A trans-kingdom T6SS effector induces the fragmentation of the mitochondrial network and activates innate immune receptor NLRX1 to promote infection
-
Sá-Pessoa, Joana
- López-Montesino, Sara
- Przybyszewska, Kornelia
-
Rodríguez-Escudero, Isabel
1
- Marshall, Helina
-
Ova, Adelia
- Schroeder, Gunnar N.
- Barabas, Peter
-
Molina, María
- Curtis, Tim
-
Cid, Víctor J.
-
Bengoechea, José A.
- 1 Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias
- 2 Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast, UK
ISSN: 2041-1723
Année de publication: 2023
Volumen: 14
Número: 1
Type: Article
D'autres publications dans: Nature Communications
Résumé
Bacteria can inhibit the growth of other bacteria by injecting effectors using a type VI secretion system (T6SS). T6SS effectors can also be injected into eukaryotic cells to facilitate bacterial survival, often by targeting the cytoskeleton. Here, we show that the trans-kingdom antimicrobial T6SS effector VgrG4 from Klebsiella pneumoniae triggers the fragmentation of the mitochondrial network. VgrG4 colocalizes with the endoplasmic reticulum (ER) protein mitofusin 2. VgrG4 induces the transfer of Ca2+ from the ER to the mitochondria, activating Drp1 (a regulator of mitochondrial fission) thus leading to mitochondrial network fragmentation. Ca2+ elevation also induces the activation of the innate immunity receptor NLRX1 to produce reactive oxygen species (ROS). NLRX1-induced ROS limits NF-κB activation by modulating the degradation of the NF-κB inhibitor IκBα. The degradation of IκBα is triggered by the ubiquitin ligase SCFβ-TrCP, which requires the modification of the cullin-1 subunit by NEDD8. VgrG4 abrogates the NEDDylation of cullin-1 by inactivation of Ubc12, the NEDD8-conjugating enzyme. Our work provides an example of T6SS manipulation of eukaryotic cells via alteration of the mitochondria.
Information sur le financement
We thank the members of the J.A.B. laboratory for their thoughtful discussions and support with this project. We thank members of the V.J.C/M.M lab, especially E. del Val and J.M. Coronas-Serna for sharing MMM1 and MDM34 constructs. Research at the V.J.C and M.M. lab was funded by Grant PID2019-105342GB-I00 from Ministerio de Ciencia e Innovación (Spain) and by Grant S2017/BMD-3691-InGEMICS-CM, funded by Comunidad de Madrid and European Structural and Investment Funds. S.L-M. was recipient of predoctoral contracts by Universidad Complutense de Madrid (UCM) (Spain) and PEJD-2019-PRE/BMD-16928 by Comunidad de Madrid. This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC, BB/T001976/1, BB/P020194/1, BB/N00700X/1, BB/V007939/1) to J.A.B.Financeurs
- European Structural
- Comunidad de Madrid Spain
-
Biotechnology and Biological Sciences Research Council
United Kingdom
- BB/V007939/1
-
Universidad Complutense de Madrid
Spain
- PEJD-2019-PRE/BMD-16928
-
Ministerio de Ciencia e Innovación
Spain
- S2017/BMD-3691-InGEMICS-CM
Références bibliographiques
- Roger, A. J., Munoz-Gomez, S. A. & Kamikawa, R. The Origin and Diversification of Mitochondria. Curr. Biol. 27, R1177–R1192 (2017).
- Friedman, J. R. & Nunnari, J. Mitochondrial form and function. Nature 505, 335–343 (2014).
- Youle, R. J. & van der Bliek, A. M. Mitochondrial fission, fusion, and stress. Science 337, 1062–1065 (2012).
- Spinelli, J. B. & Haigis, M. C. The multifaceted contributions of mitochondria to cellular metabolism. Nat. Cell Biol. 20, 745–754 (2018).
- Giorgi, C., Marchi, S. & Pinton, P. The machineries, regulation and cellular functions of mitochondrial calcium. Nat. Rev. Mol. Cell Biol. 19, 713–730 (2018).
- West, A. P., Shadel, G. S. & Ghosh, S. Mitochondria in innate immune responses. Nat. Rev. Immunol. 11, 389–402 (2011).
- Marchi, S., Morroni, G., Pinton, P. & Galluzzi, L. Control of host mitochondria by bacterial pathogens. Trends Microbiol. https://doi.org/10.1016/j.tim.2021.09.010 (2021).
- Hernandez, R. E., Gallegos-Monterrosa, R. & Coulthurst, S. J. Type VI secretion system effector proteins: Effective weapons for bacterial competitiveness. Cell Microbiol 22, e13241 (2020).
- Monjaras Feria, J. & Valvano, M. A. An Overview of Anti-Eukaryotic T6SS Effectors. Front Cell Infect. Microbiol 10, 584751 (2020).
- Holt, K. E. et al. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proc. Natl Acad. Sci. USA 112, E3574–E3581 (2015).
- Lery, L. M. et al. Comparative analysis of Klebsiella pneumoniae genomes identifies a phospholipase D family protein as a novel virulence factor. BMC Biol. 12, 41-7007 7012-7041, https://doi.org/10.1186/1741-7007-12-41 (2014).
- Storey, D. et al. Klebsiella pneumoniae type VI secretion system-mediated microbial competition is PhoPQ controlled and reactive oxygen species dependent. PLoS Pathog. 16, e1007969 (2020).
- Kroemer, G., Galluzzi, L. & Brenner, C. Mitochondrial membrane permeabilization in cell death. Physiol. Rev. 87, 99–163 (2007).
- Bleazard, W. et al. The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast. Nat. Cell Biol. 1, 298–304 (1999).
- Tieu, Q. & Nunnari, J. Mdv1p is a WD repeat protein that interacts with the dynamin-related GTPase, Dnm1p, to trigger mitochondrial division. J. Cell Biol. 151, 353–366 (2000).
- Mozdy, A., McCaffery, J. & Shaw, J. Dnm1p GTPase-mediated mitochondrial fission is a multi-step process requiring the novel integral membrane component Fis1p. J. Cell Biol. 151, 367–380 (2000).
- Cerveny, K. L., Studer, S. L., Jensen, R. E. & Sesaki, H. Yeast mitochondrial division and distribution require the cortical num1 protein. Dev. Cell 12, 363–375 (2007).
- Naylor, K. et al. Mdv1 interacts with assembled dnm1 to promote mitochondrial division. J. Biol. Chem. 281, 2177–2183 (2006).
- Bhar, D., Karren, M. A., Babst, M. & Shaw, J. M. Dimeric Dnm1-G385D interacts with Mdv1 on mitochondria and can be stimulated to assemble into fission complexes containing Mdv1 and Fis1. J. Biol. Chem. 281, 17312–17320 (2006).
- Achleitner, G. et al. Association between the endoplasmic reticulum and mitochondria of yeast facilitates interorganelle transport of phospholipids through membrane contact. Eur. J. Biochem 264, 545–553 (1999).
- Rizzuto, R. et al. Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science 280, 1763–1766 (1998).
- Westermann, B. Mitochondrial fusion and fission in cell life and death. Nat. Rev. Mol. Cell Biol. 11, 872–884 (2010).
- Lee, H. & Yoon, Y. Mitochondrial fission: regulation and ER connection. Mol. Cells 37, 89–94 (2014).
- Lang, A., John Peter, A. T. & Kornmann, B. ER-mitochondria contact sites in yeast: beyond the myths of ERMES. Curr. Opin. Cell Biol. 35, 7–12 (2015).
- Bengoechea, J. A. & Sa Pessoa, J. Klebsiella pneumoniae infection biology: living to counteract host defences. FEMS Microbiol. Rev. 43, 123–144 (2019).
- Cortes, G., Alvarez, D., Saus, C. & Alberti, S. Role of lung epithelial cells in defense against Klebsiella pneumoniae pneumonia. Infect. Immun. 70, 1075–1080 (2002).
- Garcia, J. T. et al. Measurement of effector protein injection by type III and type IV secretion systems by using a 13-residue phosphorylatable glycogen synthase kinase tag. Infect. Immun. 74, 5645–5657 (2006).
- Kwon, D., Park, E., Sesaki, H. & Kang, S. J. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) suppresses STING-mediated DNA sensing pathway through inducing mitochondrial fission. Biochem Biophys. Res Commun. 493, 737–743 (2017).
- Gong, Y. et al. Newcastle disease virus degrades SIRT3 via PINK1-PRKN-dependent mitophagy to reprogram energy metabolism in infected cells. Autophagy 18, 1503–1521 (2022).
- Ishihara, N., Jofuku, A., Eura, Y. & Mihara, K. Regulation of mitochondrial morphology by membrane potential, and DRP1-dependent division and FZO1-dependent fusion reaction in mammalian cells. Biochem. Biophys. Res. Commun. 301, 891–898 (2003).
- Hemel, I., Engelen, B. P. H., Luber, N. & Gerards, M. A hitchhiker’s guide to mitochondrial quantification. Mitochondrion 59, 216–224 (2021).
- Wolke, S., Ackermann, N. & Heesemann, J. The Yersinia enterocolitica type 3 secretion system (T3SS) as toolbox for studying the cell biological effects of bacterial Rho GTPase modulating T3SS effector proteins. Cell. Microbiol. 13, 1339–1357 (2011).
- Chan, D. C. Mitochondrial fusion and fission in mammals. Annu Rev. Cell Dev. Biol. 22, 79–99 (2006).
- Breitzig, M. T., Alleyn, M. D., Lockey, R. F. & Kolliputi, N. A mitochondrial delicacy: dynamin-related protein 1 and mitochondrial dynamics. Am. J. Physiol. Cell Physiol. 315, C80–C90 (2018).
- Mishra, P. & Chan, D. C. Metabolic regulation of mitochondrial dynamics. J. Cell Biol. 212, 379–387 (2016).
- Cassidy-Stone, A. et al. Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization. Dev. Cell 14, 193–204 (2008).
- Bordt, E. A. et al. The Putative Drp1 Inhibitor mdivi-1 Is a Reversible Mitochondrial Complex I Inhibitor that Modulates Reactive Oxygen Species. Dev. Cell 40, 583–594 e586 (2017).
- Riley, J. S. & Tait, S. W. Mitochondrial DNA in inflammation and immunity. EMBO Rep. 21, e49799 (2020).
- Massa, D., Baran, M., Bengoechea, J. A. & Bowie, A. G. PYHIN1 regulates pro-inflammatory cytokine induction rather than innate immune DNA sensing in airway epithelial cells. J. Biol. Chem. 295, 4438–4450 (2020).
- Haag, S. M. et al. Targeting STING with covalent small-molecule inhibitors. Nature 559, 269–273 (2018).
- Zorova, L. D. et al. Mitochondrial membrane potential. Anal. Biochem. 552, 50–59 (2018).
- Hung, V. et al. Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation. Elife 6, e24463 (2017).
- Rhee, H. W. et al. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science 339, 1328–1331 (2013).
- Rizzuto, R. et al. Ca(2+) transfer from the ER to mitochondria: when, how and why. Biochim. Biophys. Acta 1787, 1342–1351 (2009).
- Naon, D. et al. Critical reappraisal confirms that Mitofusin 2 is an endoplasmic reticulum-mitochondria tether. Proc. Natl Acad. Sci. USA 113, 11249–11254 (2016).
- Szabadkai, G. et al. Mitochondrial dynamics and Ca2+ signaling. Biochim. Biophys. Acta 1763, 442–449 (2006).
- Edelman, J. L., Kajimura, M., Woldemussie, E. & Sachs, G. Differential effects of carbachol on calcium entry and release in CHO cells expressing the m3 muscarinic receptor. Cell Calcium 16, 181–193 (1994).
- Balaban, R. S., Nemoto, S. & Finkel, T. Mitochondria, oxidants, and aging. Cell 120, 483–495 (2005).
- Arnoult, D. et al. An N-terminal addressing sequence targets NLRX1 to the mitochondrial matrix. J. Cell Sci. 122, 3161–3168 (2009).
- Collier-Hyams, L. S., Sloane, V., Batten, B. C. & Neish, A. S. Cutting edge: bacterial modulation of epithelial signaling via changes in neddylation of cullin-1. J. Immunol. 175, 4194–4198 (2005).
- Moranta, D. et al. Klebsiella pneumoniae capsule polysaccharide impedes the expression of beta-defensins by airway epithelial cells. Infect. Immun. 78, 1135–1146 (2010).
- Regueiro, V. et al. Klebsiella pneumoniae subverts the activation of inflammatory responses in a NOD1-dependent manner. Cell. Microbiol. 13, 135–153 (2011).
- Frank, C. G. et al. Klebsiella pneumoniae targets an EGF receptor-dependent pathway to subvert inflammation. Cell. Microbiol. 15, 1212–1233 (2013).
- Hayden, M. S. & Ghosh, S. Shared principles in NF-kappaB signaling. Cell 132, 344–362 (2008).
- Liu, Y. C. Ubiquitin ligases and the immune response. Annu Rev. Immunol. 22, 81–127 (2004).
- Read, M. A. et al. Nedd8 modification of cul-1 activates SCF(beta(TrCP))-dependent ubiquitination of IkappaBalpha. Mol. Cell Biol. 20, 2326–2333 (2000).
- Barford, D. The role of cysteine residues as redox-sensitive regulatory switches. Curr. Opin. Struct. Biol. 14, 679–686 (2004).
- Pan, Z. Q., Kentsis, A., Dias, D. C., Yamoah, K. & Wu, K. Nedd8 on cullin: building an expressway to protein destruction. Oncogene 23, 1985–1997 (2004).
- Kumar, A. et al. Commensal bacteria modulate cullin-dependent signaling via generation of reactive oxygen species. EMBO J. 26, 4457–4466 (2007).
- Navon-Venezia, S., Kondratyeva, K. & Carattoli, A. Klebsiella pneumoniae: a major worldwide source and shuttle for antibiotic resistance. FEMS Microbiol. Rev. 41, 252–275 (2017).
- Spinola-Amilibia, M. et al. The structure of VgrG1 from Pseudomonas aeruginosa, the needle tip of the bacterial type VI secretion system. Acta Crystallogr. Sect. D., Struct. Biol. 72, 22–33 (2016).
- Chang, C. R. & Blackstone, C. Dynamic regulation of mitochondrial fission through modification of the dynamin-related protein Drp1. Ann. N. Y Acad. Sci. 1201, 34–39 (2010).
- Jain, P., Luo, Z. Q. & Blanke, S. R. Helicobacter pylori vacuolating cytotoxin A (VacA) engages the mitochondrial fission machinery to induce host cell death. Proc. Natl Acad. Sci. USA 108, 16032–16037 (2011).
- Escoll, P. et al. Legionella pneumophila Modulates Mitochondrial Dynamics to Trigger Metabolic Repurposing of Infected Macrophages. Cell Host Microbe 22, 302–316 e307 (2017).
- Stavru, F., Bouillaud, F., Sartori, A., Ricquier, D. & Cossart, P. Listeria monocytogenes transiently alters mitochondrial dynamics during infection. Proc. Natl Acad. Sci. USA 108, 3612–3617 (2011).
- Stavru, F., Palmer, A. E., Wang, C., Youle, R. J. & Cossart, P. Atypical mitochondrial fission upon bacterial infection. Proc. Natl Acad. Sci. USA 110, 16003–16008 (2013).
- Ivin, M. et al. Natural killer cell-intrinsic type I IFN signaling controls Klebsiella pneumoniae growth during lung infection. PLoS Pathog. 13, e1006696 (2017).
- Soares, F. et al. NLRX1 does not inhibit MAVS-dependent antiviral signalling. Innate Immun. 19, 438–448 (2013).
- Tattoli, I. et al. NLRX1 is a mitochondrial NOD-like receptor that amplifies NF-kappaB and JNK pathways by inducing reactive oxygen species production. EMBO Rep. 9, 293–300 (2008).
- Allen, I. C. et al. NLRX1 protein attenuates inflammatory responses to infection by interfering with the RIG-I-MAVS and TRAF6-NF-kappaB signaling pathways. Immunity 34, 854–865 (2011).
- Moore, C. B. et al. NLRX1 is a regulator of mitochondrial antiviral immunity. Nature 451, 573–577 (2008).
- Abdul-Sater, A. A. et al. Enhancement of reactive oxygen species production and chlamydial infection by the mitochondrial Nod-like family member NLRX1. J. Biol. Chem. 285, 41637–41645 (2010).
- Ribet, D. & Cossart, P. Ubiquitin, SUMO, and NEDD8: Key Targets of Bacterial Pathogens. Trends Cell Biol. 28, 926–940 (2018).
- Sa-Pessoa, J. et al. Klebsiella pneumoniae Reduces SUMOylation To Limit Host Defense Responses. mBio 11, e01733–20 (2020).
- Murphy, M. P. & Hartley, R. C. Mitochondria as a therapeutic target for common pathologies. Nat. Rev. Drug Disco. 17, 865–886 (2018).
- Fernandez-Acero, T. et al. Expression of Human PTEN-L in a Yeast Heterologous Model Unveils Specific N-Terminal Motifs Controlling PTEN-L Subcellular Localization and Function. Cells 8, https://doi.org/10.3390/cells8121512 (2019).
- Rodriguez-Escudero, I. et al. Reconstitution of the mammalian PI3K/PTEN/Akt pathway in yeast. Biochem J. 390, 613–623 (2005).
- Karbowski, M. et al. Spatial and temporal association of Bax with mitochondrial fission sites, Drp1, and Mfn2 during apoptosis. J. Cell Biol. 159, 931–938 (2002).
- Wieckowski, M. R., Giorgi, C., Lebiedzinska, M., Duszynski, J. & Pinton, P. Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. Nat. Protoc. 4, 1582–1590 (2009).
- Schroeder, G. N. et al. Legionella pneumophila Effector LpdA Is a Palmitoylated Phospholipase D Virulence Factor. Infect. Immun. 83, 3989–4002 (2015).