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Microbes exploit death-induced nutrient release by gut epithelial cells

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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Fig. 1: Regulated mammalian cell death enhances bacterial growth.
Fig. 2: Mammalian cell death nutrients promote pflB expression and growth in Salmonella.
Fig. 3: PflB promotes Salmonella fitness during foodborne infection.
Fig. 4: Intestinal epithelial cell apoptosis fuels Salmonella growth in vivo.

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.

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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.).

Author information

Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Kodi S. Ravichandran.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature thanks Andreas Baumler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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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 (be), unpaired two-tailed Student’s t-test (f) or two-way ANOVA with Tukey’s multiple comparisons test (g)

Source data.

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)

Source data.

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, dg)

Source data.

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)

Source data.

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)

Source data.

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

Source data.

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)

Source data.

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

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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)

Source data.

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)

Source data.

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)

Source data.

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, eg), two-tailed Mann–Whitney U-test with each tissue analysed separately (d, h)

Source data.

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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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