Abstract
Regulated cell death is an integral part of life, and has broad effects on organism development and homeostasis1. Malfunctions within the regulated cell death process, including the clearance of dying cells, can manifest in diverse pathologies throughout various tissues including the gastrointestinal tract2. A long appreciated, yet elusively defined relationship exists between cell death and gastrointestinal pathologies with an underlying microbial component3,4,5,6, but the direct effect of dying mammalian cells on bacterial growth is unclear. Here we advance a concept that several Enterobacteriaceae, including patient-derived clinical isolates, have an efficient growth strategy to exploit soluble factors that are released from dying gut epithelial cells. Mammalian nutrients released after caspase-3/7-dependent apoptosis boosts the growth of multiple Enterobacteriaceae and is observed using primary mouse colonic tissue, mouse and human cell lines, several apoptotic triggers, and in conventional as well as germ-free mice in vivo. The mammalian cell death nutrients induce a core transcriptional response in pathogenic Salmonella, and we identify the pyruvate formate-lyase-encoding pflB gene as a key driver of bacterial colonization in three contexts: a foodborne infection model, a TNF- and A20-dependent cell death model, and a chemotherapy-induced mucositis model. These findings introduce a new layer to the complex host–pathogen interaction, in which death-induced nutrient release acts as a source of fuel for intestinal bacteria, with implications for gut inflammation and cytotoxic chemotherapy treatment.
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Data availability
RNA-seq data have been submitted to the Gene Expression Omnibus (GEO) under accession number GSE175947 and GSE178167. Other data or unique materials generated that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.
References
Boada-Romero, E., Martinez, J., Heckmann, B. L. & Green, D. R. The clearance of dead cells by efferocytosis. Nat. Rev. Mol. Cell Biol. 21, 398–414 (2020).
Doran, A. C., Yurdagul, A. Jr & Tabas, I. Efferocytosis in health and disease. Nat. Rev. Immunol. 20, 254–267 (2020).
Bäumler, A. J. & Sperandio, V. Interactions between the microbiota and pathogenic bacteria in the gut. Nature 535, 85–93 (2016).
Charbonneau, M. R. et al. A microbial perspective of human developmental biology. Nature 535, 48–55 (2016).
Storek, K. M. & Monack, D. M. Bacterial recognition pathways that lead to inflammasome activation. Immunol. Rev. 265, 112–129 (2015).
Honda, K. & Littman, D. R. The microbiota in adaptive immune homeostasis and disease. Nature 535, 75–84 (2016).
Ke, F. F. S. et al. Embryogenesis and adult life in the absence of intrinsic apoptosis effectors BAX, BAK, and BOK. Cell 173, 1217–1230.e17 (2018).
Vince, J. E. & Silke, J. The intersection of cell death and inflammasome activation. Cell. Mol. Life Sci. 73, 2349–2367 (2016).
Schwarzer, R., Jiao, H., Wachsmuth, L., Tresch, A. & Pasparakis, M. FADD and caspase-8 regulate gut homeostasis and inflammation by controlling MLKL- and GSDMD-mediated death of intestinal epithelial cells. Immunity 52, 978–993.e6 (2020).
Iwamoto, M., Koji, T., Makiyama, K., Kobayashi, N. & Nakane, P. K. Apoptosis of crypt epithelial cells in ulcerative colitis. J. Pathol. 180, 152–159 (1996).
Lane, E. R., Zisman, T. L. & Suskind, D. L. The microbiota in inflammatory bowel disease: current and therapeutic insights. J. Inflamm. Res. 10, 63–73 (2017).
Boussios, S., Pentheroudakis, G., Katsanos, K. & Pavlidis, N. Systemic treatment-induced gastrointestinal toxicity: incidence, clinical presentation and management. Ann. Gastroenterol. 25, 106–118 (2012).
Elting, L. S. et al. The burdens of cancer therapy. Clinical and economic outcomes of chemotherapy-induced mucositis. Cancer 98, 1531–1539 (2003).
Shin, N. R., Whon, T. W. & Bae, J. W. Proteobacteria: microbial signature of dysbiosis in gut microbiota. Trends Biotechnol. 33, 496–503 (2015).
Medina, C. B. et al. Metabolites released from apoptotic cells act as tissue messengers. Nature 580, 130–135 (2020).
Saavedra, P. H. V. et al. Apoptosis of intestinal epithelial cells restricts Clostridium difficile infection in a model of pseudomembranous colitis. Nat. Commun. 9, 4846 (2018).
Christgen, S. et al. Identification of the PANoptosome: a molecular platform triggering pyroptosis, apoptosis, and necroptosis (PANoptosis). Front. Cell. Infect. Microbiol. 10, 237 (2020).
Aaes, T. L. et al. Immunodominant AH1 antigen-deficient necroptotic, but not apoptotic, murine cancer cells induce antitumor protection. J. Immunol. 204, 775–787 (2020).
Rasko, D. A. et al. The pangenome structure of Escherichia coli: comparative genomic analysis of E. coli commensal and pathogenic isolates. J. Bacteriol. 190, 6881–6893 (2008).
Hayden, M. K. et al. Prevention of colonization and infection by Klebsiella pneumoniae carbapenemase-producing enterobacteriaceae in long-term acute-care hospitals. Clin. Infect. Dis. 60, 1153–1161 (2015).
Darfeuille-Michaud, A. et al. Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn’s disease. Gastroenterology 115, 1405–1413 (1998).
Cougnoux, A. et al. Bacterial genotoxin colibactin promotes colon tumour growth by inducing a senescence-associated secretory phenotype. Gut 63, 1932–1942 (2014).
Spaulding, C. N. et al. Selective depletion of uropathogenic E. coli from the gut by a FimH antagonist. Nature 546, 528–532 (2017).
Dell, C. L., Neely, M. N. & Olson, E. R. Altered pH and lysine signalling mutants of cadC, a gene encoding a membrane-bound transcriptional activator of the Escherichia coli cadBA operon. Mol. Microbiol. 14, 7–16 (1994).
Knappe, J. & Sawers, G. A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli. FEMS Microbiol. Lett. 6, 383–398 (1990).
Chen, J. et al. Shikonin and its analogs inhibit cancer cell glycolysis by targeting tumor pyruvate kinase-M2. Oncogene 30, 4297–4306 (2011).
Chekeni, F. B. et al. Pannexin 1 channels mediate ‘find-me’ signal release and membrane permeability during apoptosis. Nature 467, 863–867 (2010).
Barthel, M. et al. Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host. Infect. Immun. 71, 2839–2858 (2003).
Choi, J. & Groisman, E. A. Acidic pH sensing in the bacterial cytoplasm is required for Salmonella virulence. Mol. Microbiol. 101, 1024–1038 (2016).
Chen, L. M., Kaniga, K. & Galán, J. E. Salmonella spp. are cytotoxic for cultured macrophages. Mol. Microbiol. 21, 1101–1115 (1996).
van der Velden, A. W., Lindgren, S. W., Worley, M. J. & Heffron, F. Salmonella pathogenicity island 1-independent induction of apoptosis in infected macrophages by Salmonella enterica serotype Typhimurium. Infect. Immun. 68, 5702–5709 (2000).
Winter, S. E. et al. Gut inflammation provides a respiratory electron acceptor for Salmonella. Nature 467, 426–429 (2010).
Sellin, M. E. et al. Epithelium-intrinsic NAIP/NLRC4 inflammasome drives infected enterocyte expulsion to restrict Salmonella replication in the intestinal mucosa. Cell Host Microbe 16, 237–248 (2014).
Knodler, L. A. et al. Noncanonical inflammasome activation of caspase-4/caspase-11 mediates epithelial defenses against enteric bacterial pathogens. Cell Host Microbe 16, 249–256 (2014).
Lee, J.-Y. et al. High-fat diet and antibiotics cooperatively impair mitochondrial bioenergetics to trigger dysbiosis that exacerbates pre-inflammatory bowel disease. Cell Host Microbe 28, 273–284.e6 (2020).
Goretsky, T. et al. p53 mediates TNF-induced epithelial cell apoptosis in IBD. Am. J. Pathol. 181, 1306–1315 (2012).
Vereecke, L. et al. Enterocyte-specific A20 deficiency sensitizes to tumor necrosis factor-induced toxicity and experimental colitis. J. Exp. Med. 207, 1513–1523 (2010).
Arnold, J. W. et al. Tumor necrosis factor-α mediates the early pathology in Salmonella infection of the gastrointestinal tract. Microb. Pathog. 14, 217–227 (1993).
Montassier, E. et al. Chemotherapy-driven dysbiosis in the intestinal microbiome. Aliment. Pharmacol. Ther. 42, 515–528 (2015).
Rigby, R. J. et al. Intestinal bacteria are necessary for doxorubicin-induced intestinal damage but not for doxorubicin-induced apoptosis. Gut Microbes 7, 414–423 (2016).
Lamkanfi, M., Kalai, M., Saelens, X., Declercq, W. & Vandenabeele, P. Caspase-1 activates nuclear factor of the κ-enhancer in B cells independently of its enzymatic activity. J. Biol. Chem. 279, 24785–24793 (2004).
Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).
Shivak, D. J. et al. A modular, Tn7-based system for making bioluminescent or fluorescent salmonella and Escherichia coli strains. Appl. Environ. Microbiol. 82, 4931–4943 (2016).
Rivera-Chávez, F. et al. Salmonella uses energy taxis to benefit from intestinal inflammation. PLoS Pathog. 9, e1003267 (2013).
Anderson, C. J., Clark, D. E., Adli, M. & Kendall, M. M. Ethanolamine signaling promotes Salmonella niche recognition and adaptation during infection. PLoS Pathog. 11, e1005278 (2015).
Rowley, C. A., Anderson, C. J. & Kendall, M. M. Ethanolamine influences human commensal Escherichia coli growth, gene expression, and competition with enterohemorrhagic E. Coli O157:H7. MBio 9, e01429-18 (2018).
Dondelinger, Y. et al. Serine 25 phosphorylation inhibits RIPK1 kinase-dependent cell death in models of infection and inflammation. Nat. Commun. 10, 1729 (2019).
Poon, I. K. H. et al. Unexpected link between an antibiotic, pannexin channels and apoptosis. Nature 507, 329–334 (2014).
Dubois, H. et al. Nlrp3 inflammasome activation and Gasdermin D-driven pyroptosis are immunopathogenic upon gastrointestinal norovirus infection. PLoS Pathog. 15, e1007709 (2019).
Acknowledgements
We thank members of the Ravichandran laboratory, M. Kendall and H. Agaisse for numerous discussions and input on this work and for critical reading of the manuscript. We thank H. Remaut for sharing the clinical E. coli isolates, A. Wullaert for Gsdmd knockout mice, and M. Bertrand for Ripk1 kinase-dead mice. We thank the Germ-Free and Gnotobiotic Mouse Facility (UGent/UZ Gent/VIB), VIB Protein Core, VIB Flow Cytometry Core, VIB Bioimaging Core, and VIB Nucleomics Core for their contributions. K.S.R. is supported by FWO (Odysseus grant G0F5716N, EOS DECODE 30837538), Special Research Fund UGent (iBOF BOF20/IBF/037), European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 835243), grants from NHLBI (P01HL120840), NIGMS (R35GM122542) and the Center for Cell Clearance/University of Virginia School of Medicine; grants to P.V. from the FWO (EOS MODEL-IDI, FWO Grant 30826052, G.0E04.16N, G.0C76.18N, G.0B71.18N, G.0B96.20N), Methusalem (BOF16/MET_V/007), iBOF20/IBF/039 ATLANTIS, Foundation against Cancer (FAF-F/2016/865, F/2020/1505), CRIG and GIGG consortia, and VIB; grants to L.V. from Foundation against cancer (2020-091) and Ghent University (iBOF A21/TT/0612); and grants to G.V.L. from Foundation against Cancer (STK 2014-142 and STK 2018-093) and FWO (G020216N). Additional support was received through the FWO Postdoctoral Fellowship (1225421N to C.J.A., and 1227220N P.M.), NIH T32 Pharmacology Training Grant (T32GM007055 to C.B.M., and B.J.B.), and Ghent University BOF grant (01P02519 to T.L.A.).
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C.J.A. and K.S.R. designed all experiments. C.J.A. performed most experiments. C.B.M. and B.J.B. assisted with, and provided conceptual advice for, multiple experiments. L.K. assisted with multiple experiments. T.L.A. assisted with immunoblotting experiments. I.L., A.G. and K.L. assisted with microscopy image acquisition and analysis. J.S.A.P. assisted with the bioinformatic analysis. P.M. provided conceptual advice for caspase-1/11 mouse experiments. G.B., V.A. and L.V. provided mice, technical assistance and conducted the germ-free mouse experiments. F.G. and P.V. provided mice and conceptual advice for caspase-3/7 mouse experiments. A.M. and G.V.L. provided mice, technical assistance, and conceptual advice for A20-deficient cell lines and mouse experiments.
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Extended data figures and tables
Extended Data Fig. 1 Primary colonocyte death ex vivo.
a, C57BL/6 colonic explant stained with haematoxylin and eosin (H&E) or indicated fluorescent markers. White arrows indicate positive TUNEL staining. Scale bars, 500 μm (H&E, TUNEL (left)) and 100 μm (TUNEL (right)). b, TUNEL-positive cells from C57BL/6 colonic explants quantified using automated slide scanning image analysis from paired samples. Live (DMSO-treated) or staurosporine (2 μM) samples were treated for 8 h ex vivo. n = 4 colonocyte explants. CD45+ and CD45+TUNEL+ positive cells from C57BL/6 colonic explants quantified using automated slide scanning image analysis from paired samples. Live (DMSO-treated) or staurosporine (2 μM) samples were treated for 8 h ex vivo. n = 4 colonocyte explants. c, Activated caspase-3 units (colorimetric assay) of C57BL/6 colonic explants. Live (DMSO-treated) or staurosporine (2 μM) samples were treated for 8 h ex vivo. n = 3 independent colonic explants. d, Activated caspase-8 units (colorimetric assay) of C57BL/6 colonic explants. Live (DMSO-treated) or staurosporine (2 μM) samples were treated for 8 h ex vivo. n = 12 independent colonic explants. e, Activated caspase-3 units (colorimetric assay) of C57BL/6 colonic explants. Live (water-treated) or Doxo (20 μg ml−1) samples were treated for 6 h ex vivo. n = 4 independent colonic explants. f, CFU of Salmonella after 9 h of aerobic growth in fresh medium + 20 μg ml−1 Doxo or C57BL/6 colonic explant supernatant following 20 μg ml−1 Doxo treatment ex vivo. n = 3. Median is shown g, CFU of Salmonella and E. coli (strain HS) after 9 h of aerobic growth in medium with or without Doxo. n = 4. Box plots are as in Fig. 1. Data are mean and s.d. *P ≤ 0.05, ***P ≤ 0.0005, two-tailed paired t-test (b–e), unpaired two-tailed Student’s t-test (f) or two-way ANOVA with Tukey’s multiple comparisons test (g)
Extended Data Fig. 2 Death triggers induce varying degrees of mammalian cell death.
a, Cartoon schematic of in vitro cell line approach. b, Schematic of the CT26:FADD system. Doxycycline is used to induce expression of the construct, while addition of B/B induces oligomerization of the FKBP12 domains. Oligomerization leads to caspase activation and subsequent apoptosis. Cell death was measured using flow cytometry 5 h after B/B addition with or without QVD treatment. Membrane integrity was measured using DNA dye Sytox blue. n = 5 per condition. Western blot of the indicated apoptotic caspases (left), necroptotic machinery (right, top), or pyroptotic caspase 1 (right, below). DDR3 cells were used as positive controls for necroptosis. Representative blots from n = 3 independent experiments. c, CT26 cell death, as measured by flow cytometry. CT26:FADD cell death with or without caspase inhibition via QVD (left, n = 5 per condition) 5 h after death induction. CT26 cell death (centre, n = 4) 24 h after 600 mJ cm−2 UV treatment. CT26 cell death (right, n = 3) 24 h after staurosporine treatment. CT26 cell death 24 h after 600 mJ cm−2 UV exposure. The DNA dye 7AAD was used to assess membrane integrity. n = 4 per condition. CT26 cell death 24 h after 1 μM staurosporine treatment. The DNA dye 7-AAD was used to monitor membrane integrity. n = 3 per condition. Western blot of the indicated apoptotic caspases (left), pyroptotic caspase 1 (right, top), or necroptotic machinery (right, bottom). DDR3 cells were used as positive controls for necroptosis. Representative blots from n = 3 independent experiments. d, Bacterial CFU after anaerobic growth in UV irradiated CT26 supernatants (n = 4). Data are mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.0001, two-way ANOVA with Tukey’s multiple comparisons test (b), one-way ANOVA with Tukey’s multiple comparisons test (c, left) Student’s t-test (c, centre, right) or multiple two-tailed Student’s t-tests (d)
Extended Data Fig. 3 Mammalian cell death induces time-dependent bacterial outgrowth.
a, CT26:FADD cells were treated overnight with doxycycline to induce construct expression and were then treated with B/B dimerizer, with or without QVD, and supernatants were collected. Periodic Salmonella growth measurements were quantified by OD600 measurements. n = 5 per condition. b, Salmonella growth. Bacterial growth was assessed via repeated OD600 measurements as indicated. c, Salmonella growth. Bacterial growth was assessed via CFU. Box plots are as in Fig. 1. d, CT26 cell death (left), as determined by flow cytometry, following 24-h treatment with 50 μM PAC-1 (apoptosis inducer) and Salmonella aerobic growth (right) in those supernatants. e, Bacterial aerobic growth (Salmonella (left), E. coli strain HS (middle) and Klebsiella (right)) in CT26 cell supernatants 24 h after 600 mJ cm−2 UV irradiation. f, Bacterial aerobic growth (Salmonella (left), E. coli strain HS (middle) and Klebsiella (right)) in CT26 cell supernatants 24 h after 1 μM staurosporine treatment. g, CT26 cell death (left) and Salmonella aerobic growth (right). CT26 cells were pre-treated for 1 h and maintained with 30 μM QVD as indicated. Data are mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, two-way ANOVA with Tukey’s multiple comparisons test (a, b, d–g)
Extended Data Fig. 4 Apoptosis enhances bacterial growth.
a, Total cell death (left) and membrane integrity (middle) of HCT116 cells 24 h after 600 mJ cm−2 UV irradiation. Corresponding Salmonella aerobic growth (right) in HCT116 supernatants as measured by OD600 values. n = 3 per condition. b, Total cell death (left) and membrane integrity (middle) of HCT116 cells 24 h after 1 μM staurosporine treatment. Corresponding Salmonella aerobic growth (right) in HCT116 supernatants. n = 3 per condition. c, Jurkat cell death characterization, as measured by flow cytometry, 4 h after 150 mJ cm−2 UV irradiation. Representative flow plot (left) and quantification of membrane integrity (middle), n = 3. Corresponding Salmonella aerobic CFU (right, n = 6). Box plots are as in Fig. 1. d, CT26 cell death after three cycles of freeze–thaw, as measured by flow cytometry. Representative flow plot (left), quantification (middle), and the corresponding Salmonella aerobic growth in the supernatants of freeze–thaw conditions (right, n = 4). Data are mean ± s.e.m. *P ≤ 0.05, ***P ≤ 0.0005, two-tailed Student’s t-test (a, b, cell death), two-way ANOVA with Tukey’s multiple comparisons test (a, b, bacterial growth curve), unpaired two-tailed Student’s t-test (c)
Extended Data Fig. 5 Mammalian death-driven bacterial growth is protein-independent.
a, Schematic of supernatant manipulations after induction of apoptosis that ruled out proteins as responsible for enhanced bacterial growth. b, Total protein levels in media or CT26 supernatants, with or without FBS, at 24 h after staurosporine treatment following indicated proteinase K or filtration strategies as determined by BCA total protein assay. n = 4 per condition. c, CFU of Salmonella. n = 3 per filter size, 9 h of aerobic growth. Median is shown. d, CFU of Salmonella. n = 6 per condition, 9 h of aerobic growth. e, CFU of Salmonella. n = 5 per condition, 9 h of aerobic growth. f, CFU of Salmonella. n = 5 per condition, 9 h of aerobic growth. g, CFU of Salmonella. n = 4 per condition, 9 h of aerobic growth. Box plots as in Fig. 1. Data are mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, multiple two-tailed Student’s t-tests (c), one-way ANOVA with multiple comparisons test (d, e) or unpaired two-tailed Student’s t-test (f, g)
Extended Data Fig. 6 RNA-seq-identified Salmonella gene regulation.
a, Schematic for Salmonella RNA-seq. Volcano plots of differentially expressed Salmonella genes. CT26:FADD (left) or HCT116 (UV) treatment (right). n = 4. Table of up and downregulated genes identified in the RNA-seq experiments described in Fig. 2 and the predicted functions and fold changes of the eight Salmonella genes conserved between the two independent RNA-seq experiments. Venn diagram of differentially regulated Salmonella genes in the two different RNA-seq experiments and the list of eight regulated genes shared between the two datasets. b, Salmonella cadB expression. n = 3 per condition. c, CFU of wild-type or ΔcadBA mutant Salmonella (CJA042) after 9 h of aerobic growth. n = 4 per condition. d, E. coli strain HS gene expression. n = 6 (stauro) and n = 3 (UV). e, CFU of wild-type or ΔpflB mutant Salmonella (CJA071) after 9 h of aerobic growth. n = 4 per condition., f, Formate concentrations in supernatants derived from live (QVD treated), staurosportine-treated, UV-irradiated, or freeze–thaw CT26 cells. n = 4 per condition. g, CT26 cell death, as measured by flow cytometry. n = 3 per condition. h, Salmonella pflB gene expression. n = 4 per condition. Box plots are as in Fig. 1. Data are mean ± s.e.m. ns, P > 0.05. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, one-way (b, f, h) or two-way (c, e) ANOVA with Tukey’s multiple comparisons test
Extended Data Fig. 7 Pannexin-1 dependent metabolites enhance Enterobacteriaceae growth.
a, Left, percentage annexin V staining and TO-PRO-3 dye uptake. n = 4. Right, WT or ΔpflB (CJA071) Salmonella CFU. n = 11 per condition. b, Salmonella CFU after 9 h of anaerobic growth. n = 4 per condition. MeMix-6 formulation used: spermidine (3.0 nM); FBP (5 nM); DHAP (0.36 μM); UDG-glucose (20 nM); GMP (21 nM); IMP (3 3nM). Middle, WT or pflB mutant (CJA071) CFU after 9 h of anaerobic growth Right, Salmonella pflB or cadB gene expression. n = 4 per condition. c, CFU after 9 h of anaerobic growth of wild-type with empty vector (LK010), pflB mutant with empty vector (LK037), or complemented pflB mutant (LK049) with or without 0.2% arabinose. n = 5. d, CFU of the indicated strain of E. coli after 7 h of anaerobic growth. n = 5. e, FBP concentrations. n = 4 per condition. Box plots are as in Fig. 1. ns, P > 0.05. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, two-way ANOVA with Sidak’s multiple comparisons test (a, b (right) c, d), one-way ANOVA with Dunnett’s multiple comparisons test compared with medium control (b (left)) or one-way ANOVA with Tukey’s multiple comparisons test (e)
Extended Data Fig. 8 Salmonella PflB does not affect host morbidity during foodborne infection.
a, Salmonella burden of WT (black) or ΔcadBA (CJA033, green). n = 7 mice from 2 cohorts. b, Salmonella burden of WT (black) or ΔpflB (CJA057, blue). n = 6 SPF mice from 2 cohorts. n = 9 germ-free mice from 2 cohorts. *P ≤ 0.05, **P ≤ 0.005 two-tailed Wilcoxon signed rank test with theoretical median of 1 using the calculated competitive index from each mouse. c, Body weight (mean ± s.e.m. is shown). n = 15 (WT), n = 10 ΔSPI-1ΔSPI-2, n = 8 ΔpflB mice from 4 cohorts. ns, P > 0.05. *P ≤ 0.05, ***P ≤ 0.0005, two-way ANOVA (body weight) or one-way ANOVA (caecal weight, colon length) with Tukey’s multiple comparisons test. d, Caecal weight. n = 15 (WT), n = 10 ΔSPI-1ΔSPI-2, n = 8 ΔpflB mice from 4 cohorts. ns, P > 0.05. *P ≤ 0.05, ***P ≤ 0.0005, one-way ANOVA with Tukey’s multiple comparisons test. e, Colon length n = 15 (WT), n = 10 ΔSPI-1ΔSPI-2, n = 8 ΔpflB mice from 4 cohorts. ns, P > 0.05. *P ≤ 0.05, ***P ≤ 0.0005, one-way ANOVA with Tukey’s multiple comparisons test. f, Salmonella burden of WT (black), ΔSPI-1ΔSPI-2 (CJA077, orange) or ΔpflB (CJA057, blue). n = 15 WT, n = 8 ΔSPI-1ΔSPI-2, n = 8 ΔpflB from 4 cohorts. ns, P > 0.05. **P ≤ 0.005, Kruskal–Wallis with Dunn’s multiple comparisons test. g, Competitive index either (black) WT Salmonella compared to ΔpflB (CJA057) or (orange) ΔSPI-1ΔSPI-2 (CJA077) compared to ΔSPI-1ΔSPI-2ΔpflB (CJA081). n = 14 mice from 4 cohorts. ns, P > 0.05. *P ≤ 0.05, two-tailed Mann–Whitney U-test. h, Cleaved caspase 3 staining in colons. Scale bars, 500 μm (top), 100 μm (zoomed in box). i, Percentage body weight of Casp3/7fl/fl control or Vil-cre+/−Casp3/7fl/fl mice after Salmonella infection (as in Fig. 3). n = 11 female Casp3/7fl/fl control mice, n = 7 female Vil-cre+/−Casp3/7fl/fl mice from 3 cohorts. Data are mean ± s.e.m. j, Caecal weight of Casp3/7fl/fl control or Vil-cre+/−Casp3/7fl/fl mice after Salmonella infection (as in Fig. 3). n = 8 female Casp3/7fl/fl control mice, n = 7 female Vil-cre+/−Casp3/7fl/fl mice from 2 cohorts. ns, P > 0.05, unpaired two-tailed Student’s t-test. k, Colon length of Casp3/7fl/fl control or Vil-cre+/−Casp3/7fl/fl mice after Salmonella infection (as in Fig. 3). n = 5 female Casp3/7fl/fl control mice, n = 5 female Vil-cre+/−Casp3/7fl/fl mice from 2 cohorts. ns, P > 0.05, unpaired two-tailed Student’s t-test. l, Salmonella burden in the indicated tissue of Casp3/7fl/fl control or Vil-cre+/−Casp3/7fl/fl mice at day 4 after infection. n = 11 female Casp3/7fl/fl control mice, n = 7 female Vil-cre+/− Casp3/7fl/fl mice from 3 cohorts. m, Competitive index of wild-type Salmonella compared to ΔpflB (CJA057) in the indicated tissue of Casp3/7fl/fl control or Vil-cre+/−Casp3/7fl/fl mice at day 4 after infection. n = 8 female Casp3/7fl/fl control mice, n = 5 female Vil-cre+/−Casp3/7fl/fl mice from 2 cohorts. ns, P > 0.05. *P ≤ 0.05, two-tailed Mann–Whitney U-test with each tissue analysed separately. Box plots are as in Fig. 1. In a and b, wild-type and mutant Salmonella connected with dotted lines come from the same mouse. The median competitive index is listed below each tissue. ns, P > 0.05. *P ≤ 0.05, two-tailed Wilcoxon signed rank test with theoretical median of 1 using the calculated competitive index from each mouse
Extended Data Fig. 9 Pyroptotic machinery does not impact PflB-dependent fitness.
a, Schematic of the in vitro model system. b, Total cell death and membrane integrity of HCT116, as measured by flow cytometry, following 24 h of infection. n = 4. c, Aerobic growth of gentamicin-resistant Salmonella (CJA001) measured by OD600. n = 8. d, Expression of pflB and cadB. n = 10. e, Salmonella burden of wild-type (black) or ΔpflB (CJA057, blue) in Casp1/11 mice. n = 7 mice from 2 cohorts. Wild-type and mutant Salmonella connected with dotted lines come from the same mouse. The median competitive index is listed below each tissue. Box plots are as in Fig. 1. Data are mean ± s.e.m. ns, P > 0.05. *P ≤ 0.05, unpaired two-tailed Student’s t-test (c, d), two-tailed Wilcoxon signed rank test with theoretical median of 1 using the calculated competitive index from each mouse (e)
Extended Data Fig. 10 TNF-induced death in A20 deficient cells.
a, Schematic of the in vitro approach. b, Immunoblotting for A20 protein in the control HCT116 or A20-knockout HCT116 cells. Blots are representative of n = 3 independent experiments. Total cell death and membrane integrity of control or A20-knockout HCT116 cells 24 h after 100 ng ml−1 human TNF stimulation. n = 3 per condition. Total cell death of control or A20-knockout HCT116 cells 24 h after 100 ng ml−1 human TNF stimulation with or without QVD. n = 4 per condition. Right, western blot of indicated apoptotic caspases. Blots are representative of three independent experiments. c, Salmonella CFU. n = 9 (no TNF), n = 13 (+TNF). d, Salmonella pflB gene expression. pflB expression was normalized to 1 in control HCT116 supernatants with and without 100 ng ml−1 human TNF. n = 7 per condition. e, Salmonella cadB gene expression. cadB expression was normalized to 1 in control HCT116 supernatants with and without 100 ng ml−1 human TNF. n = 7 per condition. f, CFU of E. coli (strain HS) and Klebsiella. n = 7 per condition per strain. Box plots are as in Fig. 1. Data are mean ± s.e.m. ns, P > 0.05. *P ≤ 0.05, ***P ≤ 0.0005, one-way ANOVA with Sidak’s multiple comparisons test (b, d, e), one-way ANOVA with Tukey’s multiple comparisons test (c) or unpaired two-tailed Student’s t-test (f)
Extended Data Fig. 11 TNF- and A20-dependent cell death enhances Enterobacteriaceae growth.
a, Schematic of in vivo infections. b, Activated caspase-3 units. Day 2: n = 4 A20fl/fl control mice, n = 6 Vil-cre+/−A20fl/fl mice from 2 cohorts. Day 3: n = 4 A20fl/fl control mice, n = 4 Vil-cre+/−A20fl/fl mice from 2 cohorts. ns, P > 0.05. *P ≤ 0.05, two-way ANOVA with Sidak’s multiple comparisons test. c, Salmonella burden in the ilea. Day 2: n = 6 A20fl/fl control mice, n = 7 Vil-cre+/−A20fl/fl mice from 2 cohorts. Day 3: n = 9 A20fl/fl control mice, n = 4 Vil-cre+/−A20fl/fl mice from 2 cohorts. d, Competitive index of WT Salmonella vs ΔpflB (CJA057) in the ilea. Day 2: n = 6 A20fl/fl control mice, n = 7 Vil-cre+/−A20fl/fl mice from 2 cohorts. Day 3: n = 7 A20fl/fl control mice, n = 4 Vil-cre+/A20fl/fl mice from 2 cohorts. e, Percentage body weight. n = 12 A20fl/fl control mice. n = 6 Vil-cre+/−A20fl/fl mice from 2 cohorts. f, Caecal weights. n = 12 A20fl/fl control mice. n = 9 Vil-cre+/−A20fl/fl mice from 2 cohorts. g, Salmonella burden. n = 12 A20fl/fl mice. n = 6 Vil-cre+/−A20fl/fl mice from 2 cohorts. Each tissue was analysed separately. Box plots are as in Fig. 1. Data are mean ± s.e.m. ns, P > 0.05. **P ≤ 0.005, ***P ≤ 0.0005, two-tailed Mann–Whitney U-test (c, d, g), two-way ANOVA with Sidak’s multiple comparisons test (e), unpaired two-tailed Student’s t-test (f)
Extended Data Fig. 12 Doxorubicin-induced death enhances Enterobacteriaceae growth in vivo.
a, Schematic of in vivo treatment. b, Representative images. Colon length (cm) n = 7 C57BL/6 and Casp3/7fl/fl control mice, n = 12 C57BL/6 and Casp3/7fl/fl mice + Doxo, n = 6 Vil-cre+/−Casp3/7fl/fl mice + Doxo from 3 cohorts. Caecal weight (g) n = 15 C57BL/6 and Casp3/7fl/fl control mice, n = 25 C57BL/6 and control Casp3/7fl/fl mice + Doxo, n = 8 Vil-cre+/− Casp3/7fl/fl mice + Doxo from 3 cohorts. Caecal weight (g) n = 12 control Panx1+/+, n = 18 control Panx1+/+ + Doxo, n = 11 Panx1−/− + Doxo mice from 6 cohorts. c, Caecal weight. n = 6 C57BL/6, n = 8 C57BL/6 mice + Doxo from 2 cohorts. Colon length. n = 6 C57BL/6, n = 8 C57BL/6 mice + Doxo from 2 cohorts. d, CFU of total Enterobacteriaceae. n = 6 C57BL/6, n = 8 C57BL/6 mice + Doxo from 2 cohorts. e, Caecal weight. n = 4 C57BL/6, n = 7 C57BL/6 mice + Doxo from 2 cohorts. f, Colon length. n = 4 C57BL/6, n = 7 C57BL/6 mice + Doxo from 2 cohorts. g, Activated caspase-3 units in the ilea. n = 4 C57BL/6, n = 7 C57BL/6 mice + Doxo from 2 cohorts. h, CFU of endogenous Enterobacteriaceae CFU in the indicated tissues of uninfected C57BL/6 mice with or without Doxo treatment, 2 days after treatment. n = 4 female C57BL/6, n = 7 female C57BL/6 mice + Doxo from 2 cohorts. The endogenous Enterobacteriaceae was identified as E. coli via 16s rRNA sequencing on purified genomic DNA as well as MALDI–TOF (Bruker) analysis on bacterial colonies. Box plots are as in Fig. 1. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, one-way ANOVA with Tukey’s multiple comparisons test (b), unpaired two-tailed Student’s t-test (c, e–g), two-tailed Mann–Whitney U-test with each tissue analysed separately (d, h)
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This file contains Supplementary Figs 1-4, which show the uncropped gel data.
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This file contains Supplementary Tables 1-3, which list the primer sequences, bacterial strains and plasmids used in the study.
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Anderson, C.J., Medina, C.B., Barron, B.J. et al. Microbes exploit death-induced nutrient release by gut epithelial cells. Nature 596, 262–267 (2021). https://doi.org/10.1038/s41586-021-03785-9
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DOI: https://doi.org/10.1038/s41586-021-03785-9
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