Apoptotic signaling clears engineered Salmonella in an organ-specific manner

Taylor J. Abele
Zachary P. Billman
Lupeng Li
Carissa K. Harvest
Alexia K. Bryan
Gabrielle R Magalski
Joseph P Lopez
Heather N. Larson
Xiao-Ming Yin
Edward A. Miao author has email address

Department of Integrative Immunobiology, Duke University School of Medicine, Durham, NC, USA 27710
Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA 27710
Department of Cell Biology, Duke University School of Medicine, Durham, NC, USA 27710
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 27599
Department of Biomedical Engineering, Duke University Pratt School of Engineering, Durham, NC, USA 27710
Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, LA, USA 70112

https://doi.org/10.7554/eLife.89210.2

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Figures and data

FliC^ON S. Typhimurium activates apoptotic backup pathways in vitro.
A) Cell death pathways activated by FliC^ON S. Typhimurium.
B) Schematic of engineered FliC^ON construct.
C-F) BMMs were infected with indicated SPI2-induced S. Typhimurium strains.
C) Western blot analysis of cytosolic and mitochondrial fractions at 5 hpi. Representative image from
three independent experiments.
D) Western blot analysis of whole cell lysates at 4 hpi. Representative image from three independent experiments.
E) LDH release at 1-8 hpi. Results representative of 3 independent experiments. Data are represented as mean ± SD of 3 technical replicates.
F-G) Immunofluorescence and brightfield. Cells were stained with PI, cleaved caspase-3/7, and Hoechst.
F) Representative image from two (brightfield, PI) or one (cleaved caspase-3/7) independent experiments at 4hpi. 60x magnification, scale bar 20µm. Arrows, pyroptotic cells. Carrots, apoptotic cells.
G) Z-stack slices from Movie S1. Gsdmd^−/− BMMs infected with FliC^ON imaged at 6 hpi. Representative Z-stack from three (brightfield, PI) or one (cleaved caspase-3/7) independent experiments. 60x magnification, Z-slices 11, 18, 21, and 26 shown. Carrots; selected examples of apoptotic bodies.

Backup apoptosis does not clear FliC^ON S. Typhimurium in the spleen.
A) Schematic of competitive index infection model.
B-H) Mice were infected with a 1:1 ratio of FliC^ON and a vector control S. Typhimurium. Bacterial burdens in the spleen were determined at the indicated timepoints.
B) Timecourse of competitive index infection in indicated mice. Mice were infected with 5x10² CFU of each strain. Ratio of vector to FliC^ON is graphed. Data representative of three (WT, Gsdmd^−/−) or two (Nlrc4^−/−, Casp1^−/−) independent experiments. Line connects mean, n=3-4 mice per genotype per timepoint. Two-way ANOVA n.s. p>0.05; *** p<0.001.
C-F) Individual burdens of vector and FliC^ON from (B). Paired vector and FliC^ON data from each mouse are connected by a line. Two-way repeated measure ANOVA. n.s. p>0.05, *p<0.05, ** p<0.01
G) Mice were infected with 5x10⁴ CFU of each strain. Bacterial burdens in the spleen were determined at 48 hpi. Ratio of vector to FliC^ON is graphed. Combined two independent experiments, line representing mean ± SD, n=7-13 mice per genotype. Kruskal-Wallis n.s. p>0.05; *** p<0.001, ****p<0.0001.
H) Individual burdens from (G). Paired vector and FliC^ON data from each mouse are connected by a line. Two-way repeated measure ANOVA. n.s. p>0.05, ****p<0.0001

Engineered BID^ON S. Typhimurium activates apoptosis in vitro.
A) Schematic of engineered BID^ON construct.
B) Pathway model showing how BID^ON leads to intrinsic apoptosis.
C-G) BMMs were infected with indicated SPI2-induced S. Typhimurium strains.
C-E) Western blot analysis of whole cell lysates at 6 hpi. Data representative of two (C) or three (D-E) independent experiments.
F) Western blot analysis of cytosolic and mitochondrial fractions at 4 hpi. Data representative from three independent experiments.
G) Brightfield at 6 hpi. Data representative of three independent experiments. 60x magnification, scale
bar 20 µm, carrot, apoptotic blebs.
H) Z-stack slices from Movie S2. WT BMMs infected with BID^ON imaged at 6 hpi. Representative Z-stack from three (brightfield, PI) or one (cleaved caspase-3/7) independent experiments. 60x magnification, Z-slices 11, 19, 22, and 24 shown. Carrots; selected examples of apoptotic bodies.

Apoptosis is induced slower than pyroptosis.
A-B) BMMs were infected with indicated SPI2-induced S. Typhimurium strains.
A) Western blot analysis of whole cell lysates. Representative of five independent experiments.
B) Immunofluorescence and brightfield. Cells were stained with PI, cleaved caspase-3/7, and Hoechst and imaged at indicated timepoints. Representative image from three (brightfield, PI) or one (cleaved caspase-3/7) independent experiments. Z-stack of the 6h timepoint are represented in Movies S1-2. Z-stack slice 19 (FliC^ON in Gsdmd^−/−) and slice 20 (BID^ON in WT) shown here. 60x magnification, scale bar 20µm. Arrows, pyroptotic cells. Carrots, apoptotic cells.

Intrinsic apoptosis does not clear engineered S. Typhimurium in the spleen.
A-D) Mice were infected with a 1:1 ratio of either FliC^ON or BID^ON and a vector control S. Typhimurium. Mice were infected with 5x10² CFU of each strain. Bacterial burdens in the spleen were determined at the indicated timepoints.
A) Timecourse competitive index infection in WT mice. Ratio of vector to either FliC^ON or BID^ON is graphed. Data is combined from three independent experiments, line connects means, n=13-14 mice per condition. Two-way ANOVA *** p<0.001, ****p<0.0001.
B) Individual burdens of vector and FliC^ON or BID^ON from (A). Paired vector and FliC^ON or BID^ON data from each mouse are connected by a line. Two-way repeated measure ANOVA n.s. p>0.05; *** p<0.001, ****p<0.0001.
C) Competitive index infection of indicated mice infected with either FliC^ON or BID^ON. Ratio of vector to either FliC^ON or BID^ON is graphed. Bacterial burdens in the spleen were determined at 48 hpi. Data is combined from three independent experiments, line representing mean ± SD, n=10-12 mice per condition. One-way ANOVA n.s. p>0.05, ****p<0.0001
D) Individual burdens of vector and FliC^ON or BID^ON from (C). Paired vector and Strain^ON data from each mouse are connected by a line. Two-way repeated measure ANOVA n.s. p>0.05, ****p<0.0001
E) Schematic of triple competitive index model.
F-G) Mice were infected simultaneously with three strains, 5x10² CFU each of Cam^R vector, Kan^R FliC^ON, and Amp^R BID^ON S. Typhimurium. Bacterial burdens in the spleen were determined at 48 hpi.
F) Triple competitive index infection of WT mice. Ratio of Cam^R vector to Kan^R FliC^ON or Amp^R BID^ON is graphed. Data is combined from three independent experiments, line representing mean ±SD, n=15. Unpaired two-tailed t-test ****p<0.0001.
G) Individual burdens of Cam^R vector, Kan^R FliC^ON, and Amp^R BID^ON from (F). Paired vector, FliC^ON, and BID^ON data from each mouse are connected by a line. Two-way repeated measure ANOVA n.s. p>0.05, ****p<0.0001.

Pyroptosis clears FliC^ON from myeloid compartment in vivo.
A) Mice were infected with 5x10⁴ CFU of each strain. Bacterial burdens in the spleen were determined at 48 hpi. Ratio of vector to FliC^ON is graphed. Combined two independent experiments, line representing mean ± SD, n=6 mice per genotype. One-way ANOVA n.s. p>0.05; ****p<0.0001.
B) Individual burdens from (A). Paired vector and FliC^ON data from each mouse are connected by a line. Two-way repeated measure ANOVA. n.s. p>0.05, ****p<0.0001

Apoptotic pathways lead to clearance in the cecum.
A-D) Mice were orally treated with 20mg streptomycin, and 24h later orally infected with 1x10⁷ CFUs total bacteria comprised of a 1:1 ratio of the indicated ampicillin resistant strain and kanamycin resistant vector (pWSK129) control S. Typhimurium, all on a flgB mutant background. Bacterial burdens in the cecum, mesenteric lymph nodes (MLN), and fecal sample were determined at 48 hpi.
A) Competitive index is graphed as a ratio of vector to either pWSK29 or FliC^ON. Data is combined from two (cecum, fecal) or one (MLN) independent experiments (MLN was not harvested in the first experiment, where we harvested the spleen, which had negligible burdens; one additional representative experiment is shown in Figure S6A-B), line representing mean ± SD, n=10 (cecum, fecal) or 5 (MLN) mice per condition. Two-way repeated measure ANOVA n.s. p>0.05, ****p<0.0001.
B) Individual burdens of vector and pWSK29 or FliC^ON from (A). Paired vector and Strain^ON data from each mouse are connected by a line. Two-way repeated measure ANOVA n.s. p>0.05, *p<0.05, ***p<0.001, ****p<0.0001.
C) Competitive index infection of WT mice infected with either SspH1^SS-HA or BID^ON. Ratio of vector to either SspH1^SS-HA or BID^ON is graphed. Data is combined from two independent experiments, line representing mean ± SD, n=10 mice per condition. Two-way repeated measure ANOVA n.s. p>0.05, ****p<0.0001.
D) Individual burdens of vector and SspH1^SS-HA or BID^ON from (C). Paired vector and Strain^ON data from each mouse are connected by a line. Two-way repeated measure ANOVA n.s. p>0.05, **p<0.01, ****p<0.0001.
E) Schematic demonstrating the ability of pyroptotic or apoptotic signaling to lead to clearance of engineered S. Typhimurium in either IECs or macrophages.

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