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Listeria monocytogenes upregulates mitochondrial calcium signalling to inhibit LC3-associated phagocytosis as a survival strategy

Nature Microbiology volume 6, pages 366–379 (2021)Cite this article

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Abstract

Mitochondria are believed to have originated ~2.5 billion years ago. As well as energy generation in cells, mitochondria have a role in defence against bacterial pathogens. Despite profound changes in mitochondrial morphology and functions following bacterial challenge, whether intracellular bacteria can hijack mitochondria to promote their survival remains elusive. We report that Listeria monocytogenes—an intracellular bacterial pathogen—suppresses LC3-associated phagocytosis (LAP) by modulation of mitochondrial Ca2+ (mtCa2+) signalling in order to survive inside cells. Invasion of macrophages by L. monocytogenes induced mtCa2+ uptake through the mtCa2+ uniporter (MCU), which in turn increased acetyl-coenzyme A (acetyl-CoA) production by pyruvate dehydrogenase. Acetylation of the LAP effector Rubicon with acetyl-CoA decreased LAP formation. Genetic ablation of MCU attenuated intracellular bacterial growth due to increased LAP formation. Our data show that modulation of mtCa2+ signalling can increase bacterial survival inside cells, and highlight the importance of mitochondrial metabolism in host–microbial interactions.

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Fig. 1: Mutations in Mcu increase bacterial killing in cells.
Fig. 2: Mutation of Mcu increases LAP formation in L. monocytogenes infection.
Fig. 3: Increased bacterial killing in McuΔmye macrophages and mice.
Fig. 4: MCU inhibits LAP and bacterial killing.
Fig. 5: Acetylation of Rubicon downregulates LAP formation and bacterial killing.
Fig. 6: Acetylation of Rubicon inhibits interaction with p22phox/NOX2.

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Acknowledgements

We thank members of the Wen laboratory for discussions, Y. Shi from the UNC Genomics Core Facility for the microarray experiment, and M. Yuan for the metabolomics assay. This work was supported by National Institutes of Health grants R01GM120496 and R01GM135234 (to H. Wen), 5P01CA120964 and 5P30CA006516 (to J.M.A.), P30CA016086 (to the UNC Lineberger Comprehensive Cancer Center), R01CA163649, R01CA210439 and R01CA216853 (to P.K.S.), R01DE026728 (to Y.L.), R37AI044828 and R35CA231620 (to D.R.G.) and R01AI107250 (to S.S.).

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Author notes

  1. These authors contributed equally: Tianliang Li, Ligang Kong.

Authors and Affiliations

  1. Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, USA

    Tianliang Li, Xinghui Li, Weipeng Gong, Bao Zhao, Stephanie Seveau & Haitao Wen

  2. Shandong Institute of Otolaryngology, Department of Otolaryngology—Head and Neck Surgery, Shandong Provincial ENT Hospital, Shandong University, Jinan, China

    Ligang Kong, Lei Xu & Haibo Wang

  3. College of Pharmacy, Medicinal Chemistry and Pharmacognosy, The Ohio State University, Columbus, OH, USA

    Sijin Wu & Xiaolin Cheng

  4. Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA

    Kuldeep S. Attri & Pankaj K. Singh

  5. Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH, USA

    Yan Li

  6. Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Lupeng Li

  7. Proteomics Core Facility, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Laura E. Herring

  8. Division of Signal Transduction, Beth Israel Deaconess Medical Center and Department of Medicine, Harvard Medical School, Boston, MA, USA

    John M. Asara

  9. Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, MI, USA

    Xiaobo Luo & Yu L. Lei

  10. Department of Biomedical Informatics, The Ohio State University, Columbus, OH, USA

    Qin Ma

  11. Center for Microbial Pathogenesis, Abigail Wexner Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA

    John S. Gunn

  12. Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Douglas R. Green

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  1. Tianliang Li
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Contributions

H. Wen and H. Wang designed the experiments, supervised the study and interpreted the data. T.L., L.K., X.L., Y.L., W.G., B.Z., L.L. and L.X. performed the experiments and provided intellectual input. L.E.H. performed the key mass spectrometry experiment. J.M.A., K.S.A. and P.K.S. performed the key metabolomics experiments and provided intellectual input. S.W. and X.C. performed computational modelling of Rubicon–p22phox interaction and provided intellectual input. Q.M. performed biostatistical analyses and provided intellectual input. X.L., Y.L.L., S.S., J.S.G. and D.R.G. contributed intellectual input and generated critical reagents. H.W. wrote the manuscript.

Corresponding authors

Correspondence to Haibo Wang or Haitao Wen.

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Extended data

Extended Data Fig. 1 MCU Is required for L. monocytogenes-induced mitochondrial Ca2+ uptake in macrophages.

a−d, THP-1 cells (a and b) or BMMs (c and d) were incubated with rhod-2 (a and c) or Fluo-4 (b and d), followed by either WT or Δhly L. monocytogenes (MOI, 10) challenge. Fluorescence signal was read using a microplate reader. e−f, Fluorescence signal-based Ca2+ measurement to monitor mtCa2+ (e) and cytosolic Ca2+ (f) in Mcufl/fl and Mcu∆mye BMMs upon L. monocytogenes challenge. g-h, mtCa2+ (g) and cytosolic Ca2+ (h) in Mcufl/fl or Mcu∆mye BMMs were measured upon ATP stimulation (100 μM). i-j, mtCa2+ (i) and cytosolic Ca2+ (j) in Mcufl/fl or Mcu∆mye BMMs were measured upon purified protein listeriolysin O (LLO) (0.5 nM) challenge. k, mtCa2+ in BMMs pretreated with Xestospongin C (5 μM) contained in normal DMEM or Ca2+ free DMEM for 2 hours, were measured upon 0.5 nM LLO protein challenge. l, mtCa2+ in BMMs pretreated with cytochalasin D (10 μM) were measured upon L. monocytogenes challenge. The results presented are presentative of three independent experiments.

Extended Data Fig. 2 McuΔmye mice show no change in global immune cell populations or activation phenotype at naïve status.

Supplementary Figure 2. McuΔmye mice show no change in global immune cell populations or activation phenotype at naïve status. a, Cartoon of the strategy to generate myeloid-specific Mcu deletion mice and the primers used for genotyping. b, Genotyping result for indicated mice. c, Immunoblotting of MCU in Mcufl/fl and McuΔmye mice. d, Gating strategy to determine the percentage of differentiated macrophages (CD11b+F4/80+) and B cells (CD19+) from peritoneal cavity of Mcufl/fl and McuΔmye mice. e, The percentage of differentiated macrophages and B cells was shown. f, Gating strategy to determine the percentage of activated macrophage (CD80+, CD86+, MHCII+ and CD206+) from peritoneal cavity of Mcufl/fl and McuΔmye mice. g, Quantification of the mean fluorescence intensity (MFI) of the activated macrophages. h, Gating strategy to determine the percentage of macrophages (CD11b+F4/80+), neutrophils (CD11b+Ly6G+), monocytes (CD11b+Ly6C+) and conventional dendritic cells (CD11b+CD11c+) from spleen of Mcufl/fl and McuΔmye mice. i, The percentage of macrophages, neutrophils, monocytes and dendritic cells was shown. j, Gating strategy to determine the percentage of B cells (CD19+) and T cells (CD3+CD4+ and CD3+CD8+) from spleen of Mcufl/fl and McuΔmye mice. k and l, The percentage of B cells (k), and T cells (l) was shown. The averages of n = 3 biologically independent samples are shown. The error bars represent the SEM. Statistical significance was determined using t test (and nonparametric tests).

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Extended Data Fig. 3 MCU deficiency in myeloid cells attenuates bacterial growth without affecting cytokine production.

a, Colony-forming units in BMMs generated from Mcufl/fl and McuΔmye mice challenged with L. monocytogenes (MOI, 10) for 1 hour followed by gentamicin treatment for indicated time before cell lysis. Intracellular bacteria were plated on brain-heart-infusion plates. b, GFP-L. monocytogenes containing cells in BMMs generated from Mcufl/fl and McuΔmye mice challenged with L. monocytogenes (MOI, 10) for indicated periods were measured by FACS analysis. c-f, BMMs (c-d) or peritoneal macrophages (e-f) from Mcufl/fl and McuΔmye mice were left untreated or challenged with L. monocytogenes (MOI, 10) for indicated periods. Gene transcripts of Il6 and Tnfa in the cells (c and e), IL-6 and TNF-α proteins in the supernatants (d and f) were measured with RT-PCR and ELISA, respectively. g, Gene transcripts of IL6 and TNFA in THP-1 cells left untreated or stimulated with L. monocytogenes (MOI, 10) for indicated periods were measured with RT-PCR. h-i, NF-κB signaling molecules including IKKα, p65 and IκBα, and MAPK signaling molecules including ERK1/2, JNK1/2 and p38, in Mcufl/fl and McuΔmye BMMs (h) or peritoneal macrophages (i) left untreated or stimulated with L. monocytogenes (MOI, 10) for indicated periods were analyzed by immunoblotting. j, Immunoblotting of NF-κB signaling molecules and MAPK signaling molecules in THP-1 cells left untreated or stimulated with L. monocytogenes (MOI, 10) for indicated periods. The averages of n = 3 biologically independent samples are shown. The error bars represent the SEM. Statistical significance was determined using t test (and nonparametric tests).

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Extended Data Fig. 4 MCU deficiency does not affect canonical autophagy.

a-e, Confocal imaging (a and d) and quantification (b and e) of the colocalization of Zymosan (red) with either LC3B puncta (green) (a and b) or LysoTracker (green) (d-e), immunoblotting of LAP-associated molecules in isolated phagosomes or total cell lysates from Mcufl/fl and Mcu∆mye BMMs in the presence of LLO (5 nM) (c). Scale bar, 2 µm. f-i, Immunofluorescence staining of LC3B in Mcufl/fl and Mcu∆mye BMMs (f and g), or THP-1 MCU-WT and MCU-KO cells (h and i) left untreated or challenged with EBSS for 2 h, or rapamycin (100 nM) for 16 h. LC3B puncta per cell (g and i) were shown. Scale bar, 2 μm. j-l, Immunoblotting of LC3B and p62 in Mcufl/fl and Mcu∆mye BMMs (j), or THP-1 MCU-WT and MCU-KO cells (k) left untreated or pretreated with bafilomycin (50 nM) for 1 h, followed by EBSS or rapamycin (100 nM) incubation for another 2 or 16 h, respectively. Immunoblotting of AMPK and mTOR signaling molecules AKT, S6K and S6 in Mcufl/fl and Mcu∆mye BMMs challenged with L. monocytogenes (MOI, 10) for indicated periods (l). The averages of n = 3 biologically independent samples are shown. The error bars represent the SEM. Statistical significance was determined using t test (and nonparametric tests).

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Extended Data Fig. 5 L. monocytogenes-induced LAP in McuΔmye macrophages depends on NOX2.

a, Confocal imaging of DQ-ovalbumin in Mcufl/fl, Mcu∆mye, Cybb−/− and McuΔmyeCybb−/− BMMs challenged with L. monocytogenes (MOI, 10) for 2 h Scale bar, 10 μm. b-c, Confocal imaging (b) and quantification (c) of the colocalization of LysoTracker (red) and L. monocytogenes (green) in Mcufl/fl, Mcu∆mye, Cybb−/− and McuΔmyeCybb−/− BMMs challenged with GFP-L. monocytogenes for indicated periods. Scale bar, 2 μm. The averages of n = 3 biologically independent samples are shown. The error bars represent the SEM. Statistical significance was determined using t test (and nonparametric tests).

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Extended Data Fig. 6 MCU promotes acetyl-CoA production.

a-d, Fold changes in intermediate metabolites of the glycolysis (a), PPP (b), HBP (c) or TCA cycle (d) in Mcufl/fl and Mcu∆mye BMMs left untreated or treated with L. monocytogenes (MOI, 10) for 2 h. The averages of n = 3 biologically independent samples are shown. The error bars represent the SEM. Statistical significance was determined using t test (and nonparametric tests).

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Extended Data Fig. 7 Acetylation of Rubicon on K549 inhibits macrophage bactericidal effect.

a-b, Venn diagram analysis across four groups of genes. c, Immunoblotting of Ac-H3, H3K4me3, H3K9me2, H3K27me3 and H3 in Mcufl/fl and Mcu∆mye BMMs left untreated or stimulated with L. monocytogenes (MOI, 10) for indicated periods. d, Colocalization of L. monocytogenes (green) and LysoTracker (red) in RUBCN-KO THP-1 cells reconstituted with either empty vector or Flag-tagged Rubicon WT or K549R mutant upon GFP-L. monocytogenes challenge. Scale bar, 2 µm. The averages of n = 3 biologically independent samples are shown. The error bars represent the SEM. Statistical significance was determined using t test (and nonparametric tests).

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Extended Data Fig. 8 Acetylation of Rubicon on K549 inhibits its interaction with p22phox/NOX2 complex.

a-b, Model showing the structural difference of RUBCNWT and Ac-RUBCN complex after 100 ns MD simulations. RUBCN, cyan cartoon; p22, yellow cartoon; Glu5 in p22, blue stick; Lys549 and Ac-Lys549 in RUBCN, red stick. c, Schematic representation of the role of MCU in LC3-associated phagocytosis and bactericidal effect.

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Li, T., Kong, L., Li, X. et al. Listeria monocytogenes upregulates mitochondrial calcium signalling to inhibit LC3-associated phagocytosis as a survival strategy. Nat Microbiol 6, 366–379 (2021). https://doi.org/10.1038/s41564-020-00843-2

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