Stress‐induced host membrane remodeling protects from infection by non‐motile bacterial pathogens
2018; Springer Nature; Volume: 37; Issue: 23 Linguagem: Inglês
10.15252/embj.201798529
ISSN1460-2075
AutoresCaroline Tawk, Giulia Nigro, Ines Rodrigues Lopes, Carmen Aguilar, Clivia Lisowski, Miguel Mano, Philippe Sansonetti, Jörg Vogel, Ana Eulálio,
Tópico(s)RNA regulation and disease
ResumoArticle2 November 2018Open Access Source DataTransparent process Stress-induced host membrane remodeling protects from infection by non-motile bacterial pathogens Caroline Tawk Caroline Tawk orcid.org/0000-0003-1781-5591 Host RNA Metabolism Group, Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany RNA Biology Group, Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany Search for more papers by this author Giulia Nigro Giulia Nigro Molecular Microbial Pathogenesis Laboratory, Institut Pasteur, Paris, France Search for more papers by this author Ines Rodrigues Lopes Ines Rodrigues Lopes Functional Genomics and RNA-based Therapeutics, UC-BIOTECH, Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal RNA & Infection Group, UC-BIOTECH, Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal Search for more papers by this author Carmen Aguilar Carmen Aguilar Host RNA Metabolism Group, Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany Search for more papers by this author Clivia Lisowski Clivia Lisowski Host RNA Metabolism Group, Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany Search for more papers by this author Miguel Mano Miguel Mano orcid.org/0000-0003-1922-4824 Functional Genomics and RNA-based Therapeutics, UC-BIOTECH, Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal Search for more papers by this author Philippe Sansonetti Philippe Sansonetti orcid.org/0000-0001-7542-4527 Molecular Microbial Pathogenesis Laboratory, Institut Pasteur, Paris, France Search for more papers by this author Jörg Vogel Jörg Vogel orcid.org/0000-0003-2220-1404 RNA Biology Group, Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany Helmholtz Institute for RNA-Based Infection Research (HIRI), Würzburg, Germany Search for more papers by this author Ana Eulalio Corresponding Author Ana Eulalio [email protected] [email protected] orcid.org/0000-0002-7355-0674 Host RNA Metabolism Group, Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany RNA & Infection Group, UC-BIOTECH, Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal Search for more papers by this author Caroline Tawk Caroline Tawk orcid.org/0000-0003-1781-5591 Host RNA Metabolism Group, Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany RNA Biology Group, Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany Search for more papers by this author Giulia Nigro Giulia Nigro Molecular Microbial Pathogenesis Laboratory, Institut Pasteur, Paris, France Search for more papers by this author Ines Rodrigues Lopes Ines Rodrigues Lopes Functional Genomics and RNA-based Therapeutics, UC-BIOTECH, Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal RNA & Infection Group, UC-BIOTECH, Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal Search for more papers by this author Carmen Aguilar Carmen Aguilar Host RNA Metabolism Group, Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany Search for more papers by this author Clivia Lisowski Clivia Lisowski Host RNA Metabolism Group, Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany Search for more papers by this author Miguel Mano Miguel Mano orcid.org/0000-0003-1922-4824 Functional Genomics and RNA-based Therapeutics, UC-BIOTECH, Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal Search for more papers by this author Philippe Sansonetti Philippe Sansonetti orcid.org/0000-0001-7542-4527 Molecular Microbial Pathogenesis Laboratory, Institut Pasteur, Paris, France Search for more papers by this author Jörg Vogel Jörg Vogel orcid.org/0000-0003-2220-1404 RNA Biology Group, Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany Helmholtz Institute for RNA-Based Infection Research (HIRI), Würzburg, Germany Search for more papers by this author Ana Eulalio Corresponding Author Ana Eulalio [email protected] [email protected] orcid.org/0000-0002-7355-0674 Host RNA Metabolism Group, Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany RNA & Infection Group, UC-BIOTECH, Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal Search for more papers by this author Author Information Caroline Tawk1,2, Giulia Nigro3, Ines Rodrigues Lopes4,5, Carmen Aguilar1, Clivia Lisowski1, Miguel Mano4, Philippe Sansonetti3, Jörg Vogel2,6 and Ana Eulalio *,*,1,5 1Host RNA Metabolism Group, Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany 2RNA Biology Group, Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany 3Molecular Microbial Pathogenesis Laboratory, Institut Pasteur, Paris, France 4Functional Genomics and RNA-based Therapeutics, UC-BIOTECH, Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal 5RNA & Infection Group, UC-BIOTECH, Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal 6Helmholtz Institute for RNA-Based Infection Research (HIRI), Würzburg, Germany *Corresponding author. Tel: +351 231249170; E-mail: [email protected]; [email protected] The EMBO Journal (2018)37:e98529https://doi.org/10.15252/embj.201798529 [The copyright line of this article was changed on 10 December 2018 after original online publication.] PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract While mucosal inflammation is a major source of stress during enteropathogen infection, it remains to be fully elucidated how the host benefits from this environment to clear the pathogen. Here, we show that host stress induced by different stimuli mimicking inflammatory conditions strongly reduces the binding of Shigella flexneri to epithelial cells. Mechanistically, stress activates acid sphingomyelinase leading to host membrane remodeling. Consequently, knockdown or pharmacological inhibition of the acid sphingomyelinase blunts the stress-dependent inhibition of Shigella binding to host cells. Interestingly, stress caused by intracellular Shigella replication also results in remodeling of the host cell membrane, in vitro and in vivo, which precludes re-infection by this and other non-motile pathogens. In contrast, Salmonella Typhimurium overcomes the shortage of permissive entry sites by gathering effectively at the remaining platforms through its flagellar motility. Overall, our findings reveal host membrane remodeling as a novel stress-responsive cell-autonomous defense mechanism that protects epithelial cells from infection by non-motile bacterial pathogens. Synopsis Stress-induced host membrane remodeling constitutes a novel cell-autonomous defensive mechanism that protects epithelial cells from infection by Shigella flexneri and other non-motile bacterial pathogens. Host oxidative stress strongly reduces S. flexneri binding to epithelial cells. Stress leads to host membrane remodeling, via activation of the acid sphingomyelinase by the MAPK p38 pathway, resulting in the formation of ceramide domains. Intracellular Shigella replication induces remodeling of the host cell membrane, in vitro and in vivo. Stress-induced host membrane remodeling precludes re-infection by non-motile pathogens; motile pathogens are able to overcome this barrier through flagellar motility. Introduction Intestinal epithelial cells (IECs) constitute a physical and biochemical barrier between host and microorganisms. While not considered professional immune cells, IECs are crucial contributors to immune surveillance and the initial inflammatory responses against infection (Sansonetti, 2004; Peterson & Artis, 2014). For example, IECs secrete pro-inflammatory cytokines and chemokines in response to infection by certain enteroinvasive bacterial pathogens such as Shigella flexneri and Salmonella Typhimurium. This, in turn, leads to a massive infiltration of professional immune cells into the sites of inflammation, from which ensues a local increase in reactive oxygen species and a profound hypoxia (Colgan & Taylor, 2010; Zeitouni et al, 2016; Arena et al, 2017). Such change in environment not only constrains the pathogen but also dramatically affects the intestinal epithelium. Interestingly, while this inflammation-related stress used to be considered as posing additional harm to the affected tissues, there is recent evidence to suggest that it makes crucial contributions to counteract infection processes at the cellular level (Chovatiya & Medzhitov, 2014). Bacterial pathogens have evolved sophisticated mechanisms to subvert and often invade the intestinal epithelium, thereby overcoming host defense mechanisms (Sansonetti, 2004; Pizarro-Cerda & Cossart, 2006; Carayol & Tran Van Nhieu, 2013). Shigella flexneri, for example, crosses the intestinal barrier by transcytosis through M-cells (Wassef et al, 1989; Perdomo et al, 1994), before invading IECs from the basolateral side (Mounier et al, 1992). For invasion, Shigella uses its type III secretion system (T3SS) to inject effector proteins into target cells to subvert host defense pathways, promoting its own internalization by a trigger mechanism that involves the formation of actin-rich membrane ruffles (Ogawa et al, 2008; Schroeder & Hilbi, 2008; Parsot, 2009). Notwithstanding the diverse active mechanisms used by bacteria to mediate binding and invasion of host cells [e.g., pili, fimbriae, adhesins/invasins, T3SS (Pizarro-Cerda & Cossart, 2006; Stones & Krachler, 2016)], it has also become clear that efficient bacterial entry requires permissive sites in the host membrane. Membrane rafts, which are highly dynamic membrane domains enriched in sphingolipids and cholesterol that mediate the compartmentalization of signaling proteins and receptors (Lingwood & Simons, 2010; Sezgin et al, 2017), have been shown to be utilized by numerous bacterial pathogens (reviewed in Refs: Lafont & van der Goot, 2005; Bagam et al, 2017). For example, Shigella uses its IpaB effector protein to bind the host raft-associated CD44 transmembrane receptor (Lafont et al, 2002); entry of Listeria monocytogenes into host cells requires the localization of the host receptors E-cadherin and HGF-R/Met in specific lipid domains (Seveau et al, 2004). In addition to receptors, plasma membrane composition itself, specifically cholesterol and sphingolipid membrane content, impacts the binding and internalization of various bacterial pathogens, including Shigella and Salmonella species (Garner et al, 2002; Lafont et al, 2002; Misselwitz et al, 2011a; Santos et al, 2013). Here, we investigated the impact of the general stress response of epithelial cells on the infection by S. flexneri and Salmonella Typhimurium. We found that induction of stress in epithelial cells by inflammatory cues and oxidative insults prevents the binding of Shigella, a non-motile pathogen, to host cells. We demonstrate that this inhibition results from extensive remodeling of the host plasma membrane following a stress-induced activation of the acid sphingomyelinase (ASM). By contrast, the related motile pathogen Salmonella can overcome this barrier, using flagellar motility to reach and accumulate at the remaining permissive entry sites. Moreover, we show that intracellular replication of Shigella activates ASM and subsequent membrane remodeling, thus suppressing re-infection by non-motile pathogens. Collectively, our findings demonstrate a role for the host stress response in protecting cells against Shigella infection and demonstrate the involvement of ASM and membrane remodeling in this process. Results Host cell response to stress inhibits Shigella infection To investigate whether host cell stress has a deleterious effect on the outcome of Shigella infection, we treated HeLa cells, an epithelial cell line commonly used to study Shigella infection, with sub-lethal concentrations of sodium arsenite (Fig 1A). Arsenite is widely used to induce oxidative stress (Bernstam & Nriagu, 2000; Liu et al, 2001). Following arsenite removal, cells were extensively washed and then infected with Shigella; infection efficiency was monitored at early, intermediate, and late stages of infection (0.5, 2, and 6 hpi, respectively; Fig 1A) by: (i) fluorescence microscopy, (ii) colony-forming unit (cfu) assays, and (iii) qRT–PCR. Interestingly, pre-treatment of cells with arsenite strongly reduced Shigella infection, at all time points tested (4.7- to 8.8-fold compared to control, cfu; Figs 1B and D, and EV1A and B). Validating these observations, Shigella infection was also inhibited by arsenite in all tested colon epithelial cells, namely HCT-8, HT-29, and Caco-2 cells (Figs 1C and D, and EV1B–D). Figure 1. Shigella infection is inhibited by host cell stress A. Schematic representation of the experimental design. B, C. Representative images of HeLa (B) or HCT-8 (C) cells infected with Shigella WT pre-treated or not with arsenite, analyzed at the indicated times post-infection. D. Cfu quantification of intracellular bacteria in HeLa and HCT-8 cells pre-treated or not with arsenite and infected with Shigella. E, F. Representative images (E) and cfu quantification (F) of intracellular bacteria in HeLa cells infected with Shigella WT after pre-treatment with TNF-α, H2O2, anisomycin, hypoxia, and corresponding controls, analyzed at 0.5 hpi. G, H. Representative images (G) and cfu quantification (H) of intracellular Shigella in HeLa cells pre-treated with arsenite, anisomycin, stressors plus NAC, and corresponding controls. Data information: Shigella infection was performed at MOI 10. Results are shown as mean ± s.e.m. of five independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001 (t-test adjusted for multiple comparison for D—HeLa; paired t-test for D—HCT-8 and F; one-way ANOVA for H). Scale bars, 50 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Shigella infection is inhibited by host cell stress A. Percentage of HeLa cells infected with Shigella after pre-treatment with arsenite or control, analyzed at 0.5, 2, and 6 hpi. B. qRT–PCR quantification of intracellular bacteria in HeLa and HCT-8 cells pre-treated or not with arsenite and infected with Shigella WT. Analysis was performed at 0.5, 2, and 6 hpi for HeLa cells and at 0.5 hpi for HCT-8 cells. Results are shown normalized to the control at 0.5 hpi. C, D. Representative images (C) and cfu quantification (D) of HT-29 or Caco-2 cells pre-treated or not with arsenite and infected with Shigella WT, analyzed at 0.5 hpi. E–G. Representative images (E), cfu (F), and qRT–PCR (G) quantification of intracellular bacteria in HeLa cells pre-treated with puromycin or cycloheximide, or control, and infected with Shigella. Analysis was performed at 0.5 hpi. H. Percentage of HeLa cells infected with Shigella WT after pre-treatment with TNF-α, H2O2, anisomycin, hypoxia, and corresponding controls, analyzed at 0.5 hpi. I. qRT–PCR quantification of intracellular bacteria in HeLa cells infected with Shigella WT after pre-treatment with TNF-α, H2O2, anisomycin, hypoxia, and corresponding controls, analyzed at 0.5 hpi. J. Percentage of 7-AAD-positive cells following treatment with arsenite, TNF-α, H2O2, amitriptyline, anisomycin, and corresponding controls. K. Growth curve of Shigella WT or Salmonella WT (OD600) in LB medium (10 h) in the presence of arsenite, anisomycin, or corresponding controls. L. Cfu quantification of intracellular bacteria in HeLa cells treated or not with NAC and infected with Shigella WT, analyzed at 0.5 hpi. Data information: Shigella infection was performed at MOI 10. Results are shown as mean ± s.e.m. of 5 (panels A, B—HCT-8, D, F, I, J, K, L) or 6 (panels B—HeLa, G, H) independent experiments; **P < 0.01, ***P < 0.001 (t-test adjusted for multiple comparison for panels A and B—HeLa; paired t-test for panels B—HCT-8, D, I, and L; one-way ANOVA for panels F–H and J). Scale bars, 50 μm. Download figure Download PowerPoint Response to environmental stress in eukaryotic cells generally dampens bulk protein synthesis due to impaired mRNA translation initiation (Holcik & Sonenberg, 2005). However, inhibition of translation by puromycin and cycloheximide did not affect Shigella infection (Fig EV1E–G), demonstrating that the effect of arsenite on Shigella infection is unrelated to translation shutdown. To understand whether inhibition of Shigella infection by cellular stress is a broad phenomenon, we tested other stress inducers, namely anisomycin, hydrogen peroxide (H2O2), hypoxia, or the inflammatory cytokine TNF-α. All the stress inducers have been widely used to mimic conditions encountered by cells during inflammation (arsenite, anisomycin) or are per se stimuli present during inflammation (hypoxia, TNF-α, and H2O2). These stimuli converge in the production of reactive oxygen species (ROS), key signaling molecules during inflammation. Consistently, pre-treatment with the various stimuli strongly inhibited Shigella infection, already at early times post-infection (0.5 hpi; Figs 1E and F, and EV1H and I). The various compounds did not affect host cell viability, at the concentrations and incubation periods tested (Fig EV1J). It should be noted that host cells were extensively washed prior to infection to remove any remaining stressors. Moreover, arsenite or anisomycin treatment did not impair Shigella growth (Fig EV1K), thus excluding direct effects on the bacteria. Importantly, treatment with the antioxidant N-acetyl-L-cysteine (NAC) reverted the inhibitory effect of arsenite or anisomycin on Shigella infection (Figs 1G and H, and EV1L), further confirming that the effect of these stressors on Shigella infection is mediated by oxidative stress. Overall, these results demonstrate that the response of epithelial cells to oxidative stress limits Shigella infection. Shigella binding to host cells is inhibited upon cellular stress The evident inhibition of Shigella infection observed at 0.5 hpi strongly indicates that host cell stress affects the early steps of Shigella interaction with host cells. Accordingly, pre-treatment of cells with arsenite or anisomycin strongly inhibited Shigella binding to HeLa cells (ca. 10.0- and 2.0-fold compared to control, cfu, respectively; Fig 2A–C and Appendix Fig S1A–E). Comparable results were obtained in HCT-8 cells (Fig 2D and E, and Appendix Fig S1F). A possible effect of the stress inducers on actin cytoskeleton integrity and dynamics was excluded, since normal induction of actin-rich membrane ruffles by wild-type (WT) Shigella was observed in cells pre-treated with arsenite or anisomycin (white arrowheads in Fig 2A and Appendix Fig S1C, respectively). Accordingly, the efficiency of ruffle formation, i.e., the percentage of ruffles induced upon bacterial contact, was similar in cells treated with stressors and control cells (Appendix Fig S1G). To uncouple Shigella binding to host cells from subsequent steps of invasion, we performed parallel experiments with the Shigella ΔipaB mutant strain, which binds efficiently to host cells but is unable to invade (Menard et al, 1993). Treatment of cells with arsenite or anisomycin inhibited binding of Shigella ΔipaB, similar to the WT bacteria (Fig 2A–E, and Appendix Fig S1B, C, E and F). Binding of Shigella ΔicsA mutant strain to host cells was also inhibited by arsenite or anisomycin treatment (Appendix Fig S1H and I), demonstrating that the effect of the stressors is independent of the role of IcsA in Shigella adhesion (Brotcke Zumsteg et al, 2014). Overall, these results show that host cell stress has a strong inhibitory effect on Shigella binding. Figure 2. Host cellular stress inhibits Shigella binding to host cells A, B. Representative images (A) and cfu quantification (B) of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre-treated or not with arsenite. Ruffle formation induced by Shigella WT in panel (A) is indicated by white arrowheads. C. Cfu quantification of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre-treated with anisomycin or DMSO (control). D, E. Representative images (D) and cfu quantification (E) of Shigella WT or ΔipaB mutant bound to HCT-8 cells pre-treated or not with arsenite. Data information: Shigella infection was performed at MOI 10 for Shigella WT or at MOI 50 for the ΔipaB mutant strain; cells were incubated with the bacteria for 25 min. Results are shown as mean ± s.e.m. of 5 (C) or 6 (B, E) independent experiments; **P > 0.01, ***P < 0.001 (paired t-test). Scale bars, 25 μm. Download figure Download PowerPoint Analysis of Shigella cell-to-cell spreading in control and arsenite or anisomycin-treated cells, by quantifying the area of Shigella infection foci using fluorescence microscopy and automated image analysis (Sunkavalli et al, 2017), excluded an effect of stress in the actin-based spreading of Shigella to neighboring cells (Appendix Fig S1J). In these experiments, infections were performed at different MOIs (MOI 10 for control, MOI 50 for anisomycin, and MOI 100 for arsenite), to achieve comparable levels of bacterial invasion. ASM-dependent membrane remodeling upon stress inhibits Shigella binding Considering that binding of Shigella to host cells is inhibited by stress and that this occurs in a relatively short timeframe (e.g., 15 min pre-treatment with TNF-α; Figs 1E and F, and EV1H and I), we reasoned that the decreased binding of Shigella could result from modifications of the cellular membrane. In line with this possibility, membrane composition, specifically the presence of sphingolipids and cholesterol at the cell surface, has been shown to be required for successful Shigella infection (Lafont et al, 2002). The turnover of sphingomyelin, a ubiquitous sphingolipid component of animal cell membranes that is enriched in membrane rafts, is mediated by sphingomyelinases. These enzymes catalyze the breakdown of sphingomyelin to ceramide and phosphocholine (Goni & Alonso, 2002). Interestingly, neutral sphingomyelinase (NSM) and ASM are activated in response to various stress stimuli, including hydrogen peroxide, hypoxia, TNF-α, and infection (Hannun & Luberto, 2000; Grassme et al, 2003; Marchesini & Hannun, 2004). To investigate whether the activation of sphingomyelinases and consequently the disruption of sphingolipid-rich membrane domains could account for the inhibition of Shigella binding to host cells observed upon stress, we used specific inhibitors of these enzymes. Inhibition of NSM by GW4869 did not rescue the impairment of Shigella infection prompted by arsenite or anisomycin (Fig EV2A–F). However, treatment with the ASM inhibitor amitriptyline partially reverted the inhibitory effect of arsenite on Shigella infection (Fig 3A–C) and fully reverted that caused by anisomycin (Fig 3D–F). Corroborating these results, knockdown of ASM blunted the effect of arsenite on Shigella infection (Fig 3G–I). Of note, in the absence of stress, the inhibition of ASM activity by amitriptyline or ASM knockdown did not affect Shigella infection (Fig EV2G–J). The effect of amitriptyline in blunting ASM enzymatic activity and ceramide production upon arsenite or anisomycin treatment was confirmed (Fig EV2K–M). In addition, the decreased ASM activity, RNA, and protein levels upon knockdown were confirmed (Fig EV2N–P). Taken together, these results show that, in conditions of cellular stress, ASM activity is responsible for the inhibition of Shigella infection. Click here to expand this figure. Figure EV2. Neutral sphingomyelinase (NSM) inhibition does not affect Shigella infection upon host cell stress A–C. Representative images (A), cfu (B), and qRT–PCR (C) quantification of intracellular Shigella in HeLa cells, pre-treated with arsenite, with arsenite plus the NSM inhibitor GW4869, or control. D–F. Representative images (D), cfu (E), and qRT–PCR (F) quantification of intracellular Shigella in HeLa cells, pre-treated with anisomycin, with anisomycin plus GW4869, or DMSO (control). G, H. Cfu (G) and qRT–PCR (H) quantification of intracellular bacteria in HeLa cells treated or not with amitriptyline and infected with Shigella WT. I, J. Cfu (I) and qRT–PCR (J) quantification of intracellular bacteria in HeLa cells transfected with ASM or control siRNA. K. ASM enzymatic activity quantification in HeLa cells, treated with arsenite, with arsenite plus the ASM inhibitor amitriptyline, or control. The ASM enzymatic activity was determined in the membrane fraction corresponding to 3.0 × 105 cells per condition. L, M. Ceramide quantification in HeLa cells, treated with arsenite (L) or anisomycin (M), with the stressors plus amitriptyline, or control. Ceramide levels are shown normalized to mock-treated cells. N. Analysis of smpd1 (ASM) expression determined by qRT–PCR in HeLa cells transfected with ASM or control siRNA. Results are shown normalized to cells transfected with control siRNA. O. Western blot analysis of ASM levels in HeLa cells transfected with ASM siRNA or control siRNA; β-actin was used as loading control. P. ASM enzymatic activity quantification in HeLa cells transfected with ASM siRNA or control siRNA, and treated or not with arsenite. The ASM enzymatic activity was determined in the membrane fraction corresponding to 3.0 × 105 cells per condition. Data information: Shigella infection was performed at MOI 10 and analyzed at 0.5 hpi. Results are shown as mean ± s.e.m. of 5 (panels J, K–M, and P), 6 (panel I), or 7 (panels B, C, E–H, and N) independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA for panels B, C, E, F, K–M; paired t-test for panels G–J and N; two-way ANOVA for panel P). Scale bars, 50 μm. Source data are available online for this figure. Download figure Download PowerPoint Figure 3. Inhibition of Shigella infection upon host cell stress is a consequence of ASM activation A–F. Representative images (A, D), cfu (B, E), and qRT–PCR (C, F) quantification of intracellular Shigella in HeLa cells pre-treated with arsenite or anisomycin, in the presence or not of amitriptyline, and corresponding controls. G–I. Representative images (G), cfu (H), and qRT–PCR (I) quantification of intracellular Shigella in HeLa cells transfected with ASM siRNA or control siRNA and pre-treated or not with arsenite prior to infection. J–M. 3D reconstruction of representative images of ASM (J, K) or ceramide (L, M) staining in HeLa cells treated with arsenite or anisomycin and corresponding controls. ASM, ceramide, and Hoechst staining were surface-converted by voxel distance. Data information: Infection was performed with Shigella WT at MOI 10 and analyzed at 0.5 hpi. Results are shown as mean ± s.e.m. of 5 (H, I) or 7 (B–F) independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). Scale bars, 50 μm (A, D, G) and 5 μm (J–M). Download figure Download PowerPoint Acid sphingomyelinase is usually associated with the lysosomal compartment, but, upon activation, it is redistributed to the outer leaflet of the plasma membrane, where it hydrolyzes sphingomyelin giving rise to ceramide-rich platforms (Grassme et al, 2001). To validate whether ASM is relocalized as part of the cell response to stress, we used confocal microscopy followed by 3D reconstruction to visualize the accumulation of ASM and ceramide at the membrane. Indeed, treatment of cells with arsenite or anisomycin induced a marked accumulation of ASM at the cell surface, in both HeLa and HCT-8 cells (Fig 3J and K, Appendix Fig S2A—HeLa; Appendix Fig S2E—HCT-8). Consistent with these findings, a strong accumulation of ceramide was also observed at the membrane of cells treated with arsenite or anisomycin (Fig 3L and M, Appendix Fig S2C—HeLa; Appendix Fig S2F—HCT-8). As expected, ASM knockdown diminished ceramide accumulation in response to arsenite (Appendix Fig S2D). The ASM staining specificity was confirmed in cells transfected with ASM siRNA, in which a considerable decrease in ASM foci was observed (Appendix Fig S2B). To further reinforce these observations, we quantified ceramide in live cells, by flow cytometry. In live cells, the ceramide antibody can only recognize extracellular/exposed epitopes and thus exclusively labels ceramide present at the cellular surface. This analysis confirmed a significant increase of ceramide in cells treated with arsenite and anisomycin, and a reversion of the increase upon amitriptyline treatment (Fig EV2L and M). Overall, these results show that the binding of Shigella to host cells in conditions of cellular stress is impaired as a consequence of the membrane remodeling induced by ASM activation and translocation to the plasma membrane. p38 MAPK is required for the inhibition of Shigella infection upon stress Previous studies have shown a strong relation between sphingomyelinases and p38 mitogen-activated protein kinase
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