Apoptotic signaling clears engineered Salmonella in an organ-specific manner
- Department of Integrative Immunobiology, Duke University School of Medicine, United States
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, United States
- Department of Cell Biology, Duke University School of Medicine, United States
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, United States
- Department of Biomedical Engineering, Duke University Pratt School of Engineering, United States
- Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, United States
Figures
Figure 1 with 2 supplements
FliCON S. Typhimurium activates apoptotic backup pathways in vitro.
(A) Cell death pathways activated by FliCON S. Typhimurium. (B) Schematic of engineered FliCON construct. (C–F) Bone marrow-derived macrophages (BMMs) were infected with indicated SPI2-induced S. …
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Figure 1—source data 1
Western blot images for Figure 1C.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig1-data1-v1.zip
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Figure 1—source data 2
Western blot images for Figure 1D.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig1-data2-v1.zip
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Figure 1—source data 3
Data for Figure 1E.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig1-data3-v1.xlsx
Figure 1—figure supplement 1
Vector control S. Typhimurium does not activate apoptotic backup pathways in vitro.
(A–E) Bone marrow-derived macrophages (BMMs) were infected with indicated SPI2-induced S. Typhimurium strains. (A) Western blot analysis of whole cell lysates at 4 hpi. Results from one experiment. …
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Figure 1—figure supplement 1—source data 1
Western blot images for Figure 1—figure supplement 1A.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Data for Figure 1—figure supplement 1B–C.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig1-figsupp1-data2-v1.xlsx
Figure 1—video 1
Engineered FliCON S. Typhimurium activates apoptosis in Gsdmd–/– bone marrow-derived macrophages (BMMs) in vitro.
Gsdmd–/– BMMs were infected with FliCON SPI2-induced S. Typhimurium strains. Cells were stained with PI, cleaved caspase-3/7, and Hoechst and imaged at 6 hpi. 60 x magnification, scale bar 20 µm. …
Figure 2 with 1 supplement
Backup apoptosis does not clear FliCON S. Typhimurium in the spleen.
(A) Schematic of competitive index infection model. (B–H) Mice were infected with a 1:1 ratio of FliCON and a vector control S. Typhimurium. Bacterial burdens in the spleen were determined at the …
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Figure 2—source data 1
Data for Figure 2B–H.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Competitive index model can be used to study the clearance of S. Typhimurium in vivo.
(A–B) Mice were infected with a 1:1 ratio of pWSK129 (‘vector’) and pWSK29 (backbone of FliCON and BIDON plasmids) S. Typhimurium. Mice were infected with 5 × 102 CFU of each strain. Bacterial …
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Figure 2—figure supplement 1—source data 1
Data for Figure 2—figure supplement 1A–D.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig2-figsupp1-data1-v1.xlsx
Figure 3 with 2 supplements
Engineered BIDON S. Typhimurium activates apoptosis in vitro.
(A) Schematic of engineered BIDON construct. (B) Pathway model showing how BIDON leads to intrinsic apoptosis. (C–G) Bone marrow-derived macrophages (BMMs) were infected with indicated SPI2-induced S…
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Figure 3—source data 1
Western blot images for Figure 3C.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig3-data1-v1.zip
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Figure 3—source data 2
Western blot images for Figure 3D.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig3-data2-v1.zip
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Figure 3—source data 3
Western blot images for Figure 3E.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig3-data3-v1.zip
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Figure 3—source data 4
Western blot images for Figure 3F.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig3-data4-v1.zip
Figure 3—figure supplement 1
Production of SspH1SS-HA-BIDBH3 construct does not cause growth defects in BIDON S. Typhimurium.
(A) OD600 growth curve in LB media. Results are combined from two independent experiments, represented as mean ± SD. (B) Bone marrow-derived macrophages (BMMs) were infected with SPI2-induced S. …
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Figure 3—figure supplement 1—source data 1
Data for Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig3-figsupp1-data1-v1.xlsx
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Figure 3—figure supplement 1—source data 2
Western blot images for Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig3-figsupp1-data2-v1.zip
Figure 3—video 1
Engineered BIDON S. Typhimurium causes apoptosis in WT bone marrow-derived macrophages (BMMs) in vitro.
WT BMMs were infected with BIDON SPI2-induced S. Typhimurium strains. Cells were stained with PI, cleaved caspase-3/7, and Hoechst and imaged at 6 hpi. 60 x magnification, scale bar 20 µm. Z-stack …
Figure 4 with 1 supplement
Apoptosis is induced slower than pyroptosis.
(A–B) Bone marrow-derived macrophages (BMMs) were infected with indicated SPI2-induced S. Typhimurium strains. (A) Western blot analysis of whole cell lysates. Representative of five independent …
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Figure 4—source data 1
Western blot images for Figure 4A.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig4-data1-v1.zip
Figure 4—figure supplement 1
Vector control S. Typhimurium does not cause regulated cell death (RCD) in vitro.
(A–B) Bone marrow-derived macrophages (BMMs) were infected with indicated SPI2-induced S. Typhimurium strains. Cells were stained with PI, cleaved caspase-3/7, and Hoechst and imaged at indicated …
Figure 5 with 1 supplement
Intrinsic apoptosis does not clear engineered S. Typhimurium in the spleen.
(A–D) Mice were infected with a 1:1 ratio of either FliCON or BIDON and a vector control S. Typhimurium. Mice were infected with 5 × 102 CFU of each strain. Bacterial burdens in the spleen were …
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Figure 5—source data 1
Data for Figure 5A–D and F–G.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
pWSK229 can be used for triple competitive index infection in vivo.
(A–B) Mice were infected simultaneously with three strains, 5 × 102 CFU each of pWSK229 (‘vector (Cam),’ Cam), pWSK129 (Kan), and pWSK29 (Amp) S. Typhimurium. Bacterial burdens in the spleen were …
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Figure 5—figure supplement 1—source data 1
Data for Figure 5—figure supplement 1A–B.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig5-figsupp1-data1-v1.xlsx
Figure 6
Pyroptosis clears FliCON from myeloid compartment in vivo.
(A) Mice were infected with 5 × 104 CFU of each strain. Bacterial burdens in the spleen were determined at 48 hpi. Ratio of vector to FliCON is graphed. Combined two independent experiments, line …
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Figure 6—source data 1
Data for Figure 6A–B.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig6-data1-v1.xlsx
Figure 7 with 1 supplement
Apoptotic pathways lead to clearance in the cecum.
(A–D) Mice were orally treated with 20 mg streptomycin, and 24 hr later orally infected with 1 × 107 CFUs total bacteria comprised of a 1:1 ratio of the indicated ampicillin-resistant strain and …
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Figure 7—source data 1
Data for Figure 7A–D.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Clearance of FliCON in the cecum is NLRC4-dependent.
(A–B) Mice were orally treated with 20 mg streptomycin, and 24 hr later orally infected with 1 × 107 CFUs total bacteria comprised of a 1:1 ratio of FliCON and vector control S. Typhimurium, all on …
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Figure 7—figure supplement 1—source data 1
Data for Figure 7—figure supplement 1A–D.
- https://cdn.elifesciences.org/articles/89210/elife-89210-fig7-figsupp1-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| gene (Mus musculus) | Bid | NCBI | NM_007544.4 | AA79-102 (BH3 domain) used for plasmid construction |
| gene (Salmonella enterica serovar Typhimurium) | SspH1 | GenBank | ACY87967.1 | AA1-137 (secretion signal) used for plasmid construction |
| strain, strain background (Salmonella enterica serovar Typhimurium, 14028s) | WT | Gift from Samuel I. Miller | ||
| strain, strain background (Salmonella enterica serovar Typhimurium, SL1344) | flgB | Gift from Kelly T. Hughes | ||
| strain, strain background (Salmonella enterica serovar Typhimurium, CS401) | flgC ΔprgH-K | Gift from Kelly T. Hughes | ||
| strain, strain background (Mus musculus, C57BL/6 J) | WT | Jax and Miao lab colony, Jax stock No. 000664 | Colony bred WT mice were always used in experiments with colony bred knockout mice. Jax-purchased mice were only used in experiments having only WT mice from Jax. | |
| strain, strain background (Mus musculus, C57BL/6 J) | Mrp8-cre | Miao lab colony, Jax stock No. 021614 | ||
| strain, strain background (Mus musculus, C57BL/6 J) | Lyz2tm1(cre)Ifo (common name LysM-cre) | Miao lab colony, Jax stock No. 004781 | ||
| strain, strain background (Mus musculus, C57BL/6 J) | Casp1fl/fl | Miao lab colony, Hu et al., 2016 | ||
| strain, strain background (Mus musculus, C57BL/6 J) | Casp1–/– | Miao lab colony, Rauch et al., 2017 | ||
| strain, strain background (Mus musculus, C57BL/6 J) | Casp1–/– Casp11129mt/129mt (referred to as Casp1/11–/–) | Miao lab colony, Kuida et al., 1995 | ||
| strain, strain background (Mus musculus, C57BL/6 J) | Gsdmd–/– | Miao lab colony, Rauch et al., 2017 | ||
| strain, strain background (Mus musculus, C57BL/6 J) | Nlrc4–/– | Miao lab colony, Mariathasan et al., 2004 | ||
| strain, strain background (Mus musculus, C57BL/6 J) | Bid–/– | Miao lab colony, Yin et al., 1999 | ||
| strain, strain background (Mus musculus, C57BL/6 J) | Il1b/Il18–/– | Miao lab colony, Shornick et al., 1996; Takeda et al., 1998 | ||
| strain, strain background (Mus musculus, C57BL/6 J) | Pycard–/–Gsdmd–/– (referred to as Pycard/Gsdmd–/–) | Miao lab colony, crossed in this paper | Produced by crossing Pycard–/– (also known as Asc–/–) (Mariathasan et al., 2004) and Gsdmd–/– mice | |
| strain, strain background (Mus musculus, C57BL/6 J) | Bid–/–Gsdmd–/– (referred to as Bid/Gsdmd–/–) | Miao lab colony, crossed in this paper | Produced by crossing Bid–/–and Gsdmd–/– mice | |
| antibody | Rabbit anti- cytochrome c monoclonal antibody | Cell Signaling Technology | 11940 | Western blot 1:750 dilution |
| antibody | Rabbit anti-GAPDH polyclonal antibody | Abcam | Ab9485 | Western blot 1:10,000 dilution |
| antibody | Rabbit anti-VDAC monoclonal antibody | Cell Signaling Technology | 4661 | Western blot 1:750 dilution |
| antibody | Rabbit anti-cleaved caspase-8 monoclonal antibody | Cell Signaling Technology | 8592 | Western blot 1:1000 dilution |
| antibody | Rat anti-BID monoclonal antibody | R&D | MAB860 | Western blot 1:500 dilution |
| antibody | Mouse anti-caspase-9 monoclonal antibody | Cell Signaling Technology | 9508 | Western blot 1:750 dilution |
| antibody | Rabbit anti-cleaved caspase-7 polyclonal antibody | Cell Signaling Technology | 9491 | Western blot 1:1000 dilution |
| antibody | Rabbit anti-cleaved caspase-3 polyclonal antibody | Cell Signaling Technology | 9661 | Western blot 1:750 dilution |
| antibody | Rabbit anti-gasdermin D monoclonal antibody | Abcam | Ab209845 | Western blot 1:1000 dilution |
| antibody | Mouse anti-HA.11 monoclonal antibody | Biolegend | MMS-101R | Western blot 1:2000 dilution |
| antibody | Goat anti-rabbit polyclonal antibody | Cell Signaling Technology | 7074 | Western blot secondary 1:2000 dilution |
| antibody | Goat anti-rat polyclonal antibody | Jackson ImmunoResearch | 112-035-062 | Western blot secondary 1:10,000 dilution |
| antibody | Goat anti-mouse polyclonal antibody | Jackson ImmunoResearch | 115-035-062 | Western blot secondary 1:10,000 dilution |
| recombinant DNA reagent | pWSK29 (“Vector”) | Wang and Kushner, 1991 | See “Materials and methods, Table 1” | |
| recombinant DNA reagent | pWSK129 (“Vector”) | Wang and Kushner, 1991 | See “Materials and methods, Table 1” | |
| recombinant DNA reagent | pDM001 (“FliCON”) | Miao et al., 2010a | See “Materials and methods, Table 1” | |
| recombinant DNA reagent | pTA007 (“BIDON”) | This paper | See “Materials and methods, Table 1” | |
| recombinant DNA reagent | pTA021 (“SspH1SS-HA”) | This paper | See “Materials and methods, Table 1” | |
| recombinant DNA reagent | pWSK229 (“CamR Vector”) | This paper | See “Materials and methods, Table 1” | |
| recombinant DNA reagent | pTA015 (“KanR FliCON”) | This paper | See “Materials and methods, Table 1” | |
| recombinant DNA reagent | pTA016 (“KanR BIDON”) | This paper | See “Materials and methods, Table 1” | |
| commercial assay or kit | Pierce ECL | ThermoFisher Scientific | 32106 | |
| commercial assay or kit | SuperSignal West Pico PLUS ECL | ThermoFisher Scientific | 34580 | |
| commercial assay or kit | SuperSignal West Femto ECL | ThermoFisher Scientific | 34095 | |
| commercial assay or kit | CytoTox 96 LDH assay | Promega | G1780 | |
| software, algorithm | Prism 9 | GraphPad | ||
| Other | Hoechst 33342 | ThermoFisher | H3570 | Immuno-flourescence, used at 2 µg/ml |
| Other | Propidium Iodide | Sigma-Aldrich | P4864 | Immuno-flourescence, used at 1 µg/ml |
| Other | NucView-488 | Biotium | 10402 | Immuno-flourescence, used at 5 µM |
Table 1
Plasmids.
| Plasmids | Alias | Resistance | Notes | Reference |
|---|---|---|---|---|
| pWSK29 | Vector | Amp | Low copy vector | Wang and Kushner, 1991 |
| pWSK129 | Vector | Kan | Low copy vector | Wang and Kushner, 1991 |
| pDM1 | FliCON | Amp | pWSK29 expressing fliC fliS from sseJ promotor | Miao et al., 2010a |
| pTA007 | BIDON or AmpR BIDON | Amp | pWSK29 expressing sspH1SS-HA-mBIDBH3 from sseJ promotor | This work |
| pTA021 | SspH1SS-HA | Amp | pWSK29 expressing sspH1SS-HA from sseJ promotor | This work |
| pWSK229 | CamR Vector | Cam | Low copy vector | This work |
| pTA015 | KanR FliCON | Kan | pWSK129 expressing fliC fliS from sseJ promotor | This work |
| pTA016 | KanR BIDON | Kan | pWSK129 expressing sspH1SS-HA-mBIDBH3 from sseJ promotor | This work |
