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Autophagy restricts Mycobacterium tuberculosis during acute infection in mice

Nature Microbiology volume 8, pages 819–832 (2023)Cite this article

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Abstract

Whether or not autophagy has a role in defence against Mycobacterium tuberculosis infection remains unresolved. Previously, conditional knockdown of the core autophagy component ATG5 in myeloid cells was reported to confer extreme susceptibility to M. tuberculosis in mice, whereas depletion of other autophagy factors had no effect on infection. We show that doubling cre gene dosage to more robustly deplete ATG16L1 or ATG7 resulted in increased M. tuberculosis growth and host susceptibility in mice, although ATG5-depleted mice are more sensitive than ATG16L1- or ATG7-depleted mice. We imaged individual macrophages infected with M. tuberculosis and identified a shift from apoptosis to rapid necrosis in autophagy-depleted cells. This effect was dependent on phagosome permeabilization by M. tuberculosis. We monitored infected cells by electron microscopy, showing that autophagy protects the host macrophage by partially reducing mycobacterial access to the cytosol. We conclude that autophagy has an important role in defence against M. tuberculosis in mammals.

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Fig. 1: Cre–Lox recombination efficiency in macrophages varies between different autophagy factors and is influenced by Cre dosage.
Fig. 2: ATG16L1 and ATG7 are required for control of M. tuberculosis at later stages of infection but the mouse phenotype is undermined by limitations of the Cre–Lox system.
Fig. 3: Atg16L1 and Atg7 mutant mice display lung lesions at later stages of infection with M. tuberculosis.
Fig. 4: Macrophages deficient in different autophagy factors are susceptible to M. tuberculosis infection.
Fig. 5: The autophagy-deficient macrophage susceptibility to M. tuberculosis is ESX-1-dependent and linked to phagosome escape.
Fig. 6: M. tuberculosis infection induces necrosis-like cell death and efferocytosis in autophagy-deficient macrophages.

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Data availability

RNA-seq data can be accessed on the GEO database, accession number GSE226503 (https://www.ncbi.nlm.nih.gov/geo/). All other data are available in the main text, source data, Extended Data figures or Supplementary Information. Source data are provided with this paper.

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References

  1. Levine, B. & Kroemer, G. Biological functions of autophagy genes: a disease perspective. Cell 176, 11–42 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Chang, C., Jensen, L. E. & Hurley, J. H. Autophagosome biogenesis comes out of the black box. Nat. Cell Biol. 23, 450–456 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Deretic, V. & Lazarou, M. A guide to membrane atg8ylation and autophagy with reflections on immunity. J. Cell Biol. 221, e202203083 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Lu, Q. et al. Homeostatic control of innate lung inflammation by Vici syndrome gene Epg5 and additional autophagy genes promotes influenza pathogenesis. Cell Host Microbe 19, 102–113 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Park, S. et al. Autophagy genes enhance murine gammaherpesvirus 68 reactivation from latency by preventing virus-induced systemic inflammation. Cell Host Microbe 19, 91–101 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Martinez, J. et al. Noncanonical autophagy inhibits the autoinflammatory, lupus-like response to dying cells. Nature 533, 115–119 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  7. Thurston, T. L. M., Wandel, M. P., Muhlinen, N., von, Foeglein, Á. & Randow, F. Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion. Nature 482, 414–418 (2012).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. Jia, J. et al. AMPK, a regulator of metabolism and autophagy, is activated by lysosomal damage via a novel galectin-directed ubiquitin signal transduction system. Mol. Cell 77, 951–969 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Global Tuberculosis Report 2020 (World Health Organization, 2020).

  10. Huang, L., Nazarova, E. V. & Russell, D. G. Mycobacterium tuberculosis: bacterial fitness within the host macrophage. Microbiol. Spectr. https://doi.org/10.1128/microbiolspec.BAI-0001-2019 (2019).

  11. Mayer-Barber, K. D. et al. Host-directed therapy of tuberculosis based on interleukin-1 and type I interferon crosstalk. Nature 511, 99–103 (2014).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gutierrez, M. G. et al. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 119, 753–766 (2004).

    Article  CAS  PubMed  Google Scholar 

  13. Kimmey, J. M. et al. Unique role for ATG5 in neutrophil-mediated immunopathology during M. tuberculosis infection. Nature 528, 565–569 (2015).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Behar, S. M. & Baehrecke, E. H. Tuberculosis: autophagy is not the answer. Nature 528, 482–483 (2015).

    Article  ADS  CAS  PubMed  Google Scholar 

  15. Watson, R. O., Manzanillo, P. S. & Cox, J. S. Extracellular M. tuberculosis DNA targets bacteria for autophagy by activating the host DNA-sensing pathway. Cell 150, 803–815 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Castillo, E. F. et al. Autophagy protects against active tuberculosis by suppressing bacterial burden and inflammation. Proc. Natl Acad. Sci. USA 109, E3168–E3176 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wallis, R. S. & Hafner, R. Advancing host-directed therapy for tuberculosis. Nat. Rev. Immunol. 15, 255–263 (2015).

    Article  CAS  PubMed  Google Scholar 

  18. McCubbrey, A. L., Allison, K. C., Lee-Sherick, A. B., Jakubzick, C. V. & Janssen, W. J. Promoter specificity and efficacy in conditional and inducible transgenic targeting of lung macrophages. Front. Immunol. 8, 1618 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Vooijs, M., Jonkers, J. & Berns, A. A highly efficient ligand‐regulated Cre recombinase mouse line shows that LoxP recombination is position dependent. EMBO Rep. 2, 292–297 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Heffner, C. S. et al. Supporting conditional mouse mutagenesis with a comprehensive cre characterization resource. Nat. Commun. 3, 1218 (2012).

    Article  ADS  PubMed  Google Scholar 

  21. Thomson, J. G., Rucker, E. B. & Piedrahita, J. A. Mutational analysis of loxP sites for efficient Cre‐mediated insertion into genomic DNA. Genesis 36, 162–167 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition). Autophagy 17, 1–382 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Ji, D. X. et al. Type I interferon-driven susceptibility to Mycobacterium tuberculosis is mediated by IL-1Ra. Nat. Microbiol. 4, 2128–2135 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Moreira-Teixeira, L. et al. Type I IFN exacerbates disease in tuberculosis-susceptible mice by inducing neutrophil-mediated lung inflammation and NETosis. Nat. Commun. 11, 5566 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. Schmidt-Supprian, M. & Rajewsky, K. Vagaries of conditional gene targeting. Nat. Immunol. 8, 665–668 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Pépin, G. et al. Cre-dependent DNA recombination activates a STING-dependent innate immune response. Nucleic Acids Res. 44, 5356–5364 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Queval, C. J., Brosch, R. & Simeone, R. The macrophage: a disputed fortress in the battle against Mycobacterium tuberculosis. Front. Microbiol. 8, 2284 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Mahamed, D. et al. Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells. eLife 6, e22028 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Lerner, T. R. et al. Mycobacterium tuberculosis replicates within necrotic human macrophages. J. Cell Biol. 216, 583–594 (2017).

  30. Tan, J. M. J. et al. An ATG16L1-dependent pathway promotes plasma membrane repair and limits Listeria monocytogenes cell-to-cell spread. Nat. Microbiol. 3, 1472–1485 (2018).

    Article  CAS  PubMed  Google Scholar 

  31. Jia, J. et al. Galectin-3 coordinates a cellular system for lysosomal repair and removal. Dev. Cell 52, 69–87 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  32. López-Jiménez, A. T. et al. The ESCRT and autophagy machineries cooperate to repair ESX-1-dependent damage at the Mycobacterium-containing vacuole but have opposite impact on containing the infection. PLoS Pathog. 14, e1007501 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Houben, D. et al. ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria. Cell Microbiol. 14, 1287–1298 (2012).

    Article  CAS  PubMed  Google Scholar 

  34. Simeone, R. et al. Phagosomal rupture by Mycobacterium tuberculosis results in toxicity and host cell death. PLoS Pathog. 8, e1002507 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Smith, J. et al. Evidence for pore formation in host cell membranes by ESX-1-secreted ESAT-6 and its role in Mycobacterium marinum escape from the vacuole. Infect. Immun. 76, 5478–5487 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Conrad, W. H. et al. Mycobacterial ESX-1 secretion system mediates host cell lysis through bacterium contact-dependent gross membrane disruptions. Proc. Natl Acad. Sci. USA 114, 1371–1376 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  37. Bell, S. L., Lopez, K. L., Cox, J. S., Patrick, K. L. & Watson, R. O. Galectin-8 senses phagosomal damage and recruits selective autophagy adapter TAX1BP1 to control Mycobacterium tuberculosis infection in macrophages. Mbio 12, e01871-20 (2021).

  38. Rosenberg, O. S. et al. Substrates control multimerization and activation of the multi-domain ATPase motor of type VII secretion. Cell 161, 501–512 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Stanley, S. A., Johndrow, J. E., Manzanillo, P. & Cox, J. S. The Type I IFN response to infection with Mycobacterium tuberculosis requires ESX-1-mediated secretion and contributes to pathogenesis. J. Immunol. 178, 3143–3152 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Watson, R. O. et al. The cytosolic sensor cGAS detects Mycobacterium tuberculosis DNA to induce type I interferons and activate autophagy. Cell Host Microbe 17, 811–819 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Beckwith, K. S. et al. Plasma membrane damage causes NLRP3 activation and pyroptosis during Mycobacterium tuberculosis infection. Nat. Commun. 11, 2270 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lim, J.-A., Zare, H., Puertollano, R. & Raben, N. Atg5flox-derived autophagy-deficient model of Pompe disease: does it tell the whole story? Mol. Ther. Methods Clin. Dev. 7, 11–14 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Koo, I. C. et al. ESX‐1‐dependent cytolysis in lysosome secretion and inflammasome activation during mycobacterial infection. Cell Microbiol. 10, 1866–1878 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Wassermann, R. et al. Mycobacterium tuberculosis differentially activates cGAS- and inflammasome-dependent intracellular immune responses through ESX-1. Cell Host Microbe 17, 799–810 (2015).

    Article  CAS  PubMed  Google Scholar 

  45. Mayer-Barber, K. D. et al. Cutting edge: caspase-1 independent IL-1β production is critical for host resistance to Mycobacterium tuberculosis and does not require TLR signaling in vivo. J. Immunol. 184, 3326–3330 (2010).

    Article  CAS  PubMed  Google Scholar 

  46. Yousefi, S. et al. Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat. Cell Biol. 8, 1124–1132 (2006).

    Article  CAS  PubMed  Google Scholar 

  47. Zhao, X. et al. Bcl-xL mediates RIPK3-dependent necrosis in M. tuberculosis-infected macrophages. Mucosal Immunol. 10, 1553–1568 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Behar, S. M. et al. Apoptosis is an innate defense function of macrophages against Mycobacterium tuberculosis. Mucosal Immunol. 4, 279–287 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Repasy, T. et al. Bacillary replication and macrophage necrosis are determinants of neutrophil recruitment in tuberculosis. Microbes Infect. 17, 564–574 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Paik, S., Kim, J. K., Chung, C. & Jo, E.-K. Autophagy: a new strategy for host-directed therapy of tuberculosis. Virulence 10, 448–459 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Flores-Valdez, M. A., Segura-Cerda, C. A. & Gaona-Bernal, J. Modulation of autophagy as a strategy for development of new vaccine candidates against tuberculosis. Mol. Immunol. 97, 16–19 (2018).

    Article  CAS  PubMed  Google Scholar 

  52. Maskey, D. et al. ATG5 is induced by DNA-damaging agents and promotes mitotic catastrophe independent of autophagy. Nat. Commun. 4, 2130 (2013).

    Article  ADS  PubMed  Google Scholar 

  53. Abram, C. L., Roberge, G. L., Hu, Y. & Lowell, C. A. Comparative analysis of the efficiency and specificity of myeloid-Cre deleting strains using ROSA-EYFP reporter mice. J. Immunol. Methods 408, 89–100 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Yu, Y.-R. A. et al. A protocol for the comprehensive flow cytometric analysis of immune cells in normal and inflamed murine non-lymphoid tissues. PLoS ONE 11, e0150606 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Chen, M. et al. Lipid mediators in innate immunity against tuberculosis: opposing roles of PGE2 and LXA4 in the induction of macrophage death. J. Exp. Med. 205, 2791–2801 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Park, J. S., Tamayo, M. H., Gonzalez‐Juarrero, M., Orme, I. M. & Ordway, D. J. Virulent clinical isolates of Mycobacterium tuberculosis grow rapidly and induce cellular necrosis but minimal apoptosis in murine macrophages. J. Leukoc. Biol. 79, 80–86 (2006).

    Article  CAS  PubMed  Google Scholar 

  57. Divangahi, M., Desjardins, D., Nunes-Alves, C., Remold, H. G. & Behar, S. M. Eicosanoid pathways regulate adaptive immunity to Mycobacterium tuberculosis. Nat. Immunol. 11, 751–758 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Martin, C. J. et al. Efferocytosis is an innate antibacterial mechanism. Cell Host Microbe 12, 289–300 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Westman, J., Grinstein, S. & Marques, P. E. Phagocytosis of necrotic debris at sites of injury and inflammation. Front. Immunol. 10, 3030 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Dallenga, T. et al. M. tuberculosis-induced necrosis of infected neutrophils promotes bacterial growth following phagocytosis by macrophages. Cell Host Microbe 22, 519–530 (2017).

    Article  CAS  PubMed  Google Scholar 

  61. Sogi, K. M., Lien, K. A., Johnson, J. R., Krogan, N. J. & Stanley, S. A. The tyrosine kinase inhibitor Gefitinib restricts Mycobacterium tuberculosis growth through increased lysosomal biogenesis and modulation of cytokine signaling. ACS Infect. Dis. 3, 564–574 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Penn, B. H. et al. An Mtb-human protein–protein interaction map identifies a switch between host antiviral and antibacterial responses. Mol. Cell 71, 637–648 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Hara, T. et al. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 441, 885–889 (2006).

    Article  ADS  CAS  PubMed  Google Scholar 

  64. Hwang, S. et al. Nondegradative role of Atg5-Atg12/ Atg16L1 autophagy protein complex in antiviral activity of interferon gamma. Cell Host Microbe. 11, 397–409 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Komatsu, M. et al. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J. Cell Biol. 169, 425–434 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We acknowledge members of the Cox, Stanley, Vance and Portnoy laboratories for helpful discussion. We thank D. A. Portnoy (University of California Berkeley) for graciously letting us use his Keyence microscope. We thank H. W. Virgin (Washington University) for providing all the mouse lines used in this study. We thank the staff of the Office of Laboratory Animal Care of the University of California Berkeley for the care provided to the animals used in this study. We thank the staff at the University of California Berkeley Electron Microscope Laboratory for advice and assistance in electron microscopy sample preparation and data collection. The sequencing for RNA-seq was carried out at the DNA Technologies and Expression Analysis Cores at the University of California Davis Genome Center, supported by NIH Shared Instrumentation grant 1S10OD010786-01. Embedding in wax, sectioning and staining for histology was performed at the University of California Davis Center for Genomic Pathology Laboratory. This work was supported by NIH grants AI162583 and AI063302 and National Institute of Allergy and Infectious Diseases Host Pathogen Map Initiative grant U19AI135990 (J.S.C.).

Author information

Author notes

  1. Guillaume R. Golovkine

    Present address: Evotec, Toulouse, France

  2. Teresa Repasy

    Present address: Bio-Rad Laboratories, Seattle, WA, USA

  3. Jonathan Budzik

    Present address: Department of Medicine, University of California, San Francisco, CA, USA

  4. Erik Van Dis

    Present address: Department of Immunology, University of Washington School of Medicine, Seattle, WA, USA

  5. Lauren M. Popov

    Present address: Novome Biotechnologies, San Francisco, CA, USA

  6. Gabriel Mitchell

    Present address: Open Innovation @ NITD, Novartis Institute for Tropical Diseases, Emeryville, CA, USA

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA

    Guillaume R. Golovkine, Allison W. Roberts, Huntly M. Morrison, Rafael Rivera-Lugo, Hannah Nilsson, Nicholas E. Garelis, Teresa Repasy, Jonathan Budzik, Erik Van Dis, Lauren M. Popov, Gabriel Mitchell & Jeffery S. Cox

  2. Department of Plant & Microbial Biology, University of California, Berkeley, CA, USA

    Rita M. McCall

  3. Department of Bioengineering, University of California, Berkeley, CA, USA

    Michael Cronce

  4. UC Berkeley—UCSF Graduate program in Bioengineering, Berkeley, CA, USA

    Michael Cronce

  5. Department of Medicine, University of California, San Francisco, CA, USA

    Jonathan Budzik

  6. Electron Microscope Laboratory, University of California, Berkeley, CA, USA

    Reena Zalpuri & Danielle Jorgens

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Contributions

J.S.C., G.R.G. and G.M. conceptualized the study. G.R.G. was responsible for conducting experiments, acquisition of data and analysis. G.R.G. and M.C. performed qPCR experiments. N.G. performed RNA-seq data analysis. G.R.G. and L.M.P. performed live imaging experiments. L.M.P., G.M. and J.S.C. provided reagents. G.R.G., A.W.R., R.M.M., H.M., T.R., J.B. and E.V.D. performed in vivo experiments. G.R.G., R.R.-L. and R.M.M. performed M. marinum experiments. G.R.G. and H.M. performed immunofluorescence experiments. G.R.G., D.J. and R.Z. conducted electron microscopy experiments. G.R.G. and H.N. performed western blot experiments. G.R.G. and J.S.C. wrote the paper.

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Correspondence to Jeffery S. Cox.

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Nature Microbiology thanks Maziar Divangahi, Vojo Deretic and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Sequences of LoxP sites, map of LoxP site insertions and measurement of Cre expression levels.

a, Sequences of LoxP sites in Atg5fl/fl, Atg16L1fl/fl and Atg7fl/fl mice. b, Map of LoxP sites and Neomycin resistance cassettes insertions in Atg5fl/fl, Atg16L1fl/fl and Atg7fl/fl mice. Grey boxes denote exons. c, Measurement of mRNA levels of Cre in bone marrow-derived macrophages differentiated for 10 days. Results are the means ± SD from 3 biological replicates. n.d. = not detected. Statistical analysis performed with two-sided t-test.

Source data

Extended Data Fig. 2 Gating strategy used to isolate lung populations in M. tuberculosis-infected mice.

Lungs from infected mice were harvested after 21 or 70 days of infection. Single cells from lung homogenates were sorted into different immune cell populations according to the strategy outlined in this figure.

Extended Data Fig. 3 Additional in vivo data on autophagy-mutant mice infected with M. tuberculosis.

Mice (n = 10-12 per group, except for LysMcre+/+ for which n = 5) were infected with approximately 100 colony-forming units (c.f.u.) of M. tuberculosis and tracked for weight change (a-c). Data is presented as means ± SD. Mice that lost 15% body weight were sacrificed are counted at −15% for subsequent time points. d-e, CFU counts from the spleens of mice (n = 2-6 per group) infected with approximately 100 c.f.u of M. tuberculosis. Spleens were harvested at 21 days postinfection (d) or 70 days postinfection (e). Data is presented as means ± SD. f-g, Proportion of immune cell populations in the lungs of Atg7 (f) and Atg16L1 (g) mutant mice infected for 70 days with M. tuberculosis. Results are the means ± SD from 2–7 mice per condition. Nt = Neutrophils, Tc = T cells, Bc = B cells, Mac = Macrophages, DC = Dendritic cells, Mono = Ly6Chi inflammatory monocytes. For statistical analysis for CFU and histology experiments, LysMcre+/- and LysMcre+/+ conditions were compared to the respective LysMwt/wt control. * P ≤ 0.05, ** P ≤ 0.01 by two-sided Mann–Whitney test.

Source data

Extended Data Fig. 4 Statistics of cell death and M. tuberculosis growth during time-course macrophage infection with M. tuberculosis.

a, b, Same data as in Fig. 4b, c but each day is presented as an individual graph. Data represents number of macrophages (a) and Mean Fluorescent Intensity of fluorescent bacteria (b). Results are the means ± SD from 4 technical replicates. For statistical analysis, LysMcre+/- and LysMcre+/+ conditions were compared to the respective LysMwt/wt control. * P ≤ 0.05 by two-sided t-test/.

Extended Data Fig. 5 Growth of M. tuberculosis in autophagy-mutant BMMs and susceptibility of Atg5-flox hemizygous macrophages.

a, BMMs from autophagy-mutant mice were infected with M. tuberculosis at a MOI of 2. CFU were enumerated at day 0, day 2 and day 4 postinfection. Results are the means ± SD from 3 technical replicates per condition. For statistical analysis, LysMcre+/- and LysMcre+/+ conditions were compared to the respective LysMwt/wt control for the day 4 condition. * P ≤ 0.05 by two-sided t-test. Grey star denotes conditions were the macrophage monolayer was disrupted. b, Macrophages from Atg5fl/wt, Atg5fl/wt LysMcre+/-, Atg5fl/wt LysMcre+/+ and Atg5fl/fl LysMcre+/+ were infected with M. tuberculosis at a MOI of 1 and macrophages were enumerated by imaging every day for 7 days. Macrophages carrying one wild-type allele of Atg5 and one recombined allele (Atg5fl/wt LysMcre+/-, Atg5fl/wt LysMcre+/+) were indistinguishable from wild-type cells. Each condition was tested in technical triplicates.

Source data

Extended Data Fig. 6 Absence of autophagy promotes phagosome escape of Mycobacteria in macrophages.

a, Illustrative images of wild-type or ΔeccC M. tuberculosis bacteria in Atg5fl/fl or Atg5fl/fl LysMcre+/+ macrophages after 24 hours of infection. Orange asterisks denote bacteria fully enclosed in phagosomes, blue asterisks show bacteria with poor membrane preservation, red asterisks denote bacteria in the cytosol. Scale bar = 0.4 µm. b, Quantification of bacteria fully enclosed in phagosomes, present directly in the cytosol or with poor membrane preservation. Data is from a single experiment. A minimum of 40 bacteria per condition were quantified. c, Actin tail formation by M. marinum expressing mCherry in Atg5fl/fl or Atg5fl/fl LysMcre+/+ macrophages was measured at 6.5 h, 8 h, 9.5 h and 11 hours postinfection. Results are the means ± SD for 15 images from two technical replicates. * P ≤ 0.05 by two-sided t-test. d, Autophagy-mutant macrophages were infected with M. tuberculosis expressing GFP or ΔeccC M. tuberculosis expressing GFP. Colocalization with Gal-3 was measured at 10 h postinfection. Results are the means ± SD from three technical replicates. For statistical analysis, LysMcre+/- and LysMcre+/+ conditions were compared to the respective LysMwt/wt control. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 by two-sided Mann–Whitney test.

Source data

Extended Data Fig. 7 Survival of uninfected autophagy-deficient macrophages and statistics of cell death during infection with ΔeccC M. tuberculosis.

a-b, Quantification of number of uninfected macrophages over time. Data is averaged from two independent experiments with each condition tested in technical duplicates. Data presented as a time course (a) or as selected time points for statistical analysis (b). Data is presented as means ± SD. c, Same data as in Fig. 5c but with selected timepoints presented as an individual graphs. Results are the means ± SD from two independent experiments with each condition tested in technical duplicates. For statistical analysis, number of macrophages in cre-positive conditions were compared to the respective LysMwt/wt control. * P ≤ 0.05 by two-sided t-test.

Source data

Extended Data Fig. 8 RNA-seq on autophagy-mutant macrophages infected with M. tuberculosis. RNA was collected from uninfected BMMs and BMMs infected with M. tuberculosis for 48 h.

a-c, Volcano plot showing differentially expressed genes in Atg5fl/fl LysMcre+/+. Grey line demarcates adj.p.value of 0.05. (a), Atg16L1fl/fl LysMcre+/+ (b) and Atg7fl/fl LysMcre+/+ (c) macrophages compared to their respective flox controls. d-f, Venn diagrams showing genes with significant transcriptional changes (adj.p.value ≤ 0.05) for the cre-positive conditions compared to their respective LysMwt/wt control. Venn diagram for all DE genes in uninfected conditions (d), all DE genes in infected conditions (e) and genes exclusively differentially expressed during infection (that is with genes with significant DE in uninfected conditions filtered out) (f). g-i, Gene enrichment analysis showing pathways enriched in M. tuberculosis-infected cells. Genes with significant transcriptional changes (adj.p.value ≤ 0.05) were used for pathway enrichment using the hypergeometric test (HGT). Datasets for enrichment included: KEGG, REACTOME, MSigDB C2_CP and C3, GO, CORUM, and HALLMARK. Top 15 pathways commonly enriched in Atg5fl/fl LysMcre+/+, Atg16L1fl/fl LysMcre+/+ and Atg7fl/fl LysMcre+/+ (g), Top 15 pathways commonly enriched in all 3 genotypes specifically during infection (h) and top 15 pathways enriched exclusively in Atg5fl/fl LysMcre+/+ macrophages (i).

Extended Data Fig. 9 M. tuberculosis infection induces necrosis-like cell death and efferocytosis in autophagy-deficient macrophages.

a, Western blot for pRIP3, RIP3, BCL-xL and β-actin on cell lysates from uninfected BMMs and BMMs infected for 48 hours with M. tuberculosis. b, Measurement by ELISA of IL-1β secretion in supernatant of macrophages infected for 48 hours with M. tuberculosis. Results are the means ± SD from two independent experiments, in which each condition was tested in technical triplicates. * P ≤ 0.05 by two-sided t-test. c-d, Atg5fl/fl LysMcre+/+ macrophages were infected with GFP-expressing M. tuberculosis and imaged every 30 minutes over a 6-day period (Supplementary video 3). Time and mode of cell death were recorded for 145 Atg5fl/fl LysMcre+/+ macrophages infected with GFP-expressing M. tuberculosis from 3 independent experiments. The average time of death by necrosis-like cell death (c) and the delay between efferocytosis and subsequent cell death (d) were measured. Center line: median; box limits: upper and lower quartiles; whiskers: 1.5x interquartile range. n = 80 and 65, respectively.

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Extended Data Fig. 10 Illustrative images and statistics of PI and CellEvent staining of macrophages infected with M. tuberculosis.

a, Illustrative images of experiment quantified in Fig. 6a, b. Autophagy-deficient macrophages were infected with M. tuberculosis at a MOI of 1. Propidium iodide and CellEvent Caspase-3/7 reagent were added to the media at the beginning of the infection. Cells were imaged every 30 minutes over a period of 8 days. Scale bar denotes 100 µm. b, Same data as in Fig. 6a, b but with selected timepoints presented as an individual graphs. Results are the means ± SD with each condition tested in technical triplicates. For statistical analysis, number of CellEvent (b) and propodium iodide (c) positive macrophages in cre-positive conditions were compared to the respective LysMwt/wt control. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 by two-sided t-test.

Supplementary information

Supplementary Information

Supplementary video legends, methods for Extended Data and references.

Reporting Summary

Supplementary Video 1

Time-lapse sequence of autophagy-deficient macrophages infected with GFP-expressing M. tuberculosis.

Supplementary Video 2

Time-lapse sequence of autophagy-deficient macrophages infected with M. tuberculosis and incubated with PI and CellEvent.

Supplementary Video 3

Illustrative video of necrosis and efferocytosis of autophagy-deficient macrophages infected with M. tuberculosis.

Source data

Source Data Fig. 1

Unprocessed gels for Fig. 1a–c and unprocessed blots for Fig. 1g,h.

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Statistical source data for Fig. 1d–f.

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Statistical source data for Fig. 2 and Extended Data Fig. 3a–c.

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Statistical source data for Fig. 3.

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Statistical source data for Fig. 4 and Extended Data Fig. 4a,b.

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Statistical source data for Fig. 5 and Extended Data Fig. 7c.

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Statistical source data for Fig. 6 and Extended Data Figs. 9c,d and 10b,c.

Source Data Extended Data Fig. 1

Statistical source data for Extended Data Fig. 1c.

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Statistical source data for Extended Data Fig. 3d–g.

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Statistical source data for Extended Data Fig. 5a,b.

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Statistical source data for Extended Data Fig. 6a–d.

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Statistical source data for Extended Data Fig. 7a,b.

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Statistical source data for Extended Data Fig. 9b.

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Unprocessed blots.

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Golovkine, G.R., Roberts, A.W., Morrison, H.M. et al. Autophagy restricts Mycobacterium tuberculosis during acute infection in mice. Nat Microbiol 8, 819–832 (2023). https://doi.org/10.1038/s41564-023-01354-6

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