Salmonella presents a global public health concern. Central to Salmonella pathogenicity is an ability to subvert host defences through strategically targeting host proteins implicated in restricting infection. Therefore, to gain insight into the host–pathogen interactions governing Salmonella infection, we performed an in vivo genome-wide mutagenesis screen to uncover key host defence proteins. This revealed an uncharacterized role of CYRI (FAM49B) in conferring host resistance to Salmonella infection. We show that CYRI binds to the small GTPase RAC1 through a conserved domain present in CYFIP proteins, which are known RAC1 effectors that stimulate actin polymerization. However, unlike CYFIP proteins, CYRI negatively regulates RAC1 signalling, thereby attenuating processes such as macropinocytosis, phagocytosis and cell migration. This enables CYRI to counteract Salmonella at various stages of infection, including bacterial entry into non-phagocytic and phagocytic cells as well as phagocyte-mediated bacterial dissemination. Intriguingly, to dampen its effects, the bacterial effector SopE, a RAC1 activator, selectively targets CYRI following infection. Together, this outlines an intricate host–pathogen signalling interplay that is crucial for determining bacterial fate. Notably, our study also outlines a role for CYRI in restricting infection mediated by Mycobacterium tuberculosis and Listeria monocytogenes. This provides evidence implicating CYRI cellular functions in host defence beyond Salmonella infection.
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The data that support the findings of this study are available from the corresponding authors on reasonable request.
All commercial and custom codes used to generate data presented in this study are available from the corresponding authors on reasonable request.
Kirk, M. D. et al. World Health Organization estimates of the global and regional disease burden of 22 foodborne bacterial, protozoal, and viral diseases, 2010: a data synthesis. PLoS Med. 12, e1001921 (2015).
Majowicz, S. E. et al. The global burden of nontyphoidal Salmonella gastroenteritis. Clin. Infect. Dis. 50, 882–889 (2010).
Prestinaci, F., Pezzotti, P. & Pantosti, A. Antimicrobial resistance: a global multifaceted phenomenon. Pathog. Glob. Health 109, 309–318 (2015).
LaRock, D. L., Chaudhary, A. & Miller, S. I. Salmonellae interactions with host processes. Nat. Rev. Microbiol. 13, 191–205 (2015).
Schlumberger, M. C. & Hardt, W. D. Salmonella type III secretion effectors: pulling the host cell’s strings. Curr. Opin. Microbiol. 9, 46–54 (2006).
Byndloss, M. X., Rivera-Chavez, F., Tsolis, R. M. & Baumler, A. J. How bacterial pathogens use type III and type IV secretion systems to facilitate their transmission. Curr. Opin. Microbiol. 35, 1–7 (2017).
Guiney, D. G. & Lesnick, M. Targeting of the actin cytoskeleton during infection by Salmonella strains. Clin. Immunol. 114, 248–255 (2005).
Hardt, W. D., Chen, L. M., Schuebel, K. E., Bustelo, X. R. & Galan, J. E. S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells. Cell 93, 815–826 (1998).
Orchard, R. C. & Alto, N. M. Mimicking GEFs: a common theme for bacterial pathogens. Cell. Microbiol. 14, 10–18 (2012).
Humphreys, D., Davidson, A., Hume, P. J. & Koronakis, V. Salmonella virulence effector SopE and host GEF ARNO cooperate to recruit and activate WAVE to trigger bacterial invasion. Cell Host Microbe 11, 129–139 (2012).
Gautreau, A. et al. Purification and architecture of the ubiquitous Wave complex. Proc. Natl Acad. Sci. USA 101, 4379–4383 (2004).
Eden, S., Rohatgi, R., Podtelejnikov, A. V., Mann, M. & Kirschner, M. W. Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature 418, 790–793 (2002).
Ismail, A. M., Padrick, S. B., Chen, B., Umetani, J. & Rosen, M. K. The WAVE regulatory complex is inhibited. Nat. Struct. Mol. Biol. 16, 561–563 (2009).
Chen, Z. et al. Structure and control of the actin regulatory WAVE complex. Nature 468, 533–538 (2010).
Chen, B. et al. Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites. eLife 6, e29795 (2017).
Francis, C. L., Ryan, T. A., Jones, B. D., Smith, S. J. & Falkow, S. Ruffles induced by Salmonella and other stimuli direct macropinocytosis of bacteria. Nature 364, 639–642 (1993).
Steele-Mortimer, O. The Salmonella-containing vacuole: moving with the times. Curr. Opin. Microbiol. 11, 38–45 (2008).
Ham, H., Sreelatha, A. & Orth, K. Manipulation of host membranes by bacterial effectors. Nat. Rev. Microbiol. 9, 635–646 (2011).
Schleker, S. et al. The current Salmonella-host interactome. Proteom. Clin. Appl. 6, 117–133 (2012).
Mostowy, S. & Shenoy, A. R. The cytoskeleton in cell-autonomous immunity: structural determinants of host defence. Nat. Rev. Immunol. 15, 559–573 (2015).
Behnsen, J., Perez-Lopez, A., Nuccio, S. P. & Raffatellu, M. Exploiting host immunity: the Salmonella paradigm. Trends Immunol. 36, 112–120 (2015).
Worley, M. J., Nieman, G. S., Geddes, K. & Heffron, F. Salmonella typhimurium disseminates within its host by manipulating the motility of infected cells. Proc. Natl Acad. Sci. USA 103, 17915–17920 (2006).
Ohl, M. E. & Miller, S. I. Salmonella: a model for bacterial pathogenesis. Annu. Rev. Med. 52, 259–274 (2001).
Fabrega, A. & Vila, J. Salmonella enterica serovar Typhimurium skills to succeed in the host: virulence and regulation. Clin. Microbiol. Rev. 26, 308–341 (2013).
Vazquez-Torres, A. et al. Extraintestinal dissemination of Salmonella by CD18-expressing phagocytes. Nature 401, 804–808 (1999).
Hendriksen, R. S. et al. Global monitoring of Salmonella serovar distribution from the world health organization global foodborne infections network country data bank: results of quality assured laboratories from 2001 to 2007. Foodborne Pathog. Dis. 8, 887–900 (2011).
Soding, J., Biegert, A. & Lupas, A. N. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, W244–W248 (2005).
Michiels, F., Habets, G. G., Stam, J. C., van der Kammen, R. A. & Collard, J. G. A role for Rac in Tiam1-induced membrane ruffling and invasion. Nature 375, 338–340 (1995).
Krauthammer, M. et al. Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma. Nat. Genet. 44, 1006–1014 (2012).
Pollard, T. D. & Cooper, J. A. Actin, a central player in cell shape and movement. Science 326, 1208–1212 (2009).
Spector, I., Shochet, N. R., Kashman, Y. & Groweiss, A. Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells. Science 219, 493–495 (1983).
May, R. C. & Machesky, L. M. Phagocytosis and the actin cytoskeleton. J. Cell Sci. 114, 1061–1077 (2001).
Krause, M. & Gautreau, A. Steering cell migration: lamellipodium dynamics and the regulation of directional persistence. Nat. Rev. Mol. Cell Biol. 15, 577–590 (2014).
Wijburg, O. L., Simmons, C. P., van Rooijen, N. & Strugnell, R. A. Dual role for macrophages in vivo in pathogenesis and control of murine Salmonella enterica var. Typhimurium infections. Eur. J. Immunol. 30, 944–953 (2000).
Richter-Dahlfors, A., Buchan, A. M. & Finlay, B. B. Murine salmonellosis studied by confocal microscopy: Salmonella typhimurium resides intracellularly inside macrophages and exerts a cytotoxic effect on phagocytes in vivo. J. Exp. Med. 186, 569–580 (1997).
Lin, D. C. & Lin, S. Actin polymerization induced by a motility-related high-affinity cytochalasin binding complex from human erythrocyte membrane. Proc. Natl Acad. Sci. USA 76, 2345–2349 (1979).
Campa, C. C. et al. Rac signal adaptation controls neutrophil mobilization from the bone marrow. Sci. Signal. 9, ra124 (2016).
Pankov, R. et al. A Rac switch regulates random versus directionally persistent cell migration. J. Cell Biol. 170, 793–802 (2005).
Schlumberger, M. C. et al. Amino acids of the bacterial toxin SopE involved in G nucleotide exchange on Cdc42. J. Biol. Chem. 278, 27149–27159 (2003).
Fiskin, E., Bionda, T., Dikic, I. & Behrends, C. Global analysis of host and bacterial ubiquitinome in response to Salmonella Typhimurium infection. Mol. Cell 62, 967–981 (2016).
de Chastellier, C. & Berche, P. Fate of Listeria monocytogenes in murine macrophages: evidence for simultaneous killing and survival of intracellular bacteria. Infect. Immun. 62, 543–553 (1994).
Cossart, P. & Lebreton, A. A trip in the “New Microbiology” with the bacterial pathogen Listeria monocytogenes. FEBS Lett. 588, 2437–2445 (2014).
Rajaram, M. V. S. et al. M. tuberculosis-initiated human mannose receptor signaling regulates macrophage recognition and vesicle trafficking by FcRγ-Chain, Grb2, and SHP-1. Cell Rep. 21, 126–140 (2017).
Ireton, K., Rigano, L. A. & Dowd, G. C. Role of host GTPases in infection by Listeria monocytogenes. Cell Microbiol. 16, 1311–1320 (2014).
Shang, W. et al. Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation. Proc. Natl Acad. Sci. USA 115, E4051–E4060 (2018).
Fort, L. et al. Fam49/CYRI interacts with Rac1 and locally suppresses protrusions. Nat. Cell Biol. 20, 1159–1171 (2018).
Queval, C. J., Brosch, R. & Simeone, R. The macrophage: a disputed fortress in the battle against Mycobacterium tuberculosis. Front. Microbiol. 8, 2284 (2017).
Haney, M. S. et al. Identification of phagocytosis regulators using magnetic genome-wide CRISPR screens. Nat. Genet. 50, 1716–1727 (2018).
Drevets, D. A. Dissemination of Listeria monocytogenes by infected phagocytes. Infect. Immun. 67, 3512–3517 (1999).
Maculins, T., Fiskin, E., Bhogaraju, S. & Dikic, I. Bacteria-host relationship: ubiquitin ligases as weapons of invasion. Cell Res. 26, 499–510 (2016).
Roy, M. F. et al. Pyruvate kinase deficiency confers susceptibility to Salmonella typhimurium infection in mice. J. Exp. Med. 204, 2949–2961 (2007).
Barthel, M. et al. Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host. Infect. Immun. 71, 2839–2858 (2003).
Eva, M. M. et al. Altered IFN-γ-mediated immunity and transcriptional expression patterns in N-ethyl-N-nitrosourea-induced STAT4 mutants confer susceptibility to acute typhoid-like disease. J. Immunol. 192, 259–270 (2014).
Marei, H. et al. Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration. Nat. Commun. 7, 10664 (2016).
Benard, V., Bohl, B. P. & Bokoch, G. M. Characterization of Rac and Cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J. Biol. Chem. 274, 13198–13204 (1999).
Marei, H., Carpy, A., Macek, B. & Malliri, A. Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors. Cell Cycle 15, 1961–1974 (2016).
Karlinsey, J. E. λ-Red genetic engineering in Salmonella enterica serovar Typhimurium. Methods Enzym. 421, 199–209 (2007).
Skarnes, W. C. et al. A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474, 337–342 (2011).
Fortin, A. et al. Recombinant congenic strains derived from A/J and C57BL/6J: a tool for genetic dissection of complex traits. Genomics 74, 21–35 (2001).
Cunnington, A. J., de Souza, J. B., Walther, M. & Riley, E. M. Malaria impairs resistance to Salmonella through heme- and heme oxygenase-dependent dysfunctional granulocyte mobilization. Nat. Med. 18, 120–127 (2011).
Sixt, M. & Lammermann, T. In vitro analysis of chemotactic leukocyte migration in 3D environments. Methods Mol. Biol. 769, 149–165 (2011).
We thank L. Larivière, E. Flamant, L. Rached-D’Astous, J. Kim, F. Pampaloni and M. Sader for technical assistance, N. Prud’homme and P. D’Arcy for mouse breeding and screening, S.-J. Pilon for histopathology scoring, and K. Koch and T. Maculins for reviewing the manuscript. We also acknowledge A. Malliri for reagents and G. White for the preparation of the PAK-CRIB peptide. This work was supported by the Canadian Institutes of Health Research (grant no. MOP133700 to D.M.), the DFG-funded Collaborative Research Centre on Selective Autophagy (grant no. SFB 1177), the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 742720), the DFG-funded Cluster of Excellence ‘Macromolecular Complexes’ (grant no. EXC115), the DFG-funded SPP 1580 program ‘Intracellular Compartments as Places of Pathogen-Host-Interactions’, and by the LOEWE program Ubiquitin Networks (Ub-Net) and the LOEWE Centre for Gene and Cell Therapy Frankfurt, which are both funded by the State of Hessen, Germany (to I.D.). Funding was provided to M.M.E. by Le Fonds de Recherche Santé du Québec and to H.M. by the Alexander von Humboldt foundation as a Humboldt research fellowship for postdoctoral researchers.
The authors declare no competing interests.
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Supplementary Figs. 1–13, Supplementary Tables 1–5, Supplementary Notes and Supplementary References.
Ubiquitylation sites quantified from three replicate SILAC-diGly proteomics experiments in HeLa Flp-In T-REx GFP–SopE cells, as described in Fig. 7.
Statistics for main and supplementary figures.
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Yuki, K.E., Marei, H., Fiskin, E. et al. CYRI/FAM49B negatively regulates RAC1-driven cytoskeletal remodelling and protects against bacterial infection. Nat Microbiol 4, 1516–1531 (2019). https://doi.org/10.1038/s41564-019-0484-8
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