Abstract
Mycobacterium tuberculosis (Mtb) survives in macrophages by evading delivery to the lysosome and promoting the accumulation of lipid bodies, which serve as a bacterial source of nutrients. We found that by inducing the microRNA (miRNA) miR-33 and its passenger strand miR-33*, Mtb inhibited integrated pathways involved in autophagy, lysosomal function and fatty acid oxidation to support bacterial replication. Silencing of miR-33 and miR-33* by genetic or pharmacological means promoted autophagy flux through derepression of key autophagy effectors (such as ATG5, ATG12, LC3B and LAMP1) and AMPK-dependent activation of the transcription factors FOXO3 and TFEB, which enhanced lipid catabolism and Mtb xenophagy. These data define a mammalian miRNA circuit used by Mtb to coordinately inhibit autophagy and reprogram host lipid metabolism to enable intracellular survival and persistence in the host.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Accession codes
References
Stanley, S.A. & Cox, J.S. Host-pathogen interactions during Mycobacterium tuberculosis infections. Curr. Top. Microbiol. Immunol. 374, 211–241 (2013).
Huang, J. & Brumell, J.H. Bacteria-autophagy interplay: a battle for survival. Nat. Rev. Microbiol. 12, 101–114 (2014).
Gutierrez, M.G. et al. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 119, 753–766 (2004).
Sakowski, E.T. et al. Ubiquilin 1 promotes IFN-γ–induced xenophagy of Mycobacterium tuberculosis. PLoS Pathog. 11, e1005076 (2015).
Stanley, S.A. et al. Identification of host-targeted small molecules that restrict intracellular Mycobacterium tuberculosis growth. PLoS Pathog. 10, e1003946 (2014).
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).
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).
Kimmey, J.M. et al. Unique role for ATG5 in neutrophil-mediated immunopathology during M. tuberculosis infection. Nature 528, 565–569 (2015).
Collins, A.C. et al. Cyclic GMP-AMP synthase is an innate immune DNA sensor for Mycobacterium tuberculosis. Cell Host Microbe 17, 820–828 (2015).
Majlessi, L. & Brosch, R. Mycobacterium tuberculosis meets the cytosol: the role of cGAS in anti-mycobacterial immunity. Cell Host Microbe 17, 733–735 (2015).
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).
Deretic, V., Saitoh, T. & Akira, S. Autophagy in infection, inflammation and immunity. Nat. Rev. Immunol. 13, 722–737 (2013).
Singh, R. & Cuervo, A.M. Autophagy in the cellular energetic balance. Cell Metab. 13, 495–504 (2011).
He, C. & Klionsky, D.J. Regulation mechanisms and signaling pathways of autophagy. Annu. Rev. Genet. 43, 67–93 (2009).
Sardiello, M. et al. A gene network regulating lysosomal biogenesis and function. Science 325, 473–477 (2009).
Rubinsztein, D.C., Codogno, P. & Levine, B. Autophagy modulation as a potential therapeutic target for diverse diseases. Nat. Rev. Drug Discov. 11, 709–730 (2012).
Sanchez, A.M. et al. The role of AMP-activated protein kinase in the coordination of skeletal muscle turnover and energy homeostasis. Am. J. Physiol. Cell Physiol. 303, C475–C485 (2012).
Russell, D.G., Cardona, P.J., Kim, M.J., Allain, S. & Altare, F. Foamy macrophages and the progression of the human tuberculosis granuloma. Nat. Immunol. 10, 943–948 (2009).
Daniel, J., Maamar, H., Deb, C., Sirakova, T.D. & Kolattukudy, P.E. Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages. PLoS Pathog. 7, e1002093 (2011).
Singh, V. et al. Mycobacterium tuberculosis–driven targeted recalibration of macrophage lipid homeostasis promotes the foamy phenotype. Cell Host Microbe 12, 669–681 (2012).
D'Avila, H. et al. Mycobacterium bovis bacillus Calmette-Guérin induces TLR2-mediated formation of lipid bodies: intracellular domains for eicosanoid synthesis in vivo. J. Immunol. 176, 3087–3097 (2006).
Dávalos, A. et al. miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling. Proc. Natl. Acad. Sci. USA 108, 9232–9237 (2011).
Najafi-Shoushtari, S.H. et al. MicroRNA-33 and the SREBP host genes cooperate to control cholesterol homeostasis. Science 328, 1566–1569 (2010).
Rayner, K.J. et al. MiR-33 contributes to the regulation of cholesterol homeostasis. Science 328, 1570–1573 (2010).
Rottiers, V. et al. MicroRNAs in metabolism and metabolic diseases. Cold Spring Harb. Symp. Quant. Biol. 76, 225–233 (2011).
Goedeke, L. et al. A regulatory role for microRNA 33* in controlling lipid metabolism gene expression. Mol. Cell. Biol. 33, 2339–2352 (2013).
Ouimet, M. et al. Autophagy regulates cholesterol efflux from macrophage foam cells via lysosomal acid lipase. Cell Metab. 13, 655–667 (2011).
Zhao, G.J. et al. NF-kappaB suppresses the expression of ATP-binding cassette transporter A1/G1 by regulating SREBP-2 and miR-33a in mice. Int. J. Cardiol. 171, e93–95 (2014).
Singh, R. et al. Autophagy regulates lipid metabolism. Nature 458, 1131–1135 (2009).
Settembre, C. et al. TFEB links autophagy to lysosomal biogenesis. Science 332, 1429–1433 (2011).
Mizushima, N., Yoshimori, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–326 (2010).
Tresse, E. et al. VCP/p97 is essential for maturation of ubiquitin-containing autophagosomes and this function is impaired by mutations that cause IBMPFD. Autophagy 6, 217–227 (2010).
Martin, C.J. et al. Efferocytosis is an innate antibacterial mechanism. Cell Host Microbe 12, 289–300 (2012).
Murphy, J., Summer, R., Wilson, A.A., Kotton, D.N. & Fine, A. The prolonged life-span of alveolar macrophages. Am. J. Respir. Cell Mol. Biol. 38, 380–385 (2008).
Okamura, K. et al. The regulatory activity of microRNA* species has substantial influence on microRNA and 3′ UTR evolution. Nat. Struct. Mol. Biol. 15, 354–363 (2008).
Voellenkle, C. et al. Deep-sequencing of endothelial cells exposed to hypoxia reveals the complexity of known and novel microRNAs. RNA 18, 472–484 (2012).
Schober, A. et al. MicroRNA-126-5p promotes endothelial proliferation and limits atherosclerosis by suppressing Dlk1. Nat. Med. 20, 368–376 (2014).
Mattos, K.A. et al. Lipid droplet formation in leprosy: Toll-like receptor-regulated organelles involved in eicosanoid formation and Mycobacterium leprae pathogenesis. J. Leukoc. Biol. 87, 371–384 (2010).
Zhao, G.J. et al. NF-κB suppresses the expression of ATP-binding cassette transporter A1/G1 by regulating SREBP-2 and miR-33a in mice. Int. J. Cardiol. 171, e93–e95 (2014).
Brzostek, A., Pawelczyk, J., Rumijowska-Galewicz, A., Dziadek, B. & Dziadek, J. Mycobacterium tuberculosis is able to accumulate and utilize cholesterol. J. Bacteriol. 191, 6584–6591 (2009).
Marrero, J., Rhee, K.Y., Schnappinger, D., Pethe, K. & Ehrt, S. Gluconeogenic carbon flow of tricarboxylic acid cycle intermediates is critical for Mycobacterium tuberculosis to establish and maintain infection. Proc. Natl. Acad. Sci. USA 107, 9819–9824 (2010).
Muñoz-Elías, E.J. & McKinney, J.D. Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat. Med. 11, 638–644 (2005).
Pandey, A.K. & Sassetti, C.M. Mycobacterial persistence requires the utilization of host cholesterol. Proc. Natl. Acad. Sci. USA 105, 4376–4380 (2008).
Rodríguez, J.G. et al. Global adaptation to a lipid environment triggers the dormancy-related phenotype of Mycobacterium tuberculosis. MBio 5, e01125–14 (2014).
van der Wel, N. et al. M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells. Cell 129, 1287–1298 (2007).
Simeone, R. et al. Phagosomal rupture by Mycobacterium tuberculosis results in toxicity and host cell death. PLoS Pathog. 8, e1002507 (2012).
Wong, K.W. & Jacobs, W.R. Jr. Critical role for NLRP3 in necrotic death triggered by Mycobacterium tuberculosis. Cell. Microbiol. 13, 1371–1384 (2011).
Pandey, A.K. et al. NOD2, RIP2 and IRF5 play a critical role in the type I interferon response to Mycobacterium tuberculosis. PLoS Pathog. 5, e1000500 (2009).
Galic, S. et al. Hematopoietic AMPK β1 reduces mouse adipose tissue macrophage inflammation and insulin resistance in obesity. J. Clin. Invest. 121, 4903–4915 (2011).
Horie, T. et al. MicroRNA-33 encoded by an intron of sterol regulatory element-binding protein 2 (Srebp2) regulates HDL in vivo. Proc. Natl. Acad. Sci. USA 107, 17321–17326 (2010).
Marchiando, A.M. et al. A deficiency in the autophagy gene Atg16L1 enhances resistance to enteric bacterial infection. Cell Host Microbe 14, 216–224 (2013).
Schoggins, J.W. et al. Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity. Nature 505, 691–695 (2014).
Moore, K.J., Rayner, K.J., Suárez, Y. & Fernández-Hernando, C. microRNAs and cholesterol metabolism. Trends Endocrinol. Metab. 21, 699–706 (2010).
Wolf, A.J. et al. Mycobacterium tuberculosis infects dendritic cells with high frequency and impairs their function in vivo. J. Immunol. 179, 2509–2519 (2007).
Listenberger, L.L. B.D. Fluorescent detection of lipid droplets and associated proteins. Curr. Protoc. Cell Biol. 24, 24.2 (2007).
Mehra, A. et al. Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking. PLoS Pathog. 9, e1003734 (2013).
Acknowledgements
We thank H. Virgin (Washington University School of Medicine) and D. MacDuff (Washington University School of Medicine) for Cgas−/− mice; K. Caldwell (New York University) for Atg16l1flox/flox mice; B. Norris for help with flow cytometry; C. O'Shaughnessy for help with mouse harvests; New York University Langone Medical Center Immune Monitoring Core for use of the XFe24 Extracellular Flux Analyzer (supported by the NYU-HHC CTSI grant UL1 TR000038 and the NYU Cancer Institute's Cancer Center Support grant P30CA016087). Supported by the US National Institutes of Health (R01 HL108182 and HL119047 to K.J.M.; R01 AI087682 and R21 AI105298 to J.A.P.), the American Heart Association (13POST14490016 to B.R., 14POST20180018 to C.v.S.), the NYU Physician-Scientist Training Program (C.P.-C.), the Potts Memorial Foundation (S.K.), Edward J. Mallinckrodt, Jr. Foundation (J.A.P.), Science Foundation Ireland (13/SIRG/2136 to F.J.S.), and the Canadian Institutes of Health Research (postdoctoral fellowship to M.O.; MOP130365 and MSH130157 to K.J.R.).
Author information
Authors and Affiliations
Contributions
M.O., S.K., J.A.P. and K.J.M. designed and analyzed the experiments. T.D.R., S.O., D.K. and K.J.R. assisted with gene expression analyses. B.R. performed immunostaining. C.v.S. performed immunoprecipitation assays. S.K., E.S., C.P.-C. and F.J.S. performed M. tuberculosis studies. K.C. and P.D.Z. provided Mir33−/− bone marrow cells. Y.L.M., K.J.M. and J.A.P. supervised the experiments. M.O., J.A.P. and K.J.M. wrote the manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Induction of the miR-33 locus following Mtb infection in human THP-1 macrophages.
qPCR quantification of hsa-miR-33a, hsa-miR-33a*, and SREBF2 from RNA collected from human THP-1 macrophages infected with Mtb for 24 hours.
Supplementary Figure 2 miR-33 and miR-33* targets in the autophagy gene network.
Putative miR-33 and miR-33* targets are indicated by stars in the diagram depicting autophagy-mediated cholesterol efflux.
Supplementary Figure 3 miR-33 and miR-33* repress protein expression of autophagy gene targets.
Relative protein quantification levels for (a) THP-1 cells treated with miR-33a as compared to control (ctrl) mimic, (b) THP-1 cells treated with miR-33a* as compared to ctrl mimic, (c) mouse macrophages treated with miR-33 as compared to ctrl mimic, and (d) mouse macrophages treated with miR-33* as compared to ctrl mimic. Band intensities for each protein were quantified and normalized to their respective loading control (HSP90 or GAPDH) and data is presented as fold-change relative to the control mimic. Data are the mean ± s.e.m. of 2-3 independent experiments. #P≤0.1, *P≤0.05, **P≤0.005 (Student’s t-test).
Supplementary Figure 4 miR-33 and miR-33* control the expression of multiple genes in the autophagy pathway
(a, b) mRNA expression profiling of a panel of mouse autophagy genes in peritoneal macrophages treated with miR-33 mimic or anti-miR33 (a) or miR-33* mimic or anti-mR33* (b). Additional genes analysed that were not included on the mouse autophagy PCR array are shown below the red dotted line. Data are expressed as fold change relative to control mimic or anti-miR and are representative of 2 independent experiments. Red stars indicate genes containing predicted miR-33 or miR-33* binding sites.
Supplementary Figure 5 AMPK-dependant activation of FOXO3 and TFEB following inhibition of miR-33 and miR-33*.
(a, b) Immunofluorescence (IF) imaging of FOXO3a and TFEB in green, F-actin (red) and Dapi (blue) in peritoneal macrophages treated with (a) anti-miR-33 or (b) anti-miR-33* and ctrl anti-miR in the presence or absence of compound C (Ampki, 5μM). Scale bar = 50μm. Arrows indicate nuclear localization of transcription factors.
Supplementary Figure 6 Inhibition of miR-33 and miR-33* promotes bacterial killing in vitro, partially through xenophagy.
Quantification of bacterial viability in WT vs cGAS-/- macrophages treated with control anti-miR, anti-miR-33, or anti-miR-33* and infected with an Mtb H37Rv strain co-expressing mCherry and anhydrotetracycline-inducible GFP. Data are from one experiment (mean ± s.e.m) representative of 2 independent experiments. #P≤0.1, *P≤0.05 (One-way ANOVA).
Supplementary Figure 7 Proposed model.
M. tuberculosis induces the miR-33 locus to enhance its survival by repressing autophagy and fatty acid oxidation, resulting in a nutrient-rich lipid niche for bacteria that escape xenophagy.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–7 and Supplementary Table 1 (PDF 1733 kb)
Rights and permissions
About this article
Cite this article
Ouimet, M., Koster, S., Sakowski, E. et al. Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism. Nat Immunol 17, 677–686 (2016). https://doi.org/10.1038/ni.3434
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ni.3434
