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Nε-fatty acylation of multiple membrane-associated proteins by Shigella IcsB effector to modulate host function

Nature Microbiologyvolume 3pages9961009 (2018) | Download Citation


Shigella flexneri, an intracellular Gram-negative bacterium causative for shigellosis, employs a type III secretion system to deliver virulence effectors into host cells. One such effector, IcsB, is critical for S. flexneri intracellular survival and pathogenesis, but its mechanism of action is unknown. Here, we discover that IcsB is an 18-carbon fatty acyltransferase catalysing lysine Nε-fatty acylation. IcsB disrupted the actin cytoskeleton in eukaryotes, resulting from Nε-fatty acylation of RhoGTPases on lysine residues in their polybasic region. Chemical proteomic profiling identified about 60 additional targets modified by IcsB during infection, which were validated by biochemical assays. Most IcsB targets are membrane-associated proteins bearing a lysine-rich polybasic region, including members of the Ras, Rho and Rab families of small GTPases. IcsB also modifies SNARE proteins and other non-GTPase substrates, suggesting an extensive interplay between S. flexneri and host membrane trafficking. IcsB is localized on the Shigella-containing vacuole to fatty-acylate its targets. Knockout of CHMP5—one of the IcsB targets and a component of the ESCRT-III complex—specifically affected S. flexneri escape from host autophagy. The unique Nε-fatty acyltransferase activity of IcsB and its altering of the fatty acylation landscape of host membrane proteomes represent an unprecedented mechanism in bacterial pathogenesis.

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  1. 1.

    Cui, J. & Shao, F. Biochemistry and cell signaling taught by bacterial effectors. Trends Biochem. Sci. 36, 532–540 (2011).

  2. 2.

    Carayol, N. & Tran Van Nhieu, G. The inside story of Shigella invasion of intestinal epithelial cells. Cold Spring Harb. Perspect. Med. 3, a016717 (2013).

  3. 3.

    Ashida, H., Mimuro, H. & Sasakawa, C. Shigella manipulates host immune responses by delivering effector proteins with specific roles. Front Immunol. 6, 219 (2015).

  4. 4.

    Mellouk, N. & Enninga, J. Cytosolic access of intracellular bacterial pathogens: the Shigella paradigm. Front. Cell. Infect. Microbiol. 6, 35 (2016).

  5. 5.

    Li, H. et al. The phosphothreonine lyase activity of a bacterial type III effector family. Science 315, 1000–1003 (2007).

  6. 6.

    Zhu, Y. et al. Structural insights into the enzymatic mechanism of the pathogenic MAPK phosphothreonine lyase. Mol. Cell 28, 899–913 (2007).

  7. 7.

    Mazurkiewicz, P. et al. SpvC is a Salmonella effector with phosphothreonine lyase activity on host mitogen-activated protein kinases. Mol. Microbiol. 67, 1371–1383 (2008).

  8. 8.

    Goto, Y. et al. Discovery of unique lanthionine synthetases reveals new mechanistic and evolutionary insights. PLoS Biol. 8, e1000339 (2010).

  9. 9.

    Allaoui, A., Mounier, J., Prevost, M. C., Sansonetti, P. J. & Parsot, C. icsB: a Shigella flexneri virulence gene necessary for the lysis of protrusions during intercellular spread. Mol. Microbiol. 6, 1605–1616 (1992).

  10. 10.

    Ogawa, M., Suzuki, T., Tatsuno, I., Abe, H. & Sasakawa, C. IcsB, secreted via the type III secretion system, is chaperoned by IpgA and required at the post-invasion stage of Shigella pathogenicity. Mol. Microbiol. 48, 913–931 (2003).

  11. 11.

    Ogawa, M. et al. Escape of intracellular Shigella from autophagy. Science 307, 727–731 (2005).

  12. 12.

    Kayath, C. A. et al. Escape of intracellular Shigella from autophagy requires binding to cholesterol through the type III effector, IcsB. Microbes Infect. 12, 956–966 (2010).

  13. 13.

    Baxt, L. A. & Goldberg, M. B. Host and bacterial proteins that repress recruitment of LC3 to Shigella early during infection. PLoS ONE 9, e94653 (2014).

  14. 14.

    Mostowy, S. et al. Entrapment of intracytosolic bacteria by septin cage-like structures. Cell Host Microbe 8, 433–444 (2010).

  15. 15.

    Campbell-Valois, F. X., Sachse, M., Sansonetti, P. J. & Parsot, C. Escape of actively secreting Shigella flexneri from ATG8/LC3-positive vacuoles formed during cell-to-cell spread is facilitated by IcsB and VirA. mBio 6, e02567-14 (2015).

  16. 16.

    Pei, J. & Grishin, N. V. The Rho GTPase inactivation domain in Vibrio cholerae MARTX toxin has a circularly permuted papain-like thiol protease fold. Proteins 77, 413–419 (2009).

  17. 17.

    Ahrens, S., Geissler, B. & Satchell, K. J. Identification of a His–Asp–Cys catalytic triad essential for function of the Rho inactivation domain (RID) of Vibrio cholerae MARTX toxin. J. Biol. Chem. 288, 1397–1408 (2013).

  18. 18.

    Shao, F., Merritt, P. M., Bao, Z., Innes, R. W. & Dixon, J. E. A Yersinia effector and a Pseudomonas avirulence protein define a family of cysteine proteases functioning in bacterial pathogenesis. Cell 109, 575–588 (2002).

  19. 19.

    Sheahan, K. L. & Satchell, K. J. Inactivation of small Rho GTPases by the multifunctional RTX toxin from Vibrio cholerae. Cell. Microbiol. 9, 1324–1335 (2007).

  20. 20.

    Lemichez, E. & Aktories, K. Hijacking of Rho GTPases during bacterial infection. Exp. Cell Res. 319, 2329–2336 (2013).

  21. 21.

    Shao, F. et al. Biochemical characterization of the Yersinia YopT protease: cleavage site and recognition elements in Rho GTPases. Proc. Natl Acad. Sci. USA 100, 904–909 (2003).

  22. 22.

    Hoffman, G. R., Nassar, N. & Cerione, R. A. Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI. Cell 100, 345–356 (2000).

  23. 23.

    Mukherjee, S. et al. Yersinia YopJ acetylates and inhibits kinase activation by blocking phosphorylation. Science 312, 1211–1214 (2006).

  24. 24.

    Lupardus, P. J., Shen, A., Bogyo, M. & Garcia, K. C. Small molecule-induced allosteric activation of the Vibrio cholerae RTX cysteine protease domain. Science 322, 265–268 (2008).

  25. 25.

    Prochazkova, K. & Satchell, K. J. Structure–function analysis of inositol hexakisphosphate-induced autoprocessing of the Vibrio cholerae multifunctional autoprocessing RTX toxin. J. Biol. Chem. 283, 23656–23664 (2008).

  26. 26.

    Calder, T. et al. Vibrio type III effector VPA1380 is related to the cysteine protease domain of large bacterial toxins. PLoS ONE 9, e104387 (2014).

  27. 27.

    Mittal, R., Peak-Chew, S. Y., Sade, R. S., Vallis, Y. & McMahon, H. T. The acetyltransferase activity of the bacterial toxin YopJ of Yersinia is activated by eukaryotic host cell inositol hexakisphosphate. J. Biol. Chem. 285, 19927–19934 (2010).

  28. 28.

    Hang, H. C. & Linder, M. E. Exploring protein lipidation with chemical biology. Chem. Rev. 111, 6341–6358 (2011).

  29. 29.

    Grammel, M. & Hang, H. C. Identification of lysine acetyltransferase substrates using bioorthogonal chemical proteomics. Methods Mol. Biol. 981, 201–210 (2013).

  30. 30.

    Charron, G. et al. Robust fluorescent detection of protein fatty-acylation with chemical reporters. J. Am. Chem. Soc. 131, 4967–4975 (2009).

  31. 31.

    Wilson, J. P., Raghavan, A. S., Yang, Y. Y., Charron, G. & Hang, H. C. Proteomic analysis of fatty-acylated proteins in mammalian cells with chemical reporters reveals S-acylation of histone H3 variants. Mol. Cell Proteom. 10, M110.001198 (2011).

  32. 32.

    Dong, N. et al. Structurally distinct bacterial TBC-like GAPs link Arf GTPase to Rab1 inactivation to counteract host defenses. Cell 150, 1029–1041 (2012).

  33. 33.

    Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).

  34. 34.

    Li, P. et al. Ubiquitination and degradation of GBPs by a Shigella effector to suppress host defence. Nature 551, 378–383 (2017).

  35. 35.

    Shim, J. H. et al. CHMP5 is essential for late endosome function and down-regulation of receptor signaling during mouse embryogenesis. J. Cell Biol. 172, 1045–1056 (2006).

  36. 36.

    Ward, D. M. et al. The role of LIP5 and CHMP5 in multivesicular body formation and HIV-1 budding in mammalian cells. J. Biol. Chem. 280, 10548–10555 (2005).

  37. 37.

    Shibutani, S. T., Saitoh, T., Nowag, H., Munz, C. & Yoshimori, T. Autophagy and autophagy-related proteins in the immune system. Nat. Immunol. 16, 1014–1024 (2015).

  38. 38.

    Choy, A. et al. The Legionella effector RavZ inhibits host autophagy through irreversible Atg8 deconjugation. Science 338, 1072–1076 (2012).

  39. 39.

    Resh, M. D. Fatty acylation of proteins: the long and the short of it. Prog. Lipid Res. 63, 120–131 (2016).

  40. 40.

    Hannoush, R. N. Synthetic protein lipidation. Curr. Opin. Chem. Biol. 28, 39–46 (2015).

  41. 41.

    Nadolski, M. J. & Linder, M. E. Protein lipidation. FEBS J. 274, 5202–5210 (2007).

  42. 42.

    Schey, K. L., Gutierrez, D. B., Wang, Z., Wei, J. & Grey, A. C. Novel fatty acid acylation of lens integral membrane protein aquaporin-0. Biochemistry 49, 9858–9865 (2010).

  43. 43.

    Stevenson, F. T., Bursten, S. L., Fanton, C., Locksley, R. M. & Lovett, D. H. The 31-kDa precursor of interleukin 1 alpha is myristoylated on specific lysines within the 16-kDa N-terminal propiece. Proc. Natl Acad. Sci. USA 90, 7245–7249 (1993).

  44. 44.

    Jiang, H. et al. SIRT6 regulates TNF-alpha secretion through hydrolysis of long-chain fatty acyl lysine. Nature 496, 110–113 (2013).

  45. 45.

    Zhang, X., Spiegelman, N. A., Nelson, O. D., Jing, H. & Lin, H. SIRT6 regulates Ras-related protein R-Ras2 by lysine defatty-acylation. eLife 6, e25158 (2017).

  46. 46.

    Zhou, Y. et al. N ε-fatty acylation of Rho GTPases by a MARTX toxin effector. Science 358, 528–531 (2017).

  47. 47.

    Dolores, J. S., Agarwal, S., Egerer, M. & Satchell, K. J. Vibrio cholerae MARTX toxin heterologous translocation of beta-lactamase and roles of individual effector domains on cytoskeleton dynamics. Mol. Microbiol. 95, 590–604 (2015).

  48. 48.

    Fres, J. M., Muller, S. & Praefcke, G. J. Purification of the CaaX-modified, dynamin-related large GTPase hGBP1 by coexpression with farnesyltransferase. J. Lipid Res. 51, 2454–2459 (2010).

  49. 49.

    Dong, N. et al. Modulation of membrane phosphoinositide dynamics by the phosphatidylinositide 4-kinase activity of the Legionella LepB effector. Nat. Microbiol. 2, 16236 (2016).

  50. 50.

    Pellegrin, S. & Mellor, H. Rho GTPase activation assays. Curr. Protoc. Cell Biol. 38, 14.8.1–14.8.19 (2008).

  51. 51.

    Hu, M., Liu, Y., Yu, K. & Liu, X. Decreasing the amount of trypsin in in-gel digestion leads to diminished chemical noise and improved protein identifications. J. Proteom. 109, 16–25 (2014).

  52. 52.

    Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

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We thank R. Isberg for providing Yersinia strains, G. Praefcke for the pRSF-FTase plasmid, and the Proteomics Resource Center at The Rockefeller University for mass spectrometry analysis. We also thank members of the Shao laboratory for technical assistance and stimulating discussions. This work was supported by the Basic Science Center Project of the National Natural Science Foundation of China (81788101), National Key Research and Development Program of China (2017YFA0505900 and 2016YFA0501500) and Strategic Priority Research Program of the Chinese Academy of Sciences (XDB08020202) to F.S. H.C.H. acknowledges support from NIH-NIGMS grant R01 GM087544. The research was also supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute and Beijing Scholar Program to F.S.

Author information

Author notes

    • Yan Zhou

    Present address: Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou, Zhejiang, China

  1. These authors contributed equally: Wang Liu, Yan Zhou, Tao Peng.


  1. College of Life Science, Peking University, Beijing, China

    • Wang Liu
  2. Peking University–Tsinghua University–National Institute of Biological Sciences Joint Graduate Program, National Institute of Biological Sciences, Beijing, China

    • Wang Liu
  3. National Institute of Biological Sciences, Beijing, China

    • Wang Liu
    • , Yan Zhou
    • , Ping Zhou
    • , Xiaojun Ding
    • , Zilin Li
    • , Haoyu Zhong
    • , Yue Xu
    • , She Chen
    •  & Feng Shao
  4. College of Life Sciences, Beijing Normal University, Beijing, China

    • Yan Zhou
  5. School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China

    • Tao Peng
  6. Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY, USA

    • Tao Peng
    •  & Howard C. Hang
  7. Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, China

    • Feng Shao


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F.S. conceived the study. Y.Z. performed initial studies on the identification of RhoGTPases as the substrate of IcsB and its fatty acyltransferase activity. W.L. established the SunTag labelling of T3SS effectors, analysed the proteomic hits of IcsB, and performed the localization and autophagy studies. P.Z. and Z.L. provided technical assistance to Y.Z. and W.L. H.Z. and Y.X. performed the plaque assay. T.P. and H.C.H. were responsible for the chemical proteomic analyses. X.D. and S.C. carried out the mass spectrometry experiments. Y.Z., W.L., T.P., S.C., H.C.H. and F.S. analysed the data. W.L., Y.Z. and F.S. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Howard C. Hang or Feng Shao.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–7.

  2. Reporting Summary

  3. Supplementary Table 1

    SILAC chemical proteomic analyses IcsB-modified proteins in IcsB-transfected cells.

  4. Supplementary Table 2

    SILAC chemical proteomic analyses IcsB-modified proteins in Shigella-infected cells.

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