A unique bacterial tactic to circumvent the cell death crosstalk induced by blockade of caspase‐8

• DOI: 10.15252/embj.2020104469
• Publication Date: 2020-07-13T10:56:46Z
• Authors: Hiroshi Ashida; Chihiro Sasakawa; Toshihiko Suzuki
• Keywords: Crosstalk; Dental research; Research center

Article13 July 2020Open Access Source DataTransparent process A unique bacterial tactic to circumvent the cell death crosstalk induced by blockade of caspase-8 Hiroshi Ashida Corresponding Author [email protected] orcid.org/0000-0003-1408-6864 Department Bacterial Infection and Host Response, Graduate School Medical Dental Sciences, Tokyo University (TMDU), Tokyo, Japan Mycology Research Center, Chiba University, Chiba, Search for more papers this author Chihiro Sasakawa orcid.org/0000-0002-6919-1832 Nippon Institute Biological Science, Toshihiko Suzuki orcid.org/0000-0003-3853-0106 Information *,1,2, Sasakawa2,3 *,1 1Department 2Medical 3Nippon *Corresponding author. Tel: [phone]; E-mail: EMBO J (2020)39:e104469https://doi.org/10.15252/embj.2020104469 See also: SJ Thygesen et al (September 2020) PDFDownload PDF article text main figures. Peer ReviewDownload a summary editorial decision including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Upon invasive infection colonic epithelium, host cells induce several types eliminate pathogens. For instance, necroptosis is RIPK-dependent lytic that serves as backup system fully intracellular pathogens when apoptosis inhibited; phenomenon has been termed "cell crosstalk". To maintain their replicative niche multiply within cells, some enteric prevent epithelial delivering effectors via type III secretion system. In study, we found Shigella hijacks dual mechanism: inhibition OspC1 effector OspD3 effector. Shigella, recognize signaling key danger signal trigger form defense. counteract defense, delivers effector, protease, degrade RIPK1 RIPK3, preventing necroptosis. We believe survival prolonging bacterium's niche. Synopsis caspase-8-mediated pathogens, defence strategy. flexneri counteracts type-III system-delivered both necroptosis, thereby maintaining its in human colon cells. The Shigella-delivered prevents apoptotic signaling. OspC1-mediated caspase-8-dependent induces defence. Shigella-secreted protease targeting RIPK3 degradation. Blockade S. securing Introduction Epithelial an intrinsic immune defense against intrusion. sacrifice infected plays important role clearance damaged elimination presentation bacteria-derived antigens adaptive (Yuan al, 2016; Jorgensen 2017). Because (e.g., apoptosis, necrosis, pyroptosis, necroptosis) depending on stage infection, intensity type, physiological state, considered be aspect intrusion (Lamkanfi Dixit, 2010; Rudel 2010). Apoptosis caspase-dependent, non-inflammatory triggered mitochondria-mediated or receptor-mediated pathway, whereas pyroptosis are forms require caspase-1/-4–GSDMD RIPK3–MLKL signaling, respectively (Kerr 1972; Holler 2000; Degterev 2005; Kayagaki 2015; Shi 2015). Indeed, between different modalities (Place Kanneganti, 2019). overcome (Mocarski Intriguingly, intestinal with such enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. (EHEC), which colonize upon epithelia, not observed (Jones 2008; Hemrajani Blasche 2013; Kobayashi Li Pearson 2013). This suggests because necessary these niches, they deploy multiple countermeasures activation pathways (Stewart Cookson, 2016). particular, deliver proteins (T3SS), enables invasion, regulation death, evasion system, allowing them efficiently epithelium (Pinaud 2018). flexneri, causative agent bacillary dysentery, invades colonizes ultimately leading severe inflammatory colitis. injects subset T3SS into bacterium invade escape from vacuole, cytoplasm (Ashida During invasion multiplication bacteria release pathogen-associated molecular patterns (PAMPs) create damage-associated (DAMPs), genotoxic stress, mitochondrial damage, oxidative stress recognizes PAMPs DAMPs at early mechanism aimed terminating (Carneiro 2009; Dupont Bergounioux 2012). However, scaffold, spread neighboring evading surveillance (Pendaries 2006; Carneiro 2012; 2014; Mou example, caspase-4-dependent pyroptotic OspC3 (Kobayashi late remain poorly understood. there various involving modalities, it difficult identify mechanisms underlying functions regulate death. interplay emerging field research crucial understanding development, homeostatic maintenance, (Fritsch 2019; Newton processes modulate each other mutual inhibitory mechanisms, stand ready serve routes event defect first-line response. inhibited pharmacological agents, genetic mutation, (Pasparakis Vandenabeele, obtained first evidence two pathways, major identified prevented Infected DAMP mechanism, helps scaffold. Results inhibits elucidate strategies counteracting HT29 WT, S325 (T3SS-deficient mutant; non-invasive), mutant strains lacking T3SS-secreted effectors. Lactate dehydrogenase (LDH) cytotoxicity assay, established indicator revealed ospD3 (ΔospD3) underwent higher rate than WT (Fig 1A). OspD three homologs, OspD1, OspD2, (Tobe 2006), but neither ΔospD1 nor ΔospD2 enhanced although ΔospD123 (ospD1, ospD2, genes triple mutant) did Figure 1. were S325, ospD deletion mutants incubated 8 h. Aliquots cellular supernatants subjected assays. *P < 0.05 (one-way ANOVA). ΔospD3 fixed TUNEL PI staining. Percentages positive (TUNEL, green; PI, red) shown graph right. nuclei stained DAPI (blue). Scale bar: 100 μm. n.s., significant; (unpaired two-tailed Student's t-test). Giemsa Arrows indicate disappeared. 20 Data information: Graphs (A B) show mean ± SD, data pooled independent experiments performed triplicates. Images (B C) representative experiments. Download figure PowerPoint Cell can morphologically classified types, non-lytic apoptosis) 2011). Hence, characterized morphology physiology ΔospD3. assays no significant difference indicating cause 1B, top). By contrast, propidium iodide (PI) staining, detects loss plasma membrane integrity, was significantly ΔospD3-infected WT-infected bottom). staining confirmed levels rupture 1C). Together, activity caspase-independent Lytic forms, caspase-dependent pyroptosis) (Jorgensen Pyroptosis accompanied caspase-1 caspase-4, resulting production IL-1β IL-18 (Cookson Brennan, 2001; Broz determine whether presence pan-caspase inhibitor z-VAD. treatment failed block ΔospD3-induced ΔospC3-induced 2013) EV1A). Consistent this, ΔospC3 caused cleavage GSDMD maturation IL-18, hallmarks EV1B). Accordingly, LDH began increase after 8-h time point (late infection), ΔospC3-infected 2-h (early infection) EV1C). had effect caspase-1, -3/7, -8 activities suggesting EV1D). suggest ΔospD3-mediated pyroptosis. Click here expand figure. EV1. necrotic indicated absence caspase (Z-VAD-fmk, 10 μM) strains. After h incubation, harvested immunoblotting. ΔospD3, Aliquot points assay. stimulated staurosporine, then Cells measurement activity. (A), (C), (D) (B) Molecular weights immunoblots kDa. available online target (pyroptosis) EV1), next focused i.e., Necroptosis programmed necrosis requires kinase followed phosphorylation MLKL Phosphorylated binds necroptotic pore, inducing involved monitored level phosphorylated during infection. When mutants, MLKL, 2A). 2. A. lysates B, C. inhibitor, (z-VAD) aliquots immunoblotting assay respectively. 0.05, D, treated siRNAs (top) (bottom), knockdown efficiency assessed (inset). (C–E) (B), (D), (E) 2 [embj2020104469-sup-0003-SDataFig2.pdf] ensure siRNA RIPK3. Treatment RIPK1/RIPK3 z-VAD, 2B C). Similarly, siRNA-mediated decreased 2D E). These imply targets measured HT29, HeLa (cervix cells), HaCaT (skin keratinocytes), HCT116 (colon cells) lines, exhibited EV2A B). Of importance, essential expressed only HeLa, HaCaT, HCT116, 293T EV2C). stably expressing (HeLa/RIPK3) GFP (HeLa/GFP), used negative control EV2D Taken together, conclude specifically EV2. ospD3, 37°C B. line (E), 0.05; (A) (B)–(D) degrading sought factors end, investigated series mutants. Levels those family member RIPK2, dramatically reduced ΔospD1, ΔospD2, ΔospD123, OspD3-dependent decrease 3A). homolog also conserved EPEC EHEC 2006). data, homologs EHEC, ΔespL ΔespL2 EV3A). unlike exhibit EV3A results use redundant inhibit degradation espL insufficient restore phosphorylation. 3. h, then, transfected Myc-tagged expression plasmids. 24 cleaved RIPK1. Multiple sequence alignment family: OspD3, EspL, EspL2. Conserved amino acids asterisks. Typical catalytic sites colored red. D. plasmids E, F. ΔospD3/D3 (ΔospD3 complemented wild-type ospD3), ΔospD3/D3CS activity-deficient mutant, cysteine residue position 64 replaced serine) (F), Graph (F) shows [embj2020104469-sup-0004-SDataFig3.pdf] EV3. cleaves C-H-D motif EPEC, (the alanine). (C) confirm degradation, empty vector ospD1, RIPK As expected, RIPK1, OspD1 OspD2 3B). hypothesized have dependent (Cys-His-Asp: C-H-D) (Pearson Importantly, EspL2 highly 3C). evaluate functional importance constructed residues alanine examined effects levels. Fig 3D, motif-substituted not, confirming motifs EV3C). further involvement serine). ΔospD3/D3, 3E). cleavage, 3E F). activity, RHIM domain obtain insight degrades truncations OspD3. characteristic domains: N-terminal domain, intermediate containing RIP homotypic interaction (RHIM), C-terminal (He Wang, Using truncations, activity-mediated (amino 532–547), cleave variants lacked EV4A). RHIMs enable interact required induction therefore acid-substituted mutations IQIG 539–542) EV4B). (RIPK1-4A) (RIPK3-4A) EV4C). RIPK3-4A (HeLa/RIPK3-4A) RIPK3-4A, lower EV4D). Based EV4. along OspD3-CS (protease Asterisks non-cleaved truncated Sequence (top). (4A 3A replacement four acids, respectively, regions RIPK1) OspD3-CS. (bottom). vector, All representatives