOI=10 was used for infections.

The infection was synchronized by centrifugation 800xg/8 min/RT (end of the centrifugation: t=0). To determine bacterial invasion, the infection was stopped after 30 min (unless indicated otherwise) by removing extracellular bacteria with 20 µg/ml Lysostaphin (AMBI, Cat. No. AMBICINL) in infection medium for 30 min. Then, host cells were washed thrice with DPBS, lysed by addition of 1 ml/well (12 well plate) or 0.5 ml/well (24 well plate) Millipore water and the number of bacteria in lysates was determined by plating serial dilutions (10^-1, 10^-2, 10^-3) on TSB agar plates. Plates were incubated overnight at 37 °C and CFU were enumerated. To determine invasion efficiency, the number of bacteria determined in tested samples were normalized to untreated controls (set to 100%).

For measuring invasion dynamics, the number of bacteria was either normalized to the 30 min time point of untreated controls or to the corresponding time points of untreated controls.

If later time points in infection were investigated, infection medium containing 2 µg/ml Lysostaphin was added to the cells until the indicated time.

For testing bacterial susceptibility to inhibitors (“survival”) or adherence, Lysostaphin treatment was omitted, and host cells were immediately lysed by addition of Millipore water or washed five times with DPBS and subsequently lysed with Millipore water, respectively. The number of bacteria in lysates was determined by CFU counting (dilutions 10^-2, 10^-3, 10^-4) on TSB agar plates.

Phagosomal escape assays

For phagosomal escape assays with amitriptyline, ARC39, PCK310 and β-toxin, HeLa cells expressing RFP-CWT and Lysenin^W20A-YFP were infected with the indicated S. aureus strain as described in the previous paragraph. Infections with S. aureus JE2 pCer and S. aureus JE2 pCer+hlb were carried out in presence of 200 ng/ml AHT.

For phagosomal escape assays, HeLa cells expressing RFP-CWT and Lysenin^W20A-YFP were infected for 10 min (early invaders) or 30 min (early and late invaders) at the indicated MOIs.

For the phagosomal escape assays with two S. aureus JE2 strains expressing different fluorescence proteins, HeLa cells expressing YFP-CWT were infected with an MOI=5 of an initial S. aureus JE2 strain (S. aureus JE2 SarAP1 Cerulean or S. aureus JE2 SarAP1 mRFP). After 12 min of infection, a second infection pulse was initiated by adding the second S. aureus JE2 strain (expressing the complementary fluorescence protein, S. aureus JE2 SarAP1 mRFP or S. aureus JE2 SarAP1 Cerulean, respectively) and centrifuging the bacteria on the host cells. To exclude that the type of fluorescence protein expressed by the S. aureus strains contributes to the experimental outcome, either strain was used for the first and second infection pulse (each combination: n=4).

Phagosomal maturation assays

To monitor phagosomal maturation, HeLa cells expressing mCherry-Rab5 and YFP-Rab7 or YFP-Rab7 and RFP-CWT were infected with S. aureus JE2 SarAP1 Cerulean.

After 3 h p.i. (unless indicated otherwise), cells were washed thrice with DPBS and fixed with 0.2% glutaraldehyde (Sigma Aldrich, Cat. No. 10333) / 4% paraformaldehyde in PBS (Morphisto, Cat. No 11762.01000) for 30 min at RT. Then, cells were washed thrice with DPBS, stained with 5µg/ml Hoechst 34580 (Thermo Fisher, Cat. No. H21486) and mounted in Mowiol [24g glycerol (Roth, Cat. No. 3783.2), 9.6 g Mowiol^®4-88 (Roth, Cat. No 0713.2), 48 ml 0.2 M TRIS-HCl pH 8.5 (Sigma Aldrich, Cat. No T1503), 24 ml Millipore water].

Samples were imaged with a Leica TCS SP5 confocal microscope (Wetzlar, Germany; Software Leica LAS AF Version 2.7.3.9723) with a 40x immersion oil objective (NA1.3) and a resolution of 1024x1024 pixels [Cerulean (Ex. 458 nm/Em. 460-520), YFP (Ex. 514 nm/Em. 520-570nm), mRFP/mCherry (Ex. 561 nm/Em.571-635nm) and Hoechst 34580 (Ex. 405 nm/Em.410-460nm). At least 10 fields of view per samples were recorded.

Image analysis was performed in Fiji (81) using a previously described macro (80) that identifies and extracts individual bacteria from images as regions of interest (ROIs, see Supp. Figure 5). Subsequently, fluorescent intensity in every channel is measured to determine recruitment of fluorescence reporters to the bacteria. Results were exported as text files and proportion of bacteria that recruited individual reporters was determined with the Flowing 2 software (Turku Bioscience Center). Phagosomal escape rates were determined as the proportion of CWT-positive bacteria of the total number of intracellular bacteria. Similarly, the proportion of Lysenin^W20A-positive escape events, the ratio of Lysenin^W20A-/CWT-positive events and all CWT-positive events was calculated.

To measure the proportion of bacteria that were associated with Rab5 and/or Rab7, the proportion of Rab5- and/or Rab7-positive membranes around bacteria of all intracellular bacteria was determined. For experiments obtained with HeLa cells expressing YFP-Rab7 and RFP-CWT, bacteria that acquired RFP-CWT were removed from the dataset. Subsequently, the proportion of Rab7-positive bacteria in relation to all CWT-negative intracellular bacteria was determined.

Live cell imaging

The indicated host cells were seeded one (HeLa) or two days (HuLEC) prior to the experiments in µ-slide 8 well live cell chambers (ibidi, Cat. No 80826-90) with a density of 0.375x10⁵ cells per well. For monitoring host cell entry, cells were washed thrice with DPBS and were then, incubated for 90 min in infection medium with 1 µM BODIPY-FL-C₁₂-sphingomyelin (Thermo Fisher Cat. No. D7711), 10 µM BODIPY-FL-C₁₂-ceramide (Santa Cruz, Cat.No. sc-503923) or 10 µM visible-range FRET probe (59). Pretreatment with the FRET probe was performed in infection medium containing 1 % (v/v) FBS. Then, cells were washed thrice with DPBS and imaging medium [RPMI 1640 w/o phenol red (Gibco^TM, Cat. No. 11835030) containing 10% (v/v) heat-inactivated (56°C/30 min) FBS (Sigma Aldrich, Cat. No. F7524) and 30 mM HEPES (Gibco^TM, Cat.No. 15630080)] applied. Samples stained with the FRET probe were imaged in imaging medium containing 1% FBS and in presence of 10 µM FRET probe. Samples were infected with S. aureus JE2 SarAP1 Cerulean (FRET probe samples) or S. aureus JE2 SarAP1 mRFP (BODIPY-FL-C12-sphingomylein/-ceramide) at an MOI=50 without synchronization by centrifugation. Infection was monitored with a Leica TCS SP5 confocal microscope (Wetzlar, Germany) in intervals of 1 min by recording BODIPY-FL (Ex. 496 nm/Em. 500-535 nm), mRFP (Ex. 561 nm/Em.571) FITC (Ex. 488 nm/Em. 500-560), Cerulean (Ex. 458 nm/Em. 460-520), BODIPY-TR (Ex. 594 nm/Em. 610-680) and FRET (Ex.488 nm/ Em:610-680) channels. Cells were recorded with a 40x immersion oil objective and a resolution of 2048x2048 pixels. For samples stained with the FRET probe, 20 µg/ml lysostaphin was added 40 min p.i. to remove extracellular bacteria. For quantification of bacteria that associate with BODIPY-FL-C₁₂-sphingomyelin/-ceramide, individual bacteria in single frames were identified, extracted as ROIs and BODIPY-FL fluorescence in ROIs was measured in Fiji (81) as described before for phagosomal escape assays. Results were extracted as text files and the proportion of bacteria associating with BODIPY-FL was determined with Flowing2 (Turku Bioscience Center).

For monitoring intracellular S. aureus infection, host cells were pretreated and infected with S. aureus JE2 SarAP1 Cerulean (HeLa RFP-CWT/Lysenin^W20A-YFP) or S. aureus JE2 SarAP1 mRFP (HuLEC) as described in “S. aureus infection”. HuLEC additionally were treated with 1 µM BODIPY-FL-C₁₂-sphingomyelin in infection medium for 90 min and washed thrice with DPBS before the indicated treatment was applied. After extracellular bacteria were removed with 20 µg/ml lysostaphin for 30 min, imaging medium containing 2 µg/ml lysostaphin was applied and infections were monitored in intervals of 5 min (HeLa RFP-CWT) or 20 min (HuLEC). Phagosomal escape was evaluated in Fiji as described above. To determine intracellular replication, the number of bacteria was determined for each individual frame as described before (80). The number of bacteria at each time point was then normalized to the number of bacteria detected in the first frame to calculate the relative replication.

ASM and NSM activity assays

Thin layer chromatography (TLC)-based ASM/NSM activity assays were adapted from previously published protocols (58). To determine cellular ASM activity, the indicated cell lines were seeded in 24 well plates with a density of 1x10⁵ cell per well one day (HeLa, EA.hy 926, 16HBE14o^-) or two days (HuLEC, HuVEC) prior to the experiment. Cells were either treated with 20 µM amitriptyline or with 10 µM ARC39 for 22 h (fresh inhibitor was applied after 18h) in infection medium. Then, cells were washed thrice and lysed by addition of 100 µL per well ASM lysis buffer [250 mM NaOAc pH 5 (Roth, Cat. No 6773.2), 0.1% Nonidet^® P40 substitute (AppliChem. Cat. No. A1694,0250), 1.3 mM EDTA 1.3 mM (Roth, Cat. No. 8040.2), 1x protease inhibitor cocktail (Sigma Aldrich, 11873580001)] for 15 min/4°C. Cells were scraped from the substratum and protein concentration in the resulting lysates was measured with a Pierce^TM bicinchoninic acid (BCA) assay kit (Thermo Fisher, Cat. No. 23227). 1 µg of protein was incubated with 100 µL ASM lysate assay buffer [200 mM NaOAc pH 5 (Roth, Cat. No 6773.2), 0.02 % Nonidet^® P40 substitute (AppliChem. Cat. No. A1694,0250), 500 mM NaCl (VWR, Cat. No. 27810.364)] containing 0.58 µM BODIPY-FL-C₁₂-Sphingomyelin (Thermo Fisher, Cat. No. D7711) for 4h at 37°C and 300 rpm.

For measuring bacterial SMase/NSM activity in S. aureus cultures, an overnight culture of the indicated strain was grown in BHI medium, centrifuged 14.000xg and the supernatant was sterile filtered with 0.2 µm filter. 100 µL of sterile supernatant was incubated with 100 µL NSM assay buffer [200 mM HEPES, pH 7.0 (Roth, Cat. No. 6763.3), 200 mM MgCl₂ (Roth, Cat.No. 2189.2), 0.05% Nonidet P-40 (AppliChem. Cat. No. A1694,0250)] for 4h/37°C/300 rpm.

The reactions were stopped by addition of 2:1 CHCl₃:MeOH (Roth, Cat. No. 3313.2 and Cat. No. 8388.6). Samples were vortexed, centrifuged at 13,000 x g for 3 min and 50-100 µL of the lower organic phase were transferred to a fresh tube. Samples were completely evaporated using a SpeedVac 5301 concentrator (Eppendorf), resuspended in 10 µL 2:1 CHCl₃/MeOH and spotted in 2.5 µL aliquots on a TLC plate (Alugram, Xtra Sil G/UV254, 0.2 mm/silica gel 60; VWR, Cat. No. 552-1006).

Plates were developed using 80:20 CHCl₃ and subsequently scanned with a Typhoon 9200 Scanner (Amersham). For quantification, intensities of the lower (SM) and the upper bands (Cer) were measured in Fiji (81) and activity was determined based on the reaction time, protein amount and SM/Cer ratios. For the flow cytometry based read out, cells seeded in a density of 0.5x10⁵ cells per well in a 24 well plate either one day (HeLa, EA.hy 926, 16HBE14o^-) or two days (HuLEC, HuVEC) prior to the experiment. Cells were treated with ARC39 and amitriptyline as described above. Then, cells were washed thrice with DPBS and incubated with 10 µM FRET probe (59) for 2h in presence of the inhibitors. Subsequently, cells were washed thrice, detached with TrypLE^TM (Gibco^TM, Cat. No. 12604013) and resuspended with 2% (v/v) FBS in DPBS. Samples were analyzed for FITC (Ex. 488 nm/Em. band pass 530/30 nm) and BODIPY-TR (Ex.: 561nm/ Em. band pass 695/40nm) fluorescence with Attune NxT flow cytometer (Thermo Fisher, Attune Cytometric Software v5.2.0). Cell populations were analyzed for FITC and BODIPY-TR mean fluorescence in Flowing 2 (Turku Bioscience Center) and FITC vs. BODIPY-TR ratios were calculated to determine arbitrary probe conversion.

Cytotoxicity Assays

For all cytotoxicity assays, HuLEC were seeded in 24 well plates with a density of 0.5x10⁵ cells per well two days prior to the experiment. Then, cells were infected with S. aureus JE2 with the indicated MOI as described in “S. aureus infection” and cytotoxicity was determined 21 h p.i. For cytotoxicity measurements upon ionomycin treatment, cells were incubated with the indicated concentration of ionomycin in infection medium for 75 min and then, cytotoxicity assays were conducted.

Lactate dehydrogenase (LDH) assay was performed with the Cytotoxicity Detection KitPLUS (LDH, Sigma Aldrich, Cat. No. 4744934001) according to manufacturer’s instructions.

For annexin V and 7-Aminoactinomycin D (7-AAD) assays, cells were washed thrice with DPBS, detached with 250 µL per well trypsin and resuspended in 250 µL per well staining buffer [1.7% (v/v) APC Annexin V (BD Pharmingen^TM, Cat. No. 550475), 1.7% (v/v) 7-AAD (BD Pharmingen^TM, Cat No. 559925), 2% (v/v) heat-inactivated (56°C/30 min) FBS (Sigma Aldrich, Cat. No. F7524), 4 mM CaCl₂ (Roth, Cat. No. 5239.1) in DPBS]. After incubation for 10 min/RT, cells were analyzed with a Attune NxT flow cytometer for 7-AAD (Ex. 488 nm/Em. band pass 695/40 nm) and APC (Ex. 637 nm/Em. band pass 670/14 nm). Cell populations were analyzed with Flowing2 (Turku Biosciences Center) and gates were adjusted according to untreated control cells. To determine the proportion of cells that remained attached to the substratum during the infection, number of cells was determined based on the number of detected single cells, flow rate and sample volume.

Statistical analysis

Statistical analysis was performed in GraphPad prism (V10.1.2). One-sample t-test was used for analysis of normalized data sets. Otherwise, one- or two-way ANOVA, dependent on the number of variables, was used in combination with suitable multiple comparisons testing. Details about sample size and deployed statistical analysis can be found in respective figure legends. All data are shown as mean ± standard deviation.