Mitochondrial dysfunction caused by outer membrane vesicles from Gram-negative bacteria activates intrinsic apoptosis and inflammation

Abstract

Sensing of microbes activates the innate immune system, depending on functional mitochondria. However, pathogenic bacteria inhibit mitochondrial activity by delivering toxins via outer membrane vesicles (OMVs). How macrophages respond to pathogenic microbes that target mitochondria remains unclear. Here, we show that macrophages exposed to OMVs from Neisseria gonorrhoeae, uropathogenic Escherichia coli and Pseudomonas aeruginosa induce mitochondrial apoptosis and NLRP3 inflammasome activation. OMVs and toxins that cause mitochondrial dysfunction trigger inhibition of host protein synthesis, which depletes the unstable BCL-2 family member MCL-1 and induces BAK-dependent mitochondrial apoptosis. In parallel with caspase-11-mediated pyroptosis, mitochondrial apoptosis and potassium ion efflux activate the NLRP3 inflammasome after OMV exposure in vitro. Importantly, in the in vivo setting, the activation and release of interleukin-1β in response to N. gonorrhoeae OMVs is regulated by mitochondrial apoptosis. Our data highlight how innate immune cells sense infections by monitoring mitochondrial health.

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Fig. 1: NOMVs kill macrophages independent of caspase-11.
Fig. 2: OMVs induce BAK-dependent death in macrophages.
Fig. 3: OMVs trigger mitochondrial dysfunction.
Fig. 4: MCL-1 and BCL-XL control macrophage survival after OMV treatment.
Fig. 5: OMVs induce IL-1β secretion depending on mitochondrial apoptosis.

Data availability

All data are available in the main text, Extended Data Figs. 1–10, Supplementary Table 1, Supplementary Videos 14 and ProteomeXchange (PXD018563). Source data are provided with this paper.

Code availability

The MetaMorph journals, which are command scripts used for the quantification of live cell imaging data, are freely available at https://cloudstor.aarnet.edu.au/plus/s/8XGL750dIgwLYDn.

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Acknowledgements

We thank A. Strasser, D. Huang and S. Masters (Walter and Eliza Hall Institute of Medical Research) and V. Dixit (Genentech) for providing the genetically modified mice; I. D. Hay (University of Auckland) for the ClearColi and uropathogenic E. coli bacterial strains; M. Ryan (Monash University) for the antibodies; D. Creek (Monash Proteomics and Metabolomics Facility) for liquid chromatography–mass spectrometry support; the Monash Micro Imaging Facility for expert advice on live cell imaging microscopy; and the Monash Animal Research Platform for providing mice. This research was supported by grants from the National Health and Medical Research Council (grant nos. 1145788, 1183070 and 1141466 to J.E.V.; 1162765 and 1181089 to K.E.L.; and 1183848 and 1163556 to T.N.). J.L. and J.E.V. are Australian National Health and Medical Research Council Principal Research and Career Development fellows. K.E.L. and T.N. are Australian Research Council (ARC) Future fellows (FT190100266 and FT170100313).

Author information

Affiliations

Authors

Contributions

P.D. performed and analysed the vesicle purification, live cell imaging, ELISA, immunoblot, proteomics and mouse experiments. S.H.C. performed the radiography, ELISA and live cell imaging experiments. M.-L.H. and J.L. performed the lipid mass spectrometry analysis. C.H. and R.B.S. performed the protein mass spectrometry analysis. M.S., J.E. and K.E.L. performed and analysed the ELISA and mouse experiments. S.D. performed the ELISA and live cell imaging experiments. B.T.K., J.E.V. and K.E.L. generated the genetically modified mice. J.E.V., K.E.L. and T.N. supervised the project.

Corresponding author

Correspondence to Thomas Naderer.

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Competing interests

B.T.K., J.E.V. and K.E.L. are currently or have been employed at the Walter and Eliza Hall Institute, which received research funding and milestone payments from AbbVie and Genentech regarding ABT-199, which was developed based on ABT-737. All other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Lipid A modification of N. gonorrhoeae OMVs.

a, b, The mass spectrum (m/z) of lipid A purified from N. gonorrhoeae (a) and NOMVs (b) and identification of the major lipid species (M1, M2, M3 and M4 with their measured and predicted molecular weight to charge ratios, m/z) that contain phosphate (blue) or phosphoethanolamine (PEA, red) headgroups as indicated in (c). Representative of at least four independent experiments.

Extended Data Fig. 2 OMVs derived from N. gonorrhoeae induce inflammation in mice independent of caspase-1 and 11.

WT and caspase-1/11 deficient (Casp1/11-/-) mice were intraperitoneally (i.p) injected with N. gonorrhoeae OMVs (NOMV, 100µg) or PBS. a, Change in total body weight over time. Mean and SD, n = 2 PBS and 5 OMV-treated mice. The statistical significance was determined by two-way ANOVA followed by Sidak’s multiple comparison test. b, Images of Casp1/11-/- mice i.p injected with PBS or NOMV 24 hours post injection. c, Serum IL-1β levels 24 hours post treatment. Mean and SEM (n = 5 mice/group). The statistical significance was determined by two-tailed unpaired t test. Source data

Extended Data Fig. 3 BAK-mediated cell death in OMV-treated macrophages.

Live-cell imaging to monitor the cell death (DRAQ7 staining) over time (ah) of BAK (Bak-/-) and BAX (Bax-/-) deficient bone-marrow derived macrophages (BMDMs) treated with (a) N. gonorrhoeae OMVs (NOMV), (b) PBS, (d) uropathogenic E. coli derived OMVs (UOMV), (e) P. aeruginosa (POMV), (f) E.coli BW25113 (EOMVs) and (h) OMVs derived from LPS-deficient clear E. coli BL21 (COMV). c, Cell death (DRAQ7 staining) of WT and Casp8/Ripk3-/- deficient BMDMs treated with PBS or NOMV determined by live-cell imaging. g, Cell death (DRAQ7 staining) of WT BMDMs treated with PBS or E. coli OMVs (EOMV) derived from strain BW25113, BL21 and clear E. coli (COMV) using live-cell imaging. Mean and SD from three biological samples. Representative of at least two independent experiments. Source data

Extended Data Fig. 4 Lipid A modifications in OMVs derived from UPEC and P. aeruginosa.

a, c, The mass spectrum (m/z) of lipid A purified from uropathogenic E. coli (UPEC) (a) and (c) P. aeruginosa bacterial cells and (b, d) their respective OMVs. e, f, Identification and delineation of the major lipid A species (M5, M6, M7, M11, M12 & M13 and their modifications M8, M9, M10, M14, M15, M16 and M17). Both unmodified and 4-amino-4-deoxy-L-arabinose-modified (L-Ara4N-modified) or palmitate-modified (C16:0) forms are shown. Blue colour shows unmodified lipid A, red colour shows (PEA and L-Ara4N-modified lipid A, and brown colour shows C16:0 modified lipid A. A minor peak at m/z 1445.86 indicates the lipid A at m/z 1615.99 losing a fatty acyl chain at position 3. Representative of three independent experiments.

Extended Data Fig. 5 N. gonorrhoeae OMVs induce metabolic reprogramming in macrophages.

a, b, Bone marrow-derived macrophages (BMDMs) treated with PBS or N. gonorrhoeae OMVs (NOMV) were analysed by proteomics to identify up (red) and down (blue) -regulated proteins involved in (a) cell death signalling and (b) metabolism. Data from three (1, 2 and 3) independent experiments. AA, amino acid; Glyc, glycolysis; FA fatty acid; NT, nucleotide; PTM, post-translational modification; DT, detoxification; RA, retinoic acid.

Extended Data Fig. 6 Imaging mitochondrial membrane potential, caspase-3/7 activation and cell death in OMV treated macrophages.

WT BMDMs treated with (a) N. gonorrhoeae OMVs (NOMV, 50 µg/mL) or (b) cycloheximide (CHX, 1 µg/mL) were simultaneously analysed for mitochondrial membrane potential (ΔΨm) using TMRM staining, caspase-3/7 activity (CellEvent) and cell death (DRAQ7) at the single cell level using live-cell imaging. Mean and SEM from n = 3 independent experiments. c, Bright field, TMRM, caspase-3/7 activity and DRAQ7 staining, including merged images, of WT BMDMs treated with NOMVs at indicated time points. Yellow arrow indicates a cell that transiently regains TMRM staining before caspase-3/7 activation and cell death. Scale bar 50 µm. Representative images from three independent experiments. Source data

Extended Data Fig. 7 OMV treated macrophages transiently regain mitochondrial membrane potential.

Bak+/- control BMDMs treated with N. gonorrhoeae OMVs (NOMV, 50 µg/mL) were stained with TMRM (ΔΨm, red), caspase-3/7 substrate (green) and DRAQ7 (cell death, magenta) at indicated time points using live-cell imaging. Scale bar 50 µm. Representative images from three independent experiments.

Extended Data Fig. 8 OMV-treated macrophages show reduced protein synthesis.

a, 35S methionine labelled proteins from bone marrow-derived macrophages (BMDMs) exposed to two independent OMVs from N. gonorrhoeae (NOMV1, NOMV2), E. coli (EOMV), uropathogenic E. coli (UOMV) and LPS-deficient clear E. coli (COMV). Cycloheximide (CHX, 1 µg/mL), no 35S methionine (no35S) and Coomassie stained gel are controls. Representative of two independent experiments. b, 35S methionine labelled proteins from BMDMs treated with oligomycin (0.5 µM), antimycin A (0.5 µg/mL), FCCP (0.5 µM), NOMV (50 µg/mL), cycloheximide (CHX, 1 µg/mL) or PBS. Representative of two independent experiments. c, Dead (DRAQ7 positive) BMDMs treated with oligomycin (0.5 µM), antimycin (0.5 µg/mL) or DMSO. Data represent mean and SD from n = 3 independent experiments. d, BMDMs treated with oligomycin (0.5 µM), antimycin (0.5 µg/mL) or PBS with or without ABT-737 (1 µM) for 24 hours. Immunoblot of MCL-1, BCL-XL and cleaved caspase-3. Ponceau staining is loading control. Representative of two independent experiments. e, Immunoblot analysis of MCL-1 (L, long isoform; S, small isoform; fN, floxed N-terminus), BCL-XL and tubulin after treatment with N. gonorrhoeae OMV (NOMV, 50 µg/mL) or cycloheximide (CHX, 1 µg/mL) four hours. Representative of three independent experiments. f, WT, Bcl-xL-/- and Mcl-1-fN BMDMs exposed to NOMVs or PBS were probed for MCL-1, BCL-XL, cleaved caspase-3 at 24 hours post treatment. Tubulin is shown as loading control. Representative of two independent experiments. g, Dead (DRAQ7 stained) WT and BCL-XL deficient (Bcl-xL-/-) BMDMs treated with E. coli OMVs (EOMVs). Mean SD, n = 3 biological samples. Representative of two independent experiments. Source data

Extended Data Fig. 9 OMV-mediated secretion of IL-1β.

a, Dead (DRAQ7 stained) wild-type (WT) bone-marrow derived macrophages (BMDMs) treated with N. gonorrhoeae OMVs (NOMV) with or without NLRP3 inhibitor (MCC950) were analysed by live-cell imaging. Mean, SD, n = 3 biological samples. Representative of two independent experiments. b, Dead WT and Nlrp3-/- BMDMs. Mean and SEM from n = 6 independent experiments. c, Secreted IL-1β levels from WT BMDMs unprimed or primed with LPS (50 ng/mL) and treated with NOMV (50 µg/mL) or nigericin (10 µM) at indicated time points. Mean, SEM, n = 3 independent experiments. d, e and f, Secreted IL-1β levels from (d) Bak+/-, Bak-/- and Bak-/-/Baxfl/fl,LysMCre (Bax/Bak-/-), (e) Casp1/11-/-, Casp-11-/- and (f) Gsdmd-/- BMDMs exposed to NOMV, E. coli (EOMV), LPS deficient clear E. coli (COMV), uropathogenic E. coli (UOMV) and P. aeruginosa (POMV) 24 hours post-treatment. Mean, SD from n = 3-4 independent experiments. g, Immunoblot analysis of full length (FL) and cleaved GSDMD (p30). Ponceau staining is loading control. Molecular weight markers (kDa) on the left. Representative of two independent experiments. h, Serum IL-1β levels after N. gonorrhoeae OMVs (100 µg) peritoneal injection 6 hours post-treatment. Data represent individual mice, mean and SEM (n = 8 mice per group). Two Bcl-xL-/- mice were excluded because of morbidity. (i) Serum IL-1β and (j) TNF-α 6 hours post E. coli LPS i.p. injection. Data represent individual mice, mean and SEM (n = 5). (k) Images of Bax/Bak-/- deficient mice i.p injected with NOMV 24 hours post treatment. Representative image of five mice. Source data

Extended Data Fig. 10 Model of the role of mitochondrial apoptotic factors activated by OMVs.

The outer membrane vesicle (OMV) released by Gram-negative bacteria contains lipopolysaccharide (LPS) which activates cytosolic caspase-11, triggering gasdermin-D cleavage and pyroptosis in macrophages in vitro. Pore-formation by gasdermin-D causes potassium (K+) efflux which is sensed by the NLRP3/caspase-1 inflammasome to secrete mature IL-1β. OMVs also traffic pore-forming toxins to mitochondria and disrupt organellar homeostasis. Mitochondrial stress eventually prevents host protein synthesis which depletes pro-survival factor MCL-1. MCL-1 together with BCL-XL inhibit pro-death factor BAK and activation of apoptotic caspases, such as caspase-3 and -7. Apoptosis leads to potassium efflux, NLRP3/caspase-1 activation and secretion of IL-1β. Caspase-3/7 can also activate caspase-8 which can trigger IL-1β secretion in an NLRP3-dependent and independent manner. Activation of NLRP3 further contributes to cell death after OMV exposure, likely independent of caspase-1, but perhaps engaging caspase-8 or unknown factors. In vivo, IL-1β secretion and inflammation are primarily regulated by BCL-XL, BAK and BAX, suggesting N. gonorrhoeae OMVs activate additional host apoptotic factors and evade caspase-11 sensing.

Supplementary information

Reporting Summary

Supplementary Table 1

WT BMDMs treated with NOMVs or PBS for 24 h were analysed by mass spectrometry to identify up- and downregulated proteins in three independent samples.

Supplementary Video 1

Bright-field and DRAQ7 staining of WT BMDMs treated with EOMVs acquired every 30 min over 48 h. The results are representative of three independent experiments.

Supplementary Video 2

Bright-field and DRAQ7 staining of caspase-11-deficient (Casp11/−) BMDMs treated with EOMVs acquired every 30 min over 48 h. The results are representative of three independent experiments.

Supplementary Video 3

Bright-field and DRAQ7 staining of WT BMDMs treated with NOMVs acquired every 30 min over 48 h. The results are representative of three independent experiments.

Supplementary Video 4

Bright-field and DRAQ7 staining of caspase-1- and caspase-11-deficient (Casp1/11/−) BMDMs treated with NOMVs acquired every 30 min over 48 h. The results are representative of three independent experiments.

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Unprocessed autoradiograph, Coomassie gel and western blot.

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Western blot and Ponceau stain of membrane.

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Unprocessed autoradiograph, Coomassie gel, western blot and Ponceau stain of membrane.

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Western blot and Ponceau stain of membrane.

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Deo, P., Chow, S.H., Han, M. et al. Mitochondrial dysfunction caused by outer membrane vesicles from Gram-negative bacteria activates intrinsic apoptosis and inflammation. Nat Microbiol 5, 1418–1427 (2020). https://doi.org/10.1038/s41564-020-0773-2

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