Orchestration of human NLRP3 inflammasome activation by Staphylococcus aureus extracellular vesicles

Xiaogang Wanga, William J. Eagena, and Jean C. Leea,1

aDivision of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115

Edited by Richard P. Novick, New York University School of Medicine, New York, NY, and approved January 2, 2020 (received for review September 11, 2019) Release of extracellular vesicles (EVs) is a common feature among presents a barrier for EV release (8). However, specific eukaryotes, archaea, and . However, the biogenesis and mechanisms involved in EV biogenesis and the complex downstream biological effects of EVs released from gram-positive downstream effects of EV release on host cells remain poorly bacteria remain poorly characterized. Here, we report that EVs characterized. purified from a community-associated methicillin-resistant Staph- S. aureus EVs package a diverse array of bacterial compo- ylococcus aureus strain were internalized into human macro- nents, including cytosolic, surface, and membrane proteins, as phages in vitro and that this process was blocked by inhibition well as adhesins, lipoproteins, and secreted PFTs (8, 10–12), and of the dynamin-dependent endocytic pathway. Human macro- many of these components have been shown to play significant phages responded to S. aureus EVs by TLR2 signaling and activa- roles in bacterial virulence (13–16). S. aureus EVs were detected + tion of NLRP3 inflammasomes through K efflux, leading to the in vivo during experimental pneumonia (10), and EVs purified recruitment of ASC and activation of -1. Cleavage of pro– from S. aureus strains have been shown to contain biologically interleukin (IL)-1β, pro-IL-18, and gasdermin-D by activated active , exhibit cytotoxicity, and elicit proinflammatory caspase-1 resulted in the cellular release of the mature mediators (8, 10, 11, 17, 18). Thus, EVs may play a previously IL-1β and IL-18 and induction of . Consistent with this unrecognized role in staphylococcal pathogenesis although the result, a dose-dependent response was detected in the mechanisms whereby this occurs are unknown. extracellular fluids of mice challenged intraperitoneally with S. NLRP3 inflammasomes, multimeric cytosolic protein com- aureus EVs. Pore-forming toxins associated with S. aureus EVs plexes formed in myeloid cells in response to various pathogenic were critical for NLRP3-dependent caspase-1 activation of human or physiological stimuli, require two distinct steps for activation , but not for TLR2 signaling. In contrast, EV-associated (19). A bacterial stimulus such as lipoprotein or lipopolysac- lipoproteins not only mediated TLR2 signaling to initiate the priming charide (LPS) triggers a priming step through TLR2- or TLR4- step of NLRP3 activation but also modulated EV biogenesis and the mediated nuclear factor κB (NF-κB) signaling, resulting in the content of EVs, resulting in alterations in IL-1β,IL-18,and production of pro–interleukin (IL)-1β and pro-IL-18 and tran- caspase-1 activity. Collectively, our study describes mechanisms by scription and posttranslational modification of NLRP3. A second which S. aureus EVs induce inflammasome activation and reveals an stimulus, such as bacterial toxins or adenosine 5′-triphosphate + unexpected role of staphylococcal lipoproteins in EV biogenesis. EVs (ATP), activates the inflammasome through K efflux, leading may serve as a novel secretory pathway for S. aureus to transport protected cargo in a concentrated form to host cells during infec- Significance tions to modulate cellular functions. The relevance and biological activities of extracellular vesicles Staphylococcus aureus | extracellular vesicles | inflammasomes | (EVs) from gram-positive bacteria are poorly understood. We lipoproteins | pore-forming toxins report that EVs released by Staphylococcus aureus are in- ternalized by human macrophages by an endocytic process, taphylococcus aureus is a primary cause of invasive human highlighting the role of EVs as a delivery system for bacterial Sinfections, such as bacteremia, endocarditis, pneumonia, and virulence determinants. Macrophages incubated with S. aureus surgical wound , leading to morbidity, mortality, and EVs undergo NLRP3 inflammasome activation that is dependent excessive healthcare costs (1). Many S. aureus isolates have de- on EV cargo, including pore-forming toxins and lipoproteins. We veloped resistance to commonly used antibiotics (2). To establish provide evidence that S. aureus lipoproteins modulate EV con- a successful and survive in a hostile host environment, tent and biogenesis, revealing a previously unrecognized role S. aureus employs a wide array of virulence determinants, in- for lipoproteins. This study advances our understanding of the cluding surface proteins and glycopolymers, as well as secreted biological activities of EVs from gram-positive bacteria and proteins, such as pore-forming toxins (PFTs), , and demonstrates their role as a vehicle for the delivery of microbial proteases. Such factors are either associated with the surface effector molecules into host cells. to facilitate colonization or secreted to the environment to Author contributions: X.W. and J.C.L. designed research; X.W. and W.J.E. performed re- damage host cells and evade innate and adaptive host immune search; X.W. and J.C.L. analyzed data; and X.W. and J.C.L. wrote the paper. mechanisms (3–5). The authors declare no competing interest. Extracellular vesicles (EVs) are nano-sized, spherical particles This article is a PNAS Direct Submission. that are enclosed by a bilayered membrane. Most cells secrete vesicles, including eukaryotes, archaea, and bacteria (6). Gen- Published under the PNAS license. Data deposition: Mass spectrometry proteomics data have been deposited in the Proteo- eration of EVs from gram-positive bacteria is a complex and meXchange Consortium, http://proteomecentral.proteomexchange.org/cgi/GetDataset poorly understood process since EVs released from the cyto- via the PRIDE partner repository (dataset identifier PXD014888). plasmic membrane must traverse a thick peptidoglycan cell wall 1To whom correspondence may be addressed. Email: [email protected]. – to reach the external environment (7 9). We demonstrated that This article contains supporting information online at https://www.pnas.org/lookup/suppl/ S. aureus alpha-type phenol-soluble modulins and autolysins pro- doi:10.1073/pnas.1915829117/-/DCSupplemental. mote EV production whereas highly cross-linked peptidoglycan First published January 27, 2020.

3174–3184 | PNAS | February 11, 2020 | vol. 117 | no. 6 www.pnas.org/cgi/doi/10.1073/pnas.1915829117 Downloaded by guest on October 2, 2021 to the recruitment of ASC and activation of caspase-1, resulting quenched at high concentrations in the and in cleavage of pro–IL-1β and pro–IL-18. NLRP3 activation is “dequenched” when the probe is diluted by membrane fusion. characterized by cellular release of the mature cytokines IL-1β Thus, R18 fluorescence reflects an increase in EV internalization and IL-18 and induction of an inflammatory cell death termed or membrane fusion. The uptake of 5 μg/mL R18-labeled EVs by pyroptosis, a host defense mechanism allowing removal of human monocyte-derived MΦs after different incubation time damaged or infected host cells (20, 21). points was assessed by confocal microscopy. Fluorescence of Inflammasome activation plays an essential role in protection R18-labeled EVs, but not the sham control, was detected within against S. aureus infections (22, 23), particularly in mounting an MΦs after 45 min (SI Appendix, Fig. S1B), and longer incubation effective innate immune response, which may determine the of cells with EVs led to a greater distribution of fluorescent EVs outcome of infection by controlling the bacterial burden and within cells. The sham-treated control showed minimal fluores- shaping the nature and magnitude of the adaptive immune cence after 90 min (SI Appendix, Fig. S1B). Consistent with the response (24). Unregulated inflammasome activation, however, results obtained from the DiO-labeling experiment, pre- may result in an exaggerated innate immune response that treatment of THP-1 cells or monocyte-derived human MΦs with leads to host tissue damage (25). S. aureus culture supernatants, dynasore, but not the other inhibitors, prevented cellular entry of containing both secreted PFTs and lipoproteins, activate EVs (SI Appendix, Fig. S1 C). Quantification of cellular fluores- inflammasomes in vitro by providing both the priming and cence confirmed a significant (>fivefold) reduction in the R18- secondary stimulus (26, 27), but culture supernatants are not EV signal in MΦs treated with dynasore (SI Appendix, Fig. S1D). representative of the in vivo environment. Of note, S. aureus Subsequently, THP-1 MΦs were pretreated with endocytosis cells, purified PFTs, or lipoproteins alone are not sufficient to inhibitors or DMSO before incubation with R18-labeled EVs, activate the NLRP3 inflammasome (27). We postulate that and fluorescence was measured over time. THP-1 cells fluo- EVs serve as a unique S. aureus secretion system that transports resced within 40 min after the addition of R18-labeled EVs (SI its protected cargo, including lipoproteins and PFTs, to host Appendix, Fig. S1E). Pretreatment of THP-1 cells with dynasore, cells. but not DMSO or CPZ, resulted in a significant reduction in the In this study, we provide evidence that EVs released by S. fluorescent signal at each time point (SI Appendix, Fig. S1E). aureus cells are internalized by human macrophages (MΦs) via Together, these data indicate that internalization of EVs in MΦs an endocytic pathway and induce NLRP3 inflammasome- is primarily dependent upon dynamin-mediated endocytosis. dependent pyroptosis and IL-1β and IL-18 production. We demonstrate that EV-associated PFTs play a critical role in S. aureus EVs Activate Caspase-1 in Human Macrophages and Induce + NLRP3 activation through K efflux whereas EV lipoproteins the Release of IL-1β and IL-18. Previous studies demonstrated that MICROBIOLOGY prime inflammasome activation through TLR2 signaling but also S. aureus cutaneous infection induces IL-1β production by acti- modulate the biogenesis and PFT content of EVs. Our study vation of NLRP3 inflammasomes in myeloid cells (22, 30). provides critical insights into the role of staphylococcal lipo- Furthermore, S. aureus EVs were shown to elicit skin barrier proteins in EV production and characterizes mechanisms by disruption in mice with characteristic atopic dermatitis-like skin which S. aureus EVs induce MΦ inflammasome activation. (31). Because staphylococcal EV cargo includes both PFTs and lipoproteins (8), we postulated that EVs may play Results a critical role in inflammasome activation during infection. To EVs Are Internalized by Human Macrophages by an Endocytic Process. test this hypothesis, THP-1 MΦs were incubated for 4 h with Although numerous studies have reported the release of EVs increasing concentrations of purified S. aureus JE2 EVs. As from S. aureus, the interactions between EVs and host cells re- shown in Fig. 2A, EV concentrations ≥0.5 μg/mL resulted in main poorly characterized. We predicted that EVs were in- cellular release of IL-1β and IL-18. In time course experiments, ternalized within MΦs and that internalization played a critical secretion of IL-1β by THP-1 MΦs was detected as soon as 4 h role in modulating the host innate immune response. To test this after treatment with 0.5 μg/mL EVs (Fig. 2B). To confirm pro- hypothesis, we labeled JE2 EVs with the 3,3′-dioctadecylox- teolytic cleavage of the precursor pro–IL-1β, THP-1 MΦs were acarbocyanine perchlorate (DiO), a highly fluorescent lipophilic incubated with 0.8 μg/mL EVs for 12 h, and cleaved IL-1β from dye that has been widely used for tracing the internalization of the culture supernatants and pro–IL-1β from cell lysates were membrane vesicles (28). The uptake of 2 μg/mL DiO-labeled detected by immunoblot. Cleaved IL-1β was induced by JE2 EVs EVs by differentiated MΦ-like THP-1 cells after different in- or positive control LPS+ATP, but not by untreated cells (Fig. cubation time points was assessed by confocal microscopy. The 2C). Because release of mature IL-1β and IL-18 depends on fluorescence of EVs, but not the sham control (SI Appendix, Fig. caspase-1–mediated cleavage of the precursor molecules, we S1A), was detected within THP-1 cells after 60 min (Fig. 1A), measured caspase-1 activity in THP-1 supernatants. Activation and longer incubation times resulted in a greater distribution of of caspase-1 was consistently induced within 4 h by EV con- fluorescent EVs within cells, peaking after ∼90 min. To determine centrations ≥0.5 μg/mL (Fig. 2D). Similar experiments per- how cellular uptake of EVs occurred, MΦs were pretreated with formed with human monocyte-derived MΦs revealed that a inhibitors of clathrin-mediated endocytosis (chlorpromazine [CPZ]), higher concentration of EVs was necessary to induce secretion of raft-mediated endocytosis (methyl-β-cyclodextrin [MβCD]), IL-1β and IL-18 (Fig. 2E). Nonetheless, both IL-1β (Fig. 2F) and the solvent dimethyl sulfoxide (DMSO), or inhibitors of dynamin- caspase-1 activity (Fig. 2G) were induced within 8 h after in- dependent endocytosis (dynasore) or -dependent endocytosis/ cubation of monocyte-derived MΦs with 5 μg/mL EVs. macropinocytosis (cytochalasin D) before incubation with DiO- To test whether EVs would induce IL-1β production in vivo, labeled EVs. Pretreatment of THP-1 cells (Fig. 1B)ormonocyte- mice were injected intraperitoneally with 65 μg of purified JE2 derived human MΦs(Fig.1C) with dynasore, but not CPZ, MβCD, EVs. Cytokine levels in mouse serum and peritoneal washes were DMSO, or cytochalasin D, inhibited cellular entry of EVs. Quan- measured by -linked immunosorbent assay (ELISA) from tification of EV-positive THP-1 cells (Fig. 1D) or cellular fluores- samples taken from 4 to 48 h postinjection. At the earliest time cence of monocyte-derived human MΦs(Fig.1E)confirmeda point (4 h), mice given EVs showed a strong proinflammatory significant (>threefold) reduction intheDiO-EVsignalinMΦs signature characterized by elevated levels of IL-6 in both the treated with dynasore. serum and peritoneal fluid (SI Appendix, Fig. S2A), consistent To validate this result, we labeled JE2 EVs with the lipophilic with the lipoprotein content of S. aureus EVs (8). IL-1β was dye octadecyl rhodamine B chloride (R18) (29); a sham-treated detected in the peritoneal fluid but not in mouse serum at the 4-h dye control was incubated without EVs. R18 fluorescence is time point (SI Appendix, Fig. S2B). The in vivo response was

Wang et al. PNAS | February 11, 2020 | vol. 117 | no. 6 | 3175 Downloaded by guest on October 2, 2021 Fig. 1. Dynamin-dependent endocytosis of S. aureus EVs by human MΦs. (A) Confocal micrographs of THP-1 cells incubated with 2 μg/mL DiO-labeled EVs for different time periods. (Scale bars: 10 μm.) (B) Confocal micrographs of THP-1 cells pretreated with or without indicated inhibitors for 1 h, followed by in- cubation for 90 min with 2 μg/mL DiO-labeled S. aureus EVs. (Scale bars: 10 μm.) (C) Confocal micrographs of human monocyte-derived MΦs pretreated with or without indicated inhibitors for 1 h, followed by incubation for 90 min with 5 μg/mL DiO-labeled S. aureus EVs. (Scale bars: 10 μm.) (D) Percentage of EV uptake by THP-1 cells treated with or without the indicated inhibitors (n = 10). (E) Relative EV uptake by monocyte-derived human MΦs treated with or without the indicated inhibitors (n = 35 to 60). Image data are representative of two independent experiments. Data from D and E were analyzed by one-way ANOVA with Dunnett’s multiple comparison test. NS, not significant; **P < 0.01, ****P < 0.0001.

rapid as cytokines were not elevated in samples collected at time response was observed in both sera and peritoneal fluid samples points later than 4 h (SI Appendix, Fig. S2 A and B). In sub- (Fig. 2H) taken from mice given S. aureus EVs. An IL-1β re- sequent experiments, mice were challenged with either 65 μgof sponse was detected in the peritoneal fluid, but not in the serum bovine serum albumin (BSA) or 10, 30, or 65 μgofS. aureus EVs, (Fig. 2I). These data suggest that S. aureus EVs activate and samples were collected after 4 h. An IL-6 dose-dependent inflammasomes in vivo in a rapid and dose-dependent manner.

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Fig. 2. Inflammasome activation in vitro and in vivo by S. aureus EVs. (A) Dose-dependent release of IL-1β and IL-18 in culture supernatants of THP-1 MΦs incubated for 4 h with purified EVs (n = 4). (B) Time course of IL-1β production by THP-1 MΦs treated with 1 μg/mL EVs (n = 4). (C) Immunoblot showing cleaved IL-1β in culture supernatants and pro-IL-1β and actin (control) in cell lysates of THP-1 MΦs treated for 12 h with 1 μg/mL EVs. Media alone and LPS+ATP were assay controls (n = 2). (D) Detection of cleaved caspase-1 in supernatants of THP-1 MΦs treated for 4 h with EVs (n = 4). RLUs, relative light units. (E) Dose-dependent release of IL-1β and IL-18 from monocyte-derived human MΦs incubated for 8 h with purified EVs (n = 4). (F) Time course of IL-1β production by monocyte-derived MΦs treated with 5 μg/mL EVs (n = 4). (G) Detection of cleaved caspase-1 in supernatants of monocyte-derived human MΦs that were incubated for 8 h with increasing concentrations of S. aureus EVs (n = 4). Data pooled from at least two independent experiments were analyzed by one-way ANOVA with Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (H) IL-6 or (I) IL-1β levels in the sera and peritoneal fluids (PFs) collected at 4 h from C57BL/6 mice injected intraperitoneally with 10, 30, or 65 μg of purified JE2 EVs or 65 μg of BSA (n = 8).

NLRP3 Inflammasomes and Caspase-1 Are Essential for S. aureus EV- ASC is a key adaptor molecule required for caspase-1 acti- Mediated Release of IL-1β and IL-18 from Human Macrophages. IL-1β vation via NLR inflammasomes (19). Upon inflammasome ac- and IL-18 release from THP-1 cells incubated with ATP+LPS or tivation, ASC is recruited from the nucleus to the where S. aureus EVs was significantly reduced when the cells were it self-aggregates and forms “specks” ∼1 μm in diameter (32, 33). pretreated with the NLRP3 inhibitor MCC950 or the caspase-1 Treatment of THP-1 MΦs with either S. aureus EVs or the inhibitor VX765 (Fig. 3A). Likewise, THP-1 cells deficient in positive control ATP induced the formation of ASC specks that NLRP3 or caspase-1 abrogated IL-1β release induced by EVs were visualized by confocal microscopy (SI Appendix, Fig. S3). (Fig. 3B) but had no effect on IL-6 release (Fig. 3C). IL-6 pro- Blockage of NLRP3 inflammasome activation by MCC950 duction is not dependent on NLRP3 activation since it is gen- resulted in a significant reduction in EV-induced ASC speck Φ erated from the interactions of EV-associated lipoproteins (or formation in THP-1 M s(SI Appendix, Fig. S3 and Fig. 3F). Taken together, our results indicate that EV-induced secretion the control TLR2 ligand Pam3CSK4) with TLR2 and either of the proinflammatory cytokines IL-1β and IL-18 by human TLR1 or TLR6, resulting in downstream NF-κB pathway acti- MΦs is dependent upon NLRP3 inflammasome activation. vation. These results were verified in monocyte-derived human MΦs by treating the cells with MCC950 or VX765 before in- EV-Associated S. aureus Pore-Forming Toxins Play a Critical Role in cubation with 5 μg/mL EVs. Consistent with the results from NLRP3 Inflammasome Activation. S. aureus EVs carry an array of THP-1 MΦs, the inhibitors significantly reduced release of IL-1β PFTs, including alpha toxin (Hla) and a family of (8), and IL-18 in monocyte-derived human MΦs (Fig. 3D) but had no which are positively regulated by the accessory gene regulator effect on IL-6 production (Fig. 3E). (agr) quorum sensing system and the SaeR/S two-component

Wang et al. PNAS | February 11, 2020 | vol. 117 | no. 6 | 3177 Downloaded by guest on October 2, 2021 Fig. 3. NLRP3 inflammasome activation by S. aureus EVs is essential for induction of cytokines IL-1β and IL-18 in human MΦs. (A) Levels of IL-1β and IL-18 in culture supernatants of THP-1 MΦs that were treated with or without NLRP3 inhibitor MCC950, caspase-1 inhibitor VX765, or DMSO for 1 h prior to in- cubation for 4 h with an LPS+ATP control or 1 μg/mL EVs (n = 4 to 8). Levels of IL-1β (B) and IL-6 (C) in culture supernatants of THP-1, THP-1 (Cas9), NLRP3- deficient THP-1 (NLRP3 KO), or caspase-1–deficient THP-1 (Caspase-1 KO) MΦs that were incubated for 4 h (IL-1β) or 12 h (IL-6) with 1 μg/mL EVs or LPS+ATP (n = 4 to 6). (D and E) Concentrations of IL-1β and IL-18 (D) or IL-6 (E) in culture supernatants of monocyte-derived human MΦs treated with or without MCC950, VX765, or DMSO for 1 h prior to incubation for 8 h (IL-1β and IL-18) or 2 h (IL-6) with 5 μg/mL EVs (n = 4 to 6). (F) Quantification of the percentage of THP-1 cells with detectable ASC specks. More than 400 cells from at least 10 different fields were counted for ASC speck formation in each treatment. Data pooled from three independent experiments were analyzed by one-way ANOVA with Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

system (34). Mutation of either regulator abrogates the expres- IL-1β and IL-18 release from THP-1 MΦs incubated for 4 h sion of most PFT genes and reduces the cytotoxicity of EVs (8). with 1 μg/mL EVs purified from Δsae, Δagr,orΔagrΔsae mutants Incubation of THP-1 MΦs with 1 μg/mL S. aureus JE2 EVs over was significantly reduced compared to that of cells incubated 24 h resulted in significant cellular toxicity as measured by lactate with WT EVs (Fig. 4B). In contrast, IL-6 production resulting dehydrogenase (LDH) release (SI Appendix, Fig. S4A). EVs from TLR2 activation by EV-associated lipoproteins was similar purified from Δsae, Δagr,orΔagrΔsae JE2 mutants showed re- for THP-1 cells incubated with EVs from the WT or mutant duced cytotoxicity toward THP-1 cells (SI Appendix, Fig. S4B), a strains (Fig. 4C). To assess proteolytic cleavage of pro–IL-1β result that was confirmed by dose-dependent treatment of THP- resulting from EV treatment, THP-1 MΦs were treated with 1 μ Δ Δ 1MΦs with EVs from the wild type (WT) or the ΔagrΔsae g/mL EVs purified from JE2 or the agr sae mutant. Cleaved β mutant (SI Appendix, Fig. S4C). Monocyte-derived human MΦs IL-1 and caspase-1 in the THP-1 culture supernatants and pro- β treated with WT JE2 EVs for 8 h also showed a dose-dependent IL-1 and caspase-1 in cell lysates were detected by immunoblot. – β release of LDH (SI Appendix, Fig. S4D), and EVs purified from Although pro IL-1 and caspase-1 were present in lysates from Δ Δ cells treated with EVs purified from JE2 and JE2ΔagrΔsae, JE2 agr sae showed reduced cytotoxicity toward these cells (SI β Appendix, Fig. S4E). These results suggest that EVs purified cleaved IL-1 and cleaved caspase-1 were detected primarily in Δ Δ cells incubated with WT EVs (Fig. 4D). These results were val- from the agr sae mutants packaged minimal amount of PFTs. Φ LDH release is a marker of pyroptosis, which is initiated by idated by treatment of monocyte-derived human M s with EVs ΔagrΔsae inflammasome-mediated caspase-1 activation and cleavage of from the WT or mutant. Only WT JE2 EVs induced MΦ release of IL-1β and IL-18 (Fig. 4 E) and caspase-1 activation gasdermin D (GSDMD) to its active form, resulting in pore (Fig. 4F). In contrast, IL-6 release was induced by monocyte- formation and cell death (35). Inhibition of NLRP3 inflamma- derived MΦs treated with EVs from either the WT or the some activation by MCC950 resulted in significant reductions in Δ Δ Φ + agr sae mutant (Fig. 4G). LDH release from THP-1 M s treated with LPS ATP or WT S. aureus JE2 produces five PFTs: LukAB, Hla, LukSF-PV, EVs (SI Appendix, Fig. S5A). Likewise, THP-1 cells deficient in HlgAB, and HlgCB, and LukAB, Hla, and LukSF-PV are the NLRP3 or caspase-1 released lower levels of LDH induced by S. most potent in inflammasome activation (36). THP-1 MΦs were aureus EVs (SI Appendix, Fig. S5B). To investigate whether EV- treated for 4 h with 1 μg/mL EVs purified from WT JE2 or single associated PFTs are critical for pyroptosis, THP-1 cells were PFT mutants (ΔlukAB, Δhla,orΔlukSF-PV). As shown in Fig. Δ Δ incubated with EVs from JE2 or the agr sae mutant or 4H, release of IL-1β from THP-1 MΦs incubated with EVs puri- + LPS ATP, and cleaved GSDMD was detected by immunoblot. fied from individual toxin mutants was significantly reduced Although cleaved GSDMD was detected in supernatants and compared to that of cells treated with WT EVs. As a follow-up lysates from THP-1 cells incubated with EVs purified from JE2 experiment, THP-1 cells were incubated with EVs purified from or JE2ΔagrΔ4Asae (Fig. ), the levels of cleaved GSDMD in cells the JE2ΔagrΔsae mutant alone or complemented with the genes treated with the WT EVs were higher than that of cells treated encoding individual PFTs: LukAB, Hla, or LukSF-PV. Com- with EVs from JE2ΔagrΔsae (SI Appendix, Fig. S5C). These plementation with lukAB or hla partially restored the production findings suggest that S. aureus EVs induce inflammasome- of IL-1β from THP-1 MΦs treated with ΔagrΔsae EVs (Fig. 4I). mediated pyroptosis in MΦs and that EV-associated PFTs en- Whereas individual PFTs contribute to NLRP3 inflammasome hance this process. activation mediated by S. aureus EVs, our results indicate that

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Fig. 4. Activation of NLRP3 inflammasomes by S. aureus EVs is dependent upon PFTs. (A) Immunoblot showing cleaved N-terminal GSDMD in culture supernatants and cell lysates of THP-1 cells incubated for 12 h with 1 μg/mL EVs from the WT or ΔagrΔsae mutant. Media alone and LPS+ATP were assay controls (n = 2). (B and C) Levels of IL-1β and IL-18 (B) or IL-6 (C) in culture supernatants of THP-1 MΦs incubated for 4 h (IL-1β and IL-18) or 12 h (IL-6) with or without EVs purified from the indicated S. aureus strains or positive controls LPS+ATP and Pam3CSK4 (TLR2 ligand) (n = 4 to 6). (D) Immunoblot showing cleaved IL-1β and cleaved caspase-1 in THP-1 culture supernatants and caspase-1 in cell lysates of THP-1 cells incubated for 12 h with 1 μg/mL EVs from the WT or ΔagrΔsae mutant. Media alone and LPS+ATP were assay controls. *, background immunoblot band (n = 2). (E–G) Levels of IL-1β and IL-18 (E), active caspase-1 (F), or IL-6 (G) in culture supernatants of human monocyte-derived MΦs incubated for 8 h (IL-1β and IL-18) or 2 h (IL-6) with 5 μg/mL EVs purified from indicated strains, LPS+ATP, or Pam3CSK4 (n = 4 to 6). (H [n = 11] and I [n = 6]) Levels of IL-1β in culture supernatants of THP-1 MΦs that were incubated for 4 h with or without EVs purified from indicated S. aureus strains. (J and K) Detection of IL-1β (J) or active caspase-1 (K) in culture supernatants of THP-1 MΦs treated with or without indicated concentrations of KCl for 1 h prior to incubation for 4 h with 1 μg/mL EVs or LPS+ATP (n = 4). (L) Concentrations of IL-1β and IL-18 in culture supernatants of human monocyte-derived MΦs that were treated with or without 130 mM KCl for 1 h prior to incubation for 8 h with 5 μg/mL EVs (n = 4). Data pooled from at least three independent experiments were analyzed by one-way ANOVA with Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

+ the constellation of PFTs produced by this USA300 strain pro- of IL-1β and IL-18 can be attributed to PFT-mediated K efflux. vides maximal NLRP3 inflammasome activation. Moreover, we show that EV-associated PFTs play an essential To assess whether EV-induced NLRP3 inflammasome acti- role in caspase-1 activation and enhance NLRP3-mediated + vation was dependent on K efflux, THP-1 MΦs were pretreated pyroptosis, but are not involved in TLR2 signaling nor in EV with increasing concentrations of potassium chloride (KCl) for 1 internalization within MΦs. h before a 4-h stimulation with S. aureus EVs. A dose-dependent inhibition of EV-mediated IL-1β release (Fig. 4J) and caspase-1 Localization of PFTs in S. aureus EVs. It is not known whether S. activity (Fig. 4K) was observed in THP-1 cells pretreated with aureus EV-associated PFTs are localized within the EV lumen or KCl. Similarly, pretreatment of monocyte-derived human MΦs present at the EV surface. In the latter case, pore formation on with 130 mM KCl blocked release of IL-1β and IL-18 induced by the MΦ membrane could occur independent of EV in- + S. aureus EVs (Fig. 4L). ternalization, inducing K -mediated inflammasome activation. A To rule out the possibility that the impaired activation of proteinase K susceptibility assay was employed to detect surface inflammasomes in MΦs was due to a defect in EV internalization, localization of three PFTs that are abundant in purified EVs (8): THP-1 MΦs were incubated for 90 min with DiO-labeled EVs Hla, LukS-PV, and LukAB. As shown by silver-stained sodium purified from the ΔagrΔsae mutant. As shown in SI Appendix,Fig. dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) S6, EVs purified from the mutant were efficiently internalized (SI Appendix, Fig. S7A), treatment of intact EVs with proteinase within THP-1 MΦs. When the THP-1 cells were pretreated with K for 60 min resulted in partial digestion of EV-associated or without inhibitors (SI Appendix,Fig.S6A), only treatment of proteins. In contrast, EV proteins were fully digested by treat- cells with dynasore significantly blocked internalization of EVs ment with proteinase K in the presence of 1% SDS, an anionic purified from the ΔagrΔsae mutant (SI Appendix,Fig.S6B), Thus, detergent that disrupts the EV membrane and exposes the EV MΦ uptake of EVs purified from both the WT and the ΔagrΔsae cargo to the environment. Proteolytic digestion of purified Hla, mutant occurred through dynamin-dependent endocytosis. LukS-PV, and LukAB occurred within 30 min, 90 min, and 60 Together, our findings reveal that S. aureus EVs induce min, respectively, as deduced from immunoblots (SI Appendix, NLRP3 inflammasome activation and that EV-induced release Fig. S7B). When EVs were incubated with proteinase K for 60

Wang et al. PNAS | February 11, 2020 | vol. 117 | no. 6 | 3179 Downloaded by guest on October 2, 2021 min, most of the EV-associated Hla remained intact and was μg/mL EVs purified from the WT or lgt mutant, and cytokines in only digested in the presence of 1% SDS (SI Appendix, Fig. S7C). the MΦ culture supernatants were measured. EVs purified from In contrast, proteinase K treatment of intact EVs in the presence the lgt mutant were defective in the induction of IL-1β, IL-18, or absence of 1% SDS completely digested EV-associated LukS- and IL-6 from THP-1 MΦs, and this defect was complemented PV (SI Appendix, Fig. S7D) and LukAB (SI Appendix, Fig. S7E) when lgt was provided in trans (Fig. 5 A and B). Unexpectedly, after 90 min and 60 min, respectively. These data suggest that S. mutation of lgt also abrogated EV-induced cleavage of caspase-1 aureus PFTs are localized on the surface and within the lumen of in THP-1 MΦs, as revealed by luminescent assays (Fig. 5C) and EVs and that the latter was protected from proteolytic digestion. by immunoblot (Fig. 5D). To validate these findings, monocyte- To determine whether LukS-PV and LukAB surface receptor derived human MΦs were similarly treated with 5 μg/mL EVs binding was essential for EV-induced cytokine production, we purified from JE2, the lgt mutant, or a complemented mutant. As utilized murine J774A.1 MΦs. LukS-PV binds to human, but not expected, mutation of lgt abrogated the induction of cytokines mouse, C5aR, and LukAB binds to human, but not mouse, IL-1β, IL-18, and IL-6 (Fig. 5 E and F), as well as caspase-1 CD11b (37, 38). Whereas WT EVs stimulated IL-6 production activation (Fig. 5G). To rule out the possibility that the im- by J774A.1 MΦs in a dose-dependent fashion (SI Appendix, Fig. paired activation of caspase-1 in MΦs was due to a defect in EV S8A), IL-1β production was not detected with EV concentrations internalization, THP-1 MΦs were incubated for 1 h with R18- up to 5 μg/mL (SI Appendix, Fig. S8B). To rule out the possibility labeled EVs purified from the WT or lgt mutant strain. As shown that impaired J774A.1 inflammasome activation was due to a in Fig. 5H, EV fluorescence within MΦs was similar for both defect in EV internalization, we incubated the J774A.1 cells for strains, indicating that EVs harvested from the lgt mutant were 90 min with DiO-labeled EVs purified from WT JE2. As shown efficiently internalized within human MΦs. We further de- in SI Appendix, Fig. S8C, EV-associated fluorescence was readily termined the cellular internalization mechanism of EVs from the detectable within J774A.1 macrophages, indicating efficient EV lgt mutant. Pretreatment of THP-1 MΦs with dynasore, but not internalization. These findings suggest that surface-associated other inhibitors, significantly inhibited the cellular entry of EVs PFT interactions with specific receptors on MΦs are (SI Appendix, Fig. S9 A and B), indicating that, like WT EVs, the essential for EV-mediated NLRP3 activation. EVs purified from the lgt mutant were internalized within MΦs through dynamin-dependent endocytosis. Together, these results EV-Associated Lipoproteins Are Critical for TLR2 Signaling and demonstrate that EV-associated lipoproteins are critical for both Caspase-1 Activation. S. aureus lipoproteins are critical for TLR2 TLR2 signaling and caspase-1 activation, but not for the in- signaling and triggering host innate immune defenses against ternalization of EVs. staphylococcal infections (39–41). The S. aureus lgt gene product catalyzes the transfer of a diacyl-glyceryl group to a cysteine Lipoproteins Modulate the Content and Biogenesis of EVs in S. aureus. residue in the lipobox of the lipoprotein signal peptide (42). An Because EVs purified from the lgt mutant were deficient in the lgt mutant is deficient in the lipidation and maturation of lipo- activation of caspase-1, we investigated whether EVs from the proteins although the proteins themselves are still produced (43). mutant strain still packaged PFTs, which are important for EV- To investigate the role of lipoproteins in EV-induced NLRP3 induced caspase-1 activation (Fig. 4 D and F). Culture super- inflammasome activation, THP-1 MΦs were incubated with 1 natants of JE2 and its lgt mutant showed similar levels of

Fig. 5. S. aureus EV-associated lipoproteins are critical for TLR2 signaling and caspase-1 activation. (A–C) Levels of IL-1β and IL-18 (A), IL-6 (B), and active caspase-1 (C) in culture supernatants of THP-1 MΦs incubated for 4 h (IL-1β, IL-18, and caspase-1) or 12 h (IL-6) with EVs purified from WT JE2, the lgt mutant, the complemented lgt mutant, or controls LPS+ATP and Pam3CSK4 (n = 4). (D) Immunoblot showing cleaved caspase-1 in culture supernatants and caspase-1 in cell lysates of THP-1 MΦs that were incubated for 12 h with 1 μg/mL EVs purified from WT JE2, the lgt mutant, the complemented lgt mutant, or LPS+ATP (n = 2). (E–G) Levels of IL-1β and IL-18 (E), IL-6 (F), and active caspase-1 (G) in culture supernatants of monocyte-derived human MΦs incubated for 8 h (IL-1β, IL- 18, and caspase-1) or 2 h (IL-6) with 5 μg/mL EVs purified from WT JE2, the lgt mutant, the complemented lgt mutant, LPS+ATP, or Pam3CSK4 (n = 4). (H) Representative images of two independent experiments for THP-1 MΦs incubated for 90 min with 1 μg/mL R18-labeled EVs purified from the WT or lgt mutant (n = 2). (Scale bars: 10 μm.) Data pooled from at least two independent experiments were analyzed by one-way ANOVA with Dunnett’s multiple comparison test. ***P < 0.001, ****P < 0.0001.

3180 | www.pnas.org/cgi/doi/10.1073/pnas.1915829117 Wang et al. Downloaded by guest on October 2, 2021 hemolytic activity against rabbit erythrocytes (Fig. 6A), which are was consistent for EVs prepared from cultures in the early or late susceptible to Hla, phenol soluble modulins, and the leukocidins phases of logarithmic growth (SI Appendix, Fig. S10A). Likewise, HlgAB and LukED (5, 44, 45). In contrast, EVs recovered from EVs prepared from both growth phases of the lgt mutant showed the lgt mutant exhibited reduced hemolytic activity compared to significant reductions in cytotoxicity compared to WT EVs when WT EVs (Fig. 6B). Likewise, THP-1 MΦs incubated with EVs incubated with THP-1 MΦs(SI Appendix, Fig. S10B). Together from the lgt mutant released less LDH than WT EV-treated these results demonstrate that lipoproteins modulate the PFT cells, and lgt complementation restored EV cytotoxicity (Fig. content of S. aureus EVs. 6C). These data suggest that the lgt mutation resulted in reduced We predicted that, if the PFT content of EVs from lgt mutant PFT cargo associated with S. aureus EVs. To validate this as- was altered, the lipoprotein defect might also have an impact on sumption, the Hla and LukS-PV content of EVs from the WT or the total protein content of EVs. This assumption was corrobo- lgt mutant was evaluated by immunoblot analysis. The Hla and rated by silver-stained SDS/PAGE analysis that showed distinct LukS-PV content was dramatically reduced in EVs purified from protein profiles for EVs purified from the WT JE2 and its lgt the lgt mutant compared to WT EVs (Fig. 6D), and the toxin mutant (Fig. 6E). To validate these data, a proteomic analysis of cargo was restored when the lgt gene was provided in trans to the EVs purified from JE2 and its lgt mutant was performed by liquid mutant strain. chromatography–tandem mass spectrometry (LC–MS/MS). A To rule out the possibility that the reduced PFT content of lgt total of 180 and 198 proteins were identified from EVs recovered EVs was dependent on the bacterial growth phase, EVs were from the WT and lgt mutant, respectively (Dataset S1). However, purified from cultures grown to log phase (4.5 h) or to late log the lgt mutation resulted in significant alterations in the EV phase (7 h), and the levels of Hla and LukS-PV in purified EVs protein content. Compared to WT EVs, more cytosolic proteins were compared. Immunoblot analysis again revealed a similar and fewer extracellular proteins, including PFTs, were identified toxin profile in the culture supernatants of WT and lgt mutant in EVs from the lgt mutant (SI Appendix, Fig. S11A). In addition, strains (SI Appendix, Fig. S10A). The remarkable reduction in 49 of 180 (27.2%) and 67 of 198 (33.8%) identified proteins were the Hla and LukS content of EVs harvested from the lgt mutant unique to EVs recovered from the WT and lgt mutant, respectively MICROBIOLOGY

Fig. 6. S. aureus lipoproteins modulate EV content and biogenesis. The hemolytic activity of S. aureus culture supernatants (A) and purified EVs (B)was assessed with rabbit erythrocytes (n = 4). (C) Release of LDH in culture supernatants of THP-1 MΦs that were incubated for 4 h with 1 μg/mL EVs purified from WT JE2, the lgt mutant, or the complemented lgt mutant (n = 4). (D) The Hla and LukS-PV content of EVs purified from the indicated strains was detected by immunoblot. Purified Hla and LukS-PV were used as controls (n = 2). (E) EVs purified from WT or lgt mutant strains were subjected to SDS/PAGE and silver staining (n = 3). kD, kilodalton. (F) The membrane fluidity of the indicated strains was determined using a pyrenedecanoic acid fluorescence probe, and the percent change in membrane fluidity was calculated (n = 14). (G) EV production from S. aureus strains JE2 and Newman and their isogenic lgt mutants was evaluated by quantification of total EV protein yield (n = 8 to 10) or (H) by EV quantification using nanoparticle tracking analysis (n = 4). (I) EV size distribution and (J), average size of EVs isolated from JE2 and its lgt mutant were measured by dynamic light scattering (n = 6). Membrane fluidity, EV protein yield, and EV particle quantification experiments were calculated from at least three independent experiments. The data were analyzed with a one-way ANOVA with Dunnett’s multiple comparison test (C and F) or with an unpaired, two-tailed Student’s t test (G, H, and J). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Wang et al. PNAS | February 11, 2020 | vol. 117 | no. 6 | 3181 Downloaded by guest on October 2, 2021 (SI Appendix,Fig.S11B). These data suggest that S. aureus pathway (58), and PFT-mediated acti- lipoproteins modulate EV biogenesis. vation of necroptosis can occur during S. aureus infection (59, Membrane fluidity plays a fundamental role in governing the 60). It is likely that multiple lytic cell death pathways are involved flexibility of membranes, which may affect membrane curvature in EV-induced MΦ cytotoxicity. (46). Because increases in membrane fluidity can promote vesi- To gain a better understanding of how EV-associated PFTs cle budding (47), we measured the membrane fluidity of WT and interact with host cells, we investigated whether prototype S. lgt mutant strains using the excimer-forming lipid technique (48). aureus PFTs (Hla, LukS-PV, and LukAB) were surface associ- Mutation of lgt resulted in a significant increase in S. aureus ated or contained within the EV lumen. Because of its relative cytoplasmic membrane fluidity, and this increase was abrogated resistance to protease treatment, EV-associated Hla appears to when the lgt gene was provided in trans to the mutant strain (Fig. be contained primarily within the EV lumen. In contrast, the 6F). Consistent with the lgt mutation resulting in heightened bulk of LukS-PV and LukAB were protease sensitive, suggesting membrane fluidity, we observed significantly increased EV yield surface localization of these PFTs. EV surface-associated PFTs (Fig. 6G) and particle numbers (Fig. 6H)inlgt mutants of either may activate inflammasomes extracellularly through pore for- strain JE2 or Newman. Moreover, EVs from the JE2 lgt mutant mation, but whether there are additional mechanisms involved in were smaller in size than WT EVs (Fig. 6 I and J). this process remains unclear. Our data show that S. aureus EVs enter MΦs via a dynamin-dependent endocytic pathway, sug- Discussion gesting that EV-associated PFTs and other cargo may be pre- Outer membrane vesicles (OMVs) secreted by gram-negative sented to host cells within endosomal vesicles. S. aureus bacteria package multiple virulence factors and are considered to cytolysins, such as LukAB and delta-toxin, are capable of dis- be important mediators in bacterial–host cell interactions. rupting the membranes of subcellular compartments (61, 62). OMVs taken up by host cells modulate innate and adaptive Membrane permeation, lysosomal damage, and mitochondrial immune responses and may lead to cell injury (28, 49–52). In disruption are cellular events that can elicit NLRP3 inflamma- addition to OMV production, many gram-negative some activation (63–66). As such, it is possible that EV-associated manipulate host cellular functions by utilizing dedicated secre- PFTs may trigger NLRP3 activation by pore formation directly at tion systems (T3SS, T4SS, and T6SS) to transport bacterial the cell membrane or following the entry of EVs into cellular products into host cells (53). In contrast, gram-positive bacteria compartments. Although S. aureus is deemed a classic extracel- lack sophisticated secretion systems characteristic of gram- lular , it is capable of survival within host cells, including negative microbes. S. aureus EV cargo includes secreted pro- macrophages (67), where it presumably secretes EVs. Intracellular teins, cell wall-anchored proteins, and membrane proteins, but it EV formation has been demonstrated for typical intracellular is cytoplasmic proteins that represent the most abundant com- microbes, like Listeria monocytogenes (68) and Mycobacterium ponent of EVs (8). Because cytoplasmic proteins lack export avium (69). signals, EVs clearly represent a unique secretory mechanism for Lipoproteins, abundant in S. aureus EVs, represent a subset of gram-positive bacteria. The concept that EV production enables bacterial membrane proteins that are anchored in the cytoplas- bacteria to encapsulate and deliver their products into host cells, mic membrane by a covalently linked N-terminal lipid moiety while protecting the cargo from detection or destruction by ex- (70). S. aureus lipoproteins are critical for bacterial nutrient ternal factors, has modified and shaped our understanding of the uptake (15), but they also play a role in host immune defense pathogenesis of infections caused by gram-positive pathogens. against infection by activating host innate immunity through Nonetheless, the biological role of EVs in S. aureus infection, as TLR2 signaling (39, 41, 71). An S. aureus lgt mutant, deficient in well as the downstream fate of EVs following bacterial release, the lipidation and maturation of lipoproteins, produces pre- remains poorly characterized. lipoproteins that are not modified by lipidation and are unable to In this report, we demonstrate that S. aureus EVs are sensed in activate TLR2 signaling (43). These prelipoproteins may be human MΦs by NLRP3 inflammasomes (SI Appendix, Fig. S12), retained transiently at the cytoplasmic membrane by the a major signaling pathway of the innate that is uncleaved signal sequence, including the characteristic lipobox, critical for host defense against bacterial infections (54). Con- but eventually are released from S. aureus cells in high amounts sistent with previous reports indicating that activation of NLRP3 due to proteolytic processing (43, 72). We characterized EVs + inflammasomes requires pore forming-mediated K efflux (26, from an lgt mutant of S. aureus because we suspected that they 27, 55, 56), NLRP3 activation by S. aureus EVs was dependent would be deficient in TLR2 signaling, which is essential for + on PFT cargo and K efflux. S. aureus EVs carry many virulence NLRP3 inflammasome activation. Predictably, EVs purified factors, including surface proteins and glycopolymers, as well as from the lgt mutant were deficient in stimulating human MΦsto secreted proteins, such as PFTs, superantigens, phenol soluble secrete IL-6, IL-1β, and IL-18. However, we did not anticipate modulins, and proteases, and EVs are toxic to a broad range of the loss of caspase-1 activity that occurred when THP-1 MΦrso host cells (8, 10–12). Our data indicate that multiple PFTs such monocyte-derived human MΦs were incubated with EVs puri- as LukAB, Hla, and LukSF-PV are involved in EV-induced fied from the lgt mutant. Upon further investigation, we observed NLRP3 inflammasome activation and that the combined action that an S. aureus lgt mutant produced more EVs than the pa- of PFTs was required for maximal activation by EVs. S. aureus rental strain and that the EVs from the lgt mutant showed an produces a diverse array of PFTs, and individual PFTs target altered protein composition with a marked reduction in PFT specific cellular receptors carried by a subset of human hemo- cargo. Thus, lipoproteins play a previously unrecognized role in poietic cells (5, 57). Whereas individual PFTs promote bacterial S. aureus EV biogenesis. Membrane proteins maintain the in- virulence, the synergistic action of multiple PFTs may be nec- tegrity, organization, and flow of materials through the bacterial essary to target sufficient numbers of host cells to result in in- membrane (73). The WT S. aureus USA300 strain expresses ∼67 flammatory signaling and pore formation (57). Pyroptosis is a lipoproteins (15), and the absence of lipoproteins resulted in an programmed lytic cell death process associated with inflamma- increase in membrane fluidity of S. aureus, as well as alterations some activation. Our data indicate that pyroptosis was activated in the protein content of EVs, their yield, and size. upon incubation of human MΦs with S. aureus EVs. However, In summary, our work provides insights into the potential role inhibition of NLRP3 inflammasomes or knockout of NLRP3 or of EVs in the molecular pathogenesis of gram-positive infections caspase-1 did not fully abrogate the EV-induced LDH release, by serving as a secretory pathway for transporting protected suggesting that other lytic cell death pathways are likely involved bacterial cargo into host cells. We demonstrate the critical role in EV-induced cell death in human MΦs. Necroptosis is another that staphylococcal lipoproteins play in EV biogenesis and the

3182 | www.pnas.org/cgi/doi/10.1073/pnas.1915829117 Wang et al. Downloaded by guest on October 2, 2021 essential role of S. aureus EV-associated PFTs in triggering Data Availability. The data supporting the findings of this study are available inflammasome activation, which modulates the host innate im- within the main text and SI Appendix. Mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium (http://proteomecentral. mune response during infection. proteomexchange.org/cgi/GetDataset) via the PRIDE partner repository with Materials and Methods the data set identifier PXD014888. The S. aureus strains used in this study are listed in SI Appendix, Table S1. The ACKNOWLEDGMENTS. Research reported in this publication was supported isolation, purification, and analysis of EV protein concentrations, particle by a Brigham Research Institute Pilot Funding Award from Brigham and numbers, and EV diameters were performed as described previously (8). Women’s Hospital and the National Institute of Allergy and Infectious Dis- Primary human monocytes were isolated from the peripheral blood of eases of the National Institutes of Health under Awards R21AI135613 and R01AI141885 (to J.C.L.). The content is solely the responsibility of the authors healthy volunteers as approved by the Institutional Review Board of Partners and does not necessarily represent the official views of the National Insti- HealthCare System. Mouse experiments were approved by the Institutional tutes of Health. We thank Drs. Romeo Ricci (Institut de Génétique et de Animal Care and Use Committee of the Brigham and Women’s Hospital. All Biologie Moléculaire et Cellulaire, Illkirch, France) for providing the NLRP3 other materials and details of experimental procedures involved in the knockout THP-1 cell line; Seth L. Masters (Inflammation Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia) for provid- generation of S. aureus mutants, cell culture, immunoblots, ELISAs, confocal ing the THP-1 (Cas9) and Caspase-1 knockout THP-1 cell lines; and Simon microscopy, membrane fluidity assays, proteinase K assays, and animal Dove (Division of Infectious Diseases, Boston Children’s Hospital and Harvard studies are provided in SI Appendix. Medical School, Boston, MA) for providing the J774A.1 cell line.

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