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Salmonella stimulates pro-inflammatory signalling through p21-activated kinases bypassing innate immune receptors

Nature Microbiology volume 3, pages 1122–1130 (2018)Cite this article

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Abstract

Microbial infections are most often countered by inflammatory responses that are initiated through the recognition of conserved microbial products by innate immune receptors and result in pathogen expulsion1,2,3,4,5,6. However, inflammation can also lead to pathology. Tissues such as the intestinal epithelium, which are exposed to microbial products, are therefore subject to stringent negative regulatory mechanisms to prevent signalling through innate immune receptors6,7,8,9,10,11. This presents a challenge to the enteric pathogen Salmonella Typhimurium, which requires intestinal inflammation to compete against the resident microbiota and to acquire the nutrients and electron acceptors that sustain its replication12,13. We show here that S. Typhimurium stimulates pro-inflammatory signalling by a unique mechanism initiated by effector proteins that are delivered by its type III protein secretion system. These effectors activate Cdc42 and the p21-activated kinase 1 (PAK1) leading to the recruitment of TNF receptor-associated factor 6 (TRAF6) and mitogen-activated protein kinase kinase kinase 7 (TAK1), and the stimulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inflammatory signalling. The removal of Cdc42, PAK1, TRAF6 or TAK1 prevented S. Typhimurium from stimulating NF-κB signalling in cultured cells. In addition, oral administration of a highly specific PAK inhibitor blocked Salmonella-induced intestinal inflammation and bacterial replication in the mouse intestine, although it resulted in a significant increase in the bacterial loads in systemic tissues. Thus, S. Typhimurium stimulates inflammatory signalling in the intestinal tract by engaging critical downstream signalling components of innate immune receptors. These findings illustrate the unique balance that emerges from host–pathogen co-evolution, in that pathogen-initiated responses that help pathogen replication are also important to prevent pathogen spread to deeper tissues.

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Fig. 1: S. Typhimurium stimulates pro-inflammatory signalling through Cdc42 and its effector kinase PAK1.
Fig. 2: PAK1–TRAF6–TAK1 mediates S. Typhimurium pro-inflammatory signalling downstream of Cdc42.
Fig. 3: S. Typhimurium stimulates TAK1-dependent PAK1 phosphorylation, which is essential for pro-inflammatory signalling.
Fig. 4: PAK-mediated pro-inflammatory signalling is required for S. Typhimurium replication within the intestine and for the host response that limits systemic infection.

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All data generated or analysed during this study are included in this published article (and the Supplementary Information).

References

  1. Nish, S. & Medzhitov, R. Host defense pathways: role of redundancy and compensation in infectious disease phenotypes. Immunity 34, 629–636 (2011).

    Article  CAS  Google Scholar 

  2. Medzhitov, R. Recognition of microorganisms and activation of the immune response. Nature 449, 819–826 (2007).

    Article  CAS  Google Scholar 

  3. Medzhitov, R. Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1, 135–145 (2001).

  4. Inohara, N. & Nunez, G. Cell death and immunity: NODs: intracellular proteins involved in inflammation and apoptosis. Nat. Rev. Immunol. 3, 371–382 (2003).

  5. Barton, G. & Medzhitov, R. Toll-like receptor signaling pathways. Science 300, 1524–1525 (2003).

    Article  CAS  Google Scholar 

  6. Creagh, E. & O’Neill, L. TLRs, NLRs and RLRs: a trinity of pathogen sensors that co-operate in innate immunity. Trends Immunol. 27, 352–357 (2006).

    Article  CAS  Google Scholar 

  7. Lee, J., Mo, J., Shen, C., Rucker, A. & Raz, E. Toll-like receptor signaling in intestinal epithelial cells contributes to colonic homoeostasis. Curr. Opin. Gastroenterol. 23, 27–31 (2007).

    Article  CAS  Google Scholar 

  8. Kelly, D., Conway, S. & Aminov, R. Commensal gut bacteria: mechanisms of immune modulation. Trends Immunol. 26, 326–333 (2005).

    Article  CAS  Google Scholar 

  9. Eckmann, L. Sensor molecules in intestinal innate immunity against bacterial infections. Curr. Opin. Gastroenterol. 22, 95–101 (2006).

    Article  CAS  Google Scholar 

  10. Shibolet, O. & Podolsky, D. TLRs in the Gut. IV. Negative regulation of Toll-like receptors and intestinal homeostasis: addition by subtraction. Am. J. Physiol. Gastrointest. Liver Physiol. 292, G1469–G1473 (2007).

    Article  CAS  Google Scholar 

  11. Lang, T. & Mansell, A. The negative regulation of Toll-like receptor and associated pathways. Immunol. Cell Biol. 85, 425–434 (2007).

    Article  CAS  Google Scholar 

  12. Stecher, B. et al. Salmonella enterica serovar Typhimurium exploits inflammation to compete with the intestinal microbiota. PLoS Biol. 5, 2177–2189 (2007).

    Article  CAS  Google Scholar 

  13. Winter, S. et al. Gut inflammation provides a respiratory electron acceptor for Salmonella. Nature 467, 426–429 (2010).

    Article  CAS  Google Scholar 

  14. Chen, L. M., Hobbie, S. & Galan, J. E. Requirement of CDC42 for Salmonella-induced cytoskeletal and nuclear responses. Science 274, 2115–2118 (1996).

    Article  CAS  Google Scholar 

  15. Hobbie, S., Chen, L. M., Davis, R. & Galán, J. E. Involvement of the mitogen-activated protein kinase pathways in the nuclear responses and cytokine production induced by Salmonella typhimurium in cultured intestinal cells. J. Immunol. 159, 5550–5559 (1997).

    CAS  PubMed  Google Scholar 

  16. Bruno, V. M. et al. Salmonella Typhimurium type III secretion effectors stimulate innate immune responses in cultured epithelial cells. PLoS Pathog. 5, e1000538 (2009).

    Article  Google Scholar 

  17. Hardt, W.-D., Chen, L.-M., Schuebel, K. E., Bustelo, X. R. & Galán, J. E. Salmonella typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells. Cell 93, 815–826 (1998).

    Article  CAS  Google Scholar 

  18. Patel, J. C. & Galan, J. E. Differential activation and function of Rho GTPases during Salmonella-host cell interactions. J. Cell Biol. 175, 453-463 (2006).

  19. Zhou, D., Chen, L. M., Hernandez, L., Shears, S. B. & Galan, J. E. A Salmonella inositol polyphosphatase acts in conjunction with other bacterial effectors to promote host cell actin cytoskeleton rearrangements and bacterial internalization. Mol. Microbiol. 39, 248–259 (2001).

    Article  CAS  Google Scholar 

  20. Chen, L. M., Bagrodia, S., Cerione, R. A. & Galan, J. E. Requirement of p21-activated kinase (PAK) for Salmonella typhimurium-induced nuclear responses. J. Exp. Med. 189, 1479–1488 (1999).

    Article  CAS  Google Scholar 

  21. Keestra, A. et al. Manipulation of small Rho GTPases is a pathogen-induced process detected by NOD1. Nature 496, 233–237 (2013).

    Article  CAS  Google Scholar 

  22. Kobayashi, K. et al. RICK/Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune systems. Nature 416, 194–199 (2002).

    Article  CAS  Google Scholar 

  23. Chin, A. et al. Involvement of receptor-interacting protein 2 in innate and adaptive immune responses. Nature 416, 190–194 (2002).

    Article  CAS  Google Scholar 

  24. Friebel, A. et al. SopE and SopE2 from Salmonella typhimurium activate different sets of RhoGTPases of the host cell. J. Biol. Chem. 276, 34035–34040 (2001).

    Article  CAS  Google Scholar 

  25. Lara-Tejero, M. et al. Role of the caspase-1 inflammasome in Salmonella typhimurium pathogenesis. J. Exp. Med. 203, 1407-1412 (2006).

  26. Hannemann, S., Gao, B. & Galán, J. Salmonella modulates host cell gene expression to promote its intracellular growth. PLoS Pathog. 9, e1003668 (2013).

    Article  Google Scholar 

  27. Dammann, K., Khare, V. & Gasche, C. Tracing PAKs from GI inflammation to cancer. Gut 63, 1173–1184 (2014).

    Article  CAS  Google Scholar 

  28. Kumar, R., Sanawar, R., Li, X. & Li, F. Structure, biochemistry, and biology of PAK kinases. Gene 605, 20–31 (2017).

    Article  CAS  Google Scholar 

  29. Radu, M., Semenova, G., Kosoff, R. & J, C. PAK signalling during the development and progression of cancer. Nat. Rev. Cancer 14, 13–25 (2014).

    Article  CAS  Google Scholar 

  30. Dammann, K. et al. PAK1 modulates a PPARγ/NF-κB cascade in intestinal inflammation. Biochim. Biophys. Acta 1853, 2349–2360 (2015).

    Article  CAS  Google Scholar 

  31. Lyons, J. et al. Integrated in vivo multiomics analysis identifies p21-activated kinase signaling as a driver of colitis. Sci. Signal. 11, eaan3580 (2018).

    Article  Google Scholar 

  32. Norris, F. A., Wilson, M. P., Wallis, T. S., Galyov, E. E. & Majerus, P. W. SopB, a protein required for virulence of Salmonella dublin, is an inositol phosphate phosphatase. Proc. Natl Acad. Sci. USA 95, 14057–14059 (1998).

    Article  CAS  Google Scholar 

  33. Knodler, L., Finlay, B. & O., S.-M. The Salmonella effector protein SopB protects epithelial cells from apoptosis by sustained activation of Akt. J. Biol. Chem. 280, 9058–9064 (2005).

    Article  CAS  Google Scholar 

  34. Steele-Mortimer, O. et al. Activation of Akt/protein kinase B in epithelial cells by the Salmonella typhimurium effector sigD. J. Biol. Chem. 275, 37718–37724 (2000).

    Article  CAS  Google Scholar 

  35. Hernandez, L. D., Hueffer, K., Wenk, M. R. & Galan, J. E. Salmonella modulates vesicular traffic by altering phosphoinositide metabolism. Science 304, 1805–1807 (2004).

    Article  CAS  Google Scholar 

  36. Prudnikova, T., Villamar-Cruz, O., Rawat, S., Cai, K. & Chernoff, J. Effects of p21-activated kinase 1 inhibition on 11q13-amplified ovarian cancer cells. Oncogene 35, 2178–2185 (2016).

    Article  CAS  Google Scholar 

  37. Joseph, G. et al. Group I Paks promote skeletal myoblast differentiation in vivo and in vitro. Mol. Cell. Biol. 37, e00222-16 (2017).

    Article  Google Scholar 

  38. Elsherif, L. et al. Potential compensation among group I PAK members in hindlimb ischemia and wound healing. PLoS ONE 9, e112239 (2014).

    Article  Google Scholar 

  39. Rennefahrt, U. et al. Specificity profiling of Pak kinases allows identification of novel phosphorylation sites. J. Biol. Chem. 282, 15667–15678 (2007).

    Article  CAS  Google Scholar 

  40. Dolan, B. et al. Rescue of fragile X syndrome phenotypes in Fmr1 KO mice by the small-molecule PAK inhibitor FRAX486. Proc. Natl Acad. Sci. USA 110, 5671–5676 (2013).

    Article  CAS  Google Scholar 

  41. Frost, J. et al. Stimulation of NFkappa B activity by multiple signaling pathways requires PAK1. J. Biol. Chem. 275, 19693–19699 (2000).

    Article  CAS  Google Scholar 

  42. Walsh, M., Lee, J., Choi, Y. & Ibrahim, M. Tumor necrosis factor receptor- associated factor 6 (TRAF6) regulation of development, function, and homeostasis of the immune system. Immunol. Rev. 266, 72–92 (2015).

    Article  CAS  Google Scholar 

  43. Ajibade, A., Wang, H. & Wang, R. Cell type-specific function of TAK1 in innate immune signaling. Trends Immunol. 34, 307–316 (2013).

    Article  CAS  Google Scholar 

  44. Shin, Y., Kim, Y. & Kim, J. Protein kinase CK2 phosphorylates and activates p21-activated kinase 1. Mol. Biol. Cell. 24, 2990–2999 (2013).

    Article  CAS  Google Scholar 

  45. McDaniel, A. et al. Pak1 regulates multiple c-Kit mediated Ras-MAPK gain-in-function phenotypes in Nf1 +/– mast cells. Blood 112, 4646–4654 (2008).

    Article  CAS  Google Scholar 

  46. 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).

    Article  CAS  Google Scholar 

  47. Sun, H., Kamanova, J., Lara-Tejero, M. & Galán, J. A family of Salmonella type III secretion effector proteins selectively targets the NF-κB signaling pathway to preserve host homeostasis. PLoS Pathog. 12, e1005484 (2016).

    Article  Google Scholar 

  48. Gibson, D. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  Google Scholar 

  49. Obert, S., O’Connor, R. J., Schmid, S. & Hearing, P. The adenovirus E4-6/7 protein transactivates the E2 promoter by inducing dimerization of a heteromeric E2F complex. Molec. Cell. Biol. 14, 1333–1346 (1994).

    Article  CAS  Google Scholar 

  50. Hoiseth, S. K. & Stocker, B. A. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291, 238–239 (1981).

    Article  CAS  Google Scholar 

  51. Galan, J. E., Ginocchio, C. & Costeas, P. Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family. J. Bacteriol. 174, 4338–4349 (1992).

    Article  CAS  Google Scholar 

  52. Galán, J. E. & Curtiss, R. III Expression of Salmonella typhimurium genes required for invasion is regulated by changes in DNA supercoiling. Infect. Immun. 58, 1879–1885 (1990).

    PubMed  PubMed Central  Google Scholar 

  53. Intestinal Epithelial Organoid Culture with IntestiCult™ Organoid Growth Medium (Mouse) Doc. #28223 (STEMCELL Technologies, 2016).

  54. Chang, S., Song, J. & Galán, J. Receptor-mediated sorting of typhoid toxin during its export from Salmonella Typhi-infected cells. Cell Host Microbe 20, 682–689 (2016).

    Article  CAS  Google Scholar 

  55. Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281-2308 (2013).ð

  56. Galán, J. E. & Curtiss, R. III Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells. Proc. Natl Acad. Sci. USA 86, 6383–6387 (1989).

    Article  Google Scholar 

  57. Kamanova, J., Sun, H., Lara-Tejero, M. & Galan, J. E. The Salmonella effector protein SopA modulates innate immune responses by targeting TRIM E3 ligase family members. PLoS Pathog. 12, e1005552 (2016).

    Article  Google Scholar 

  58. Liu, X., Gao, B., Novik, V. & Galan, J. E. Quantitative proteomics of intracellular Campylobacter jejuni reveals metabolic reprogramming. PLoS Pathog. 8, e1002562 (2012).

    Article  CAS  Google Scholar 

  59. Perkins, D. N., Pappin, D. J., Creasy, D. M. & Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551-3567 (1999).

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Acknowledgements

We thank J. Chernoff for providing the PAK1–/– mice and members of the Galán laboratory for careful reading of the manuscript. This work was supported by the National Institute of Allergy and Infectious Diseases grant AI055472 (to J.E.G.).

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  1. Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, USA

    Hui Sun, Jana Kamanova, Maria Lara-Tejero & Jorge E. Galán

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Contributions

H.S. and J.E.G. designed the research and analysed data. H.S., J.K. and M.L.-T. performed the research. H.S. and J.E.G. wrote the manuscript with input from all the authors.

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Correspondence to Jorge E. Galán.

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Supplementary information

Supplementary Information

Supplementary Figures 1–20, Supplementary Tables 5–7.

Reporting Summary

Supplementary Table 1

LC–MS/MS analyses of CDC42-interacting proteins in uninfected cells.

Supplementary Table 2

LC–MS/MS analyses of CDC42-interacting proteins in S. Typhimurium infected cells.

Supplementary Table 3

LC-MS/MS analyses of PAK1-interacting proteins in uninfected cells.

Supplementary Table 4

LC–MS/MS analyses of PAK1-interacting proteins in S. Typhimurium-infected cells.

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Sun, H., Kamanova, J., Lara-Tejero, M. et al. Salmonella stimulates pro-inflammatory signalling through p21-activated kinases bypassing innate immune receptors. Nat Microbiol 3, 1122–1130 (2018). https://doi.org/10.1038/s41564-018-0246-z

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