Listeria hijacks host mitophagy through a novel mitophagy receptor to evade killing

Abstract

Cells use mitophagy to remove damaged or unwanted mitochondria to maintain homeostasis. Here we report that the intracellular bacterial pathogen Listeria monocytogenes exploits host mitophagy to evade killing. We found that L. monocytogenes induced mitophagy in macrophages through the virulence factor listeriolysin O (LLO). We discovered that NLRX1, the only Nod-like receptor (NLR) family member with a mitochondrial targeting sequence, contains an LC3-interacting region (LIR) and directly associated with LC3 through the LIR. NLRX1 and its LIR motif were essential for L. monocytogenes–induced mitophagy. NLRX1 deficiency and use of a mitophagy inhibitor both increased mitochondrial production of reactive oxygen species and thereby suppressed the survival of L. monocytogenes. Mechanistically, L. monocytogenes and LLO induced oligomerization of NLRX1 to promote binding of its LIR motif to LC3 for induction of mitophagy. Our study identifies NLRX1 as a novel mitophagy receptor and discovers a previously unappreciated strategy used by pathogens to hijack a host cell homeostasis system for their survival.

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Fig. 1: L. monocytogenes induces mitophagy in macrophages.
Fig. 2: NLRX1 is a novel mitophagy receptor.
Fig. 3: NLRX1 is essential for L. monocytogenes–induced mitophagy.
Fig. 4: L. monocytogenes hijacks host mitophagy for its survival both in vitro and in vivo.
Fig. 5: L. monocytogenes–induced mitophagy decreases mitochondrial ROS production and promotes bacterial survival.
Fig. 6: Conventional autophagy is required for L. monocytogenes–induced mitophagy.
Fig. 7: L. monocytogenes induces mitophagy mainly through LLO.
Fig. 8: L. monocytogenes induces NLRX1 oligomerization for mitophagy activation through release of LRR-domain-mediated autoinhibition.

Data availability

All summary or representative data generated and supporting the findings of this study are available within the paper. Full scans of all blots and gels are shown in Supplementary Dataset 1. All primary data are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (91842306, 81430036, 81830018, 91429307, 91542119 and 31470179), the National Key R&D Program of China (2018YFA0507402) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19000000). GFP-LC3 vector was provided by Q. Chen (Nan Kai University). Bacterial strains of DP-L4317 and DP-L6173 were provided by D.A. Portnoy (University of California).

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Contributions

Y.Q. and Y.Z. designed the experiments and wrote the manuscript; Y.Z. and Y.Y. conducted the experiments and analyzed the data. X.Q., G.W., Z.H., S.C., H.G. and Z.W. helped with experiments. S.E.G., H.S., J.W. and N.Y. provided reagents. Y.Q. supervised the study.

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Correspondence to Youcun Qian.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 CCCP and L.m induce mitophagy response in mouse PMs.

a,b, Q-PCR analysis of mtDNA/nDNA (a) (n=3) and western blot analysis of mitochondria markers TIM23 and HSP60 (b) in mouse PMs after CCCP stimulation (30μM) for the indicated time points. c,d, Mitochondria membrane potential (Δψm) determined by TMRM staining after L.m infection (c) or CCCP (30μM) stimulation (d). (n=3). e, Q-PCR analysis of mtDNA/nDNA in mouse PMs infected with L.m at MOI 5 with or without CQ (n=3). f, Western blot analysis of mitochondria markers TIM23 and HSP60 treated as in (e). g, Light microscopy analysis of mouse PM morphology after L.m infection at the indicated time points at MOI 5. TNF+ZVAD as a positive control for cell death. Scale bars, 100μm. h,i, Western blot analysis of cleaved caspase-3 (h) or caspase-1 (i) after L.m infection at the indicated time points at MOI 5 in PMs. TNF+CHX (TC) and LPS+ATP (L+A) as positive controls. j. LDH release from mouse PMs after L.m infection at MOI 5 for the indicated time points or CCCP treatment (n=4). TNF+ZVAD (TZ) as a positive control. k, Q-PCR analysis of mtDNA/nDNA of mouse PMs infected with L.m and treated with or without Mdivi-1 (20μM) for 3h (n=3). l, Δψm after L.m infection or CCCP stimulation for 6h in the THP1 macrophages (n=3). m, Q-PCR analysis of mtDNA/nDNA in THP1 macrophages infected with L.m at MOI 5 with or without CQ (n=3). n, Western blot analysis of mitochondria markers TIM23 and HSP60 treated as in (m). o, Q-PCR analysis of mtDNA/nDNA of THP1 macrophages infected with L.m and treated with or without Mdivi-1 for 3h (n=3). Data are representative of three (a-l) or two independent experiments (m-o) and represent mean ± SEM (a,c-e,j-m,o). Two-sided Student’s t-test was used to measure significance.

Supplementary Figure 2 Mitophagy induction was analyzed after S.Typhimurium, E.coli or C. rodentium infection.

a, Q-PCR analysis of mtDNA/nDNA after Salmonella enterica Typhimurium (S.T) infection in mouse PMs at MOI 5 for the indicated time points (n=3). b,c, Western blot analysis of mitochondria markers TIM23 and HSP60 (b) in mouse PMs after S.T infection as in (a) and densitometric quantification of TIM23/GAPDH and HSP60/GAPDH ratios (c) of (b). (n=3 independent experiments). d, Q-PCR analysis of mtDNA/nDNA after E.coli infection in mouse PMs at MOI 30 for the indicated time points (n=3). e,f, Western blot analysis of mitochondria markers TIM23 and HSP60 (e) in mouse PMs after E.coli infection as in (d) and densitometric quantification of TIM23/GAPDH and HSP60/GAPDH ratios (f) of (e). (n=3 independent experiments). g, Q-PCR analysis of mtDNA/nDNA after C. rodentium infection in mouse PMs at MOI 30 for the indicated time points (n=3). h,i, Western blot analysis of mitochondria markers TIM23 and HSP60 (h) in mouse PMs after C. rodentium infection as in (g) and densitometric quantification of TIM23/GAPDH and HSP60/GAPDH ratios (i) of (g). (n=3 independent experiments). j,k, TMRM staining (j) or Q-PCR analysis of mtDNA/nDNA (k) in HeLa cells after L.m (MOI 50) or S.T (MOI 50) infection for the indicated time points. (n=3). l, Q-PCR analysis of mtDNA/nDNA in HT29 cells at the indicated time points after treatment as in (k) (n=3). Data are representative of three (a-i) or two (j-l) independent experiments and represent mean ± SEM (a,c-d,f-g,i-l). Two-sided Student’s t-test was used to measure significance.

Supplementary Figure 3 L.m may induce mitophagy through a previously unknown pathway.

a, Western blot analysis of PINK1 accumulation in THP1 macrophages after L.m (MOI 5) infection or CCCP stimulation. b, Parkin subcellular location in mouse PMs after L.m (MOI 5) infection or CCCP (30μM) stimulation for 6h. c, Confocal microscopy analysis of mitochondrial marker HSP60 co-localized with ubiquitin (Ub) in mouse PMs after L.m infection at MOI 5 or CCCP stimulation for 3h. Scale bars, 5μm. d, Quantification of the Pearson’s co-localization coefficient between HSP60 and Ub as shown in (c). 30 cells were counted in each group from two different experiments. e, Q-PCR analysis of mtDNA/nDNA in mouse PMs that were treated with siRNAs of Pink1 or Prkn and then infected with L.m (MOI 5) or CCCP (30μM) stimulation for 3h (n=3). f,g, PINK1 (f) (n=3) or Parkin (g) Knock-down efficiency in (e). h,i, Q-PCR analysis of PINK1 mRNA in THP1 macrophages (h) or mouse PMs (i) infected with L.m (MOI 5) for 3h or CCCP stimulation for 3h. (n=3). j, Q-PCR analysis of mtDNA/nDNA in mouse PMs that were treated with siRNAs for the indicated mitophagy receptors and then infected with L.m (MOI 5) or CCCP stimulation. (n=3). k, The gene knock-down efficiency in (j) (n=3). Data are representative of three (a-b,e-k) or two (c-d) independent experiments and represent mean ± SEM (d-f,h-k). Two-sided Student’s t-test was used to measure significance.

Supplementary Figure 4 NLRX1 is partially required for CCCP-induced mitophagy.

a, Confocal microscopy analysis of LC3-GFP co-localized with wild-type NLRX1-HA (NLRX1-WT-HA) or NLRX1-dLIR-HA overexpressed in GFP-LC3 stable HeLa cells without or with CCCP (10µM) stimulation for 2h. Representative microscopic images with higher magnification of the indicated area in square. Scale bars, 10µm. b, Quantification of pearson’s co-localization coefficient between LC3-GFP and wild-type NLRX1-HA or NLRX1-dLIR-HA as shown in (a). 30 cells were counted in each group from two different experiments. c, Representative microscopic images showing intracellular distribution of wild-type NLRX1-HA or NLRX1-dLIR-HA with mitochondria marker HSP60 in HeLa cells. Scale bars, 5µm. d, Q-PCR analysis of mtDNA/nDNA in mouse PMs treated with CCCP (30µM) for the indicated time points (n=3). e, Western blot analysis of mitochondria marker TIM23 treated as in (d). f, Densitometric quantification of TIM23/GAPDH ratios of (e). (n=3 independent experiments). g, Western blot analysis of LC3-II after glucose and serum starvation treatment with or without CQ for the indicated time points. h, Densitometric quantification of LC3-II/GAPDH ratios of (g). (n=3 independent experiments). Data are representative of three (d-h) or two (a-c) independent experiments and represent mean ± SEM (b,d,f,h). Two-sided Student’s t-test was used to measure significance.

Supplementary Figure 5 NLRX1 does not affect the production of type I IFN or inflammatory cytokines after L.m infection.

a-c, ELISA analysis of TNF in spleen (a) (day 0, n=3 and day 3, n=6) and serum (b) (day 3, n=6), and IL-1β in spleen (c) (day 0, n=4 and day 3, n=6) from Nlrx1fl/fl or Nlrx1fl/flLys2-cre mice after intraperitoneally L.m infection for 3 days. d-g, Q-PCR analysis of Tnf in spleen (d) and liver (e), Il6 in spleen (f), and Ifnb in spleen (g) from Nlrx1fl/fl or Nlrx1fl/flLys2-cre mice after L.m infection for 3 days. (day0, n=4 and day3, n=6). h, Western blot analysis of p-IκBα and p-P65 in WT or Nlrx1–/– PMs infected with L.m (MOI 5) for the indicated time points. i-m, Q-PCR analysis of Tnf (i) (n=3), Il6 (j) (n=3) and Ifnb (l) (n=3) or ELISA analysis of IL-6 (k) (n=4) and IL-1β (m) (n=4) of WT or Nlrx1–/– PMs after L.m infection for the indicated time points. Data are representative of three independent experiments (a-m) and represent mean ± SEM (a-g,i-m). Two-sided Student’s t-test was used to measure significance.

Supplementary Figure 6 LLO secreted from L.m induces mitophagy in macrophages but not in epithelial cells.

a-c, Q-PCR analysis of mtDNA/nDNA in mouse PMs, treated with or without isolated L.m DNA or RNA (1μg/ml) (a), transfected with or without isolated L.m DNA or RNA (1μg/ml) by Lipofectmine 2000 (b), or treated with or without HKLM (heat killed L.m) (MOI 5) (c) for the indicated time points. (n=3). d-g, Δψm measured by TMRM staining (d,f) or Q-PCR analysis of mtDNA/nDNA (e,g) in HeLa cells (d,e) or HT29 cells (f,g) with or without LLO stimulation (100ng/ml) for the indicated time points. (n=3). h, Q-PCR analysis of mtDNA/nDNA in mouse PMs infected with L.m-EGDe Δhly bacteria complemented with LLO expression (L.m-EGDe CΔhly) at MOI 5 (n=3). i,j, Intracellular L.m loads in J774 cells (i) or mouse PMs (j) infected with L.m-EGDe or L.m-EGDe Δhly strain (MOI 5 or MOI 30) for the indicated time points. (n=3). k, Q-PCR analysis of mtDNA/nDNA levels in mouse PMs infected with L.m-EGDe Δhly strain (MOI 30) at indicated time points (n=3). l, Intracellular L.m loads in wild-type (WT) or Nlrx1–/– PMs infected with L.m-EGDe strain (MOI 5) for 6h (n=3). m, Bacteria loads in the liver and spleen from WT or Nlrx1–/– mice i.p. infected with 2.5x105 CFU L.m-EGDe strain for 3 days (n=3). n, mtROS in WT or Nlrx1–/– PMs infected with L.m-EGDe or L.m-EGDe Δhly for 6h (n=3). Data are representative of three (a-c) or two (d-n) independent experiments and represent mean ± SEM (a-n). two-sided Student’s t-test was used to measure significance.

Supplementary Figure 7 LLO induces calcium influx to promote mitochondria damage and mitophagy.

a-b, Δψm analyzed by TMRM staining (a) or Q-PCR analysis of mtDNA/nDNA (b) in mouse PMs after LLO treatment in the present of BAPTA-AM (25µM) stimulation or KCl (90mM) medium. (n=3). c, The Fluo-4 intensity over time determined by FACS in mouse PMs after LLO treatment (LLO was added at 180s) with or without BAPTA-AM. d,e, Δψm (d) (Mock, n=3 and LLO, n=4) and Q-PCR of mtDNA/nDNA (e) (n=3) in siNC and siMcu mouse PMs after LLO treatment. f, Mcu Knock-down efficiency in (d,e) (n=3). g,h, Q-PCR of NLRX1 mRNA (g) (n=4) and western blot analysis of NLRX1 (h) in the indicated cell lines. i, Western blot analysis of NLRX1 in HeLa, HeLa with NLRX1 overexpression (HeLa-NLRX1) and THP1 macrophages. j, Q-PCR analysis of mtDNA/nDNA in the indicated cells as in (i) treated with LLO (n=3). Data are representative of two (a-j) independent experiments and represent mean ± SEM (a-b,d-g,j). Two-sided Student’s t-test was used to measure significance.

Supplementary Figure 8 ER-tagged NLRX1-dLRR is sufficient to induce ER-phagy.

a, Confocal microscopy analysis of ER marker CLIMP-63 co-localized with Flag-tagged dmt-NLRX1, ret-dmt-NLRX1, ret-dmt-NLRX1-dLRR or ret-dmt-NLRX1-dLRR-dLIR in U2OS cells. Scale bars, 5μm. b, Quantification of the Pearson’s co-localization coefficient between CLIMP-63 and the individual Flag-tag proteins as shown in (a). 20 cells were counted in each group from two different experiments. c, Oligomerization of ret-dmt-NLRX1 or ret-dmt-NLRX1-dLRR overexpressed in 293T cells. d, Western blot analysis of ER marker CLIMP-63 after overexpression of the indicated proteins as in (a). e, Densitometric quantification of CLIMP-63/Actin ratios of (d). (n=3 independent experiments). f, Confocal microscopy analysis of LC3 co-localized with Flag-tag ret-dmt-NLRX1, ret-dmt-NLRX1-dLRR or ret-dmt-NLRX1-dLRR-dLIR in U2OS cells. Representative images are shown. Scale bars, 5μm. g, Quantification of the Pearson’s co-localization coefficient between LC3 and the individual Flag-tag protein as shown in (f). Cells were counted in each group from two different experiments. (n=20 in ret-dmt-NLRX1 or ret-dmt-NLRX1-dLRR-dLIR group; n=30 in ret-dmt-NLRX1-dLRR group). h, The model for L.m-induced mitophagy through a novel mitophagy receptor NLRX1. In quiescent macrophages, NLRX1 localized on mitochondria is maintained in a monomeric autoinhibition state by its LRR associated with NACHT. L.m infection or LLO stimulation decreases mitochondria membrane potential, releases its LRR suppression and induces NLRX1 oligomerization to expose its LIR motif to associate with LC3 for mitophagy induction. Data are representative of two (a-c,f,g) or three (d,e) independent experiments and represent mean ± SEM (b,e,g). Two-sided Student’s t-test was used to measure significance.

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Zhang, Y., Yao, Y., Qiu, X. et al. Listeria hijacks host mitophagy through a novel mitophagy receptor to evade killing. Nat Immunol 20, 433–446 (2019). https://doi.org/10.1038/s41590-019-0324-2

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