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Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa

Abstract

Secreted proteins are crucial to the arsenal of bacterial pathogens. Although optimal activity of these proteins is likely to require precise regulation of release, the signalling events that trigger secretion are poorly understood. Here, we identify a threonine phosphorylation event that post-translationally regulates the Hcp secretion island-I-encoded type VI secretion system of Pseudomonas aeruginosa (H-T6SS). We show that a serine–threonine kinase, PpkA, is required for assembly of the H-T6SS and for secretion of Hcp1. PpkA activity is antagonized by PppA, a Ser–Thr phosphatase. These proteins exhibit reciprocal effects on the H-T6SS by acting on an FHA domain-containing protein, termed Fha1. Colocalization experiments with the T6S AAA+ family protein, ClpV1, indicate that Fha1 is a core scaffolding protein of the H-T6SS. Mutations affecting this H-T6S regulatory pathway provide a molecular explanation for the variation in Hcp1 secretion among clinical P. aeruginosa isolates. This mechanism of triggering secretion may be general, as many T6SSs contain orthologues of these proteins. Post-translational regulation of protein secretion by Thr phosphorylation is unprecedented in bacteria, and is likely to reflect the requirement for T6S to respond rapidly and reversibly to its environment.

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Figure 1: PpkA and pppA reciprocally regulate Hcp1 secretion and H-T6SS assembly.
Figure 2: Phosphorylation of Fha1 at Thr 362 by PpkA is required for Hcp1 secretion.
Figure 3: Fha1 and ClpV1 colocalize to H-T6S foci and Fha1 is required for ClpV1 recruitment.
Figure 4: Levels of PpkA and PppA activity underlie variations in H-T6SS activation states found in clinical isolates.

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References

  1. Economou, A. et al. Secretion by numbers: protein traffic in prokaryotes. Mol. Microbiol. 62, 308–319 (2006).

    Article  CAS  Google Scholar 

  2. Yahr, T. L. A critical new pathway for toxin secretion? N. Engl. J. Med. 355, 1171–1172 (2006).

    Article  CAS  Google Scholar 

  3. Bladergroen, M. R., Badelt, K. & Spaink, H. P. Infection-blocking genes of a symbiotic Rhizobium leguminosarum strain that are involved in temperature-dependent protein secretion. Mol. Plant Microbe Interact. 16, 53–64 (2003).

    Article  CAS  Google Scholar 

  4. Pukatzki, S. et al. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc. Natl Acad. Sci. USA 103, 1528–1533 (2006).

    Article  CAS  Google Scholar 

  5. Dudley, E. G., Thomson, N. R., Parkhill, J., Morin, N. P. & Nataro, J. P. Proteomic and microarray characterization of the AggR regulon identifies a pheU pathogenicity island in enteroaggregative Escherichia coli. Mol. Microbiol. 61, 1267–1282 (2006).

    Article  CAS  Google Scholar 

  6. Mougous, J. D. et al. A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 312, 1526–1530 (2006).

    Article  CAS  Google Scholar 

  7. Parsons, D. A. & Heffron, F. sciS, an icmF homolog in Salmonella enterica serovar Typhimurium, limits intracellular replication and decreases virulence. Infect. Immun. 73, 4338–4345 (2005).

    Article  CAS  Google Scholar 

  8. Rao, P. S., Yamada, Y., Tan, Y. P. & Leung, K. Y. Use of proteomics to identify novel virulence determinants that are required for Edwardsiella tarda pathogenesis. Mol. Microbiol. 53, 573–586 (2004).

    Article  CAS  Google Scholar 

  9. Ventre, I. et al. Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes. Proc. Natl Acad. Sci. USA 103, 171–176 (2006).

    Article  CAS  Google Scholar 

  10. Goodman, A. L. et al. A signaling network reciprocally regulates genes associated with acute infection and chronic persistence in Pseudomonas aeruginosa. Dev. Cell 7, 745–754 (2004).

    Article  CAS  Google Scholar 

  11. Laskowski, M. A., Osborn, E. & Kazmierczak, B. I. A novel sensor kinase-response regulator hybrid regulates type III secretion and is required for virulence in Pseudomonas aeruginosa. Mol. Microbiol. 54, 1090–1103 (2004).

    Article  CAS  Google Scholar 

  12. Yahr, T. L. & Greenberg, E. P. The genetic basis for the commitment to chronic versus acute infection in Pseudomonas aeruginosa. Mol. Cell 16, 497–498 (2004).

    Article  Google Scholar 

  13. Potvin, E. et al. In vivo functional genomics of Pseudomonas aeruginosa for high-throughput screening of new virulence factors and antibacterial targets. Environ. Microbiol. 5, 1294–1308 (2003).

    Article  CAS  Google Scholar 

  14. Motley, S. T. & Lory, S. Functional characterization of a serine/threonine protein kinase of Pseudomonas aeruginosa. Infect. Immun. 67, 5386–5394 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Schlieker, C., Zentgraf, H., Dersch, P. & Mogk, A. ClpV, a unique Hsp100/Clp member of pathogenic proteobacteria. Biol. Chem. 386, 1115–1127 (2005).

    Article  CAS  Google Scholar 

  16. Pallen, M., Chaudhuri, R. & Khan, A. Bacterial FHA domains: neglected players in the phospho-threonine signalling game? Trends Microbiol. 10, 556–563 (2002).

    Article  CAS  Google Scholar 

  17. Hammet, A. et al. FHA domains as phospho-threonine binding modules in cell signaling. IUBMB Life 55, 23–27 (2003).

    Article  CAS  Google Scholar 

  18. Grundner, C., Gay, L. M. & Alber, T. Mycobacterium tuberculosis serine/threonine kinases PknB, PknD, PknE, and PknF phosphorylate multiple FHA domains. Protein Sci. 14, 1918–1921 (2005).

    Article  CAS  Google Scholar 

  19. Alderwick, L. J. et al. Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA 103, 2558–2563 (2006).

    Article  CAS  Google Scholar 

  20. Villarino, A. et al. Proteomic identification of M. tuberculosis protein kinase substrates: PknB recruits GarA, a FHA domain-containing protein, through activation loop-mediated interactions. J. Mol. Biol. 350, 953–963 (2005).

    Article  CAS  Google Scholar 

  21. Molle, V. et al. An FHA phosphoprotein recognition domain mediates protein EmbR phosphorylation by PknH, a Ser/Thr protein kinase from Mycobacterium tuberculosis. Biochemistry 42, 15300–15309 (2003).

    Article  CAS  Google Scholar 

  22. Molle, V. et al. Two FHA domains on an ABC transporter, Rv1747, mediate its phosphorylation by PknF, a Ser/Thr protein kinase from Mycobacterium tuberculosis. FEMS Microbiol. Lett. 234, 215–223 (2004).

    Article  CAS  Google Scholar 

  23. Durocher, D. et al. The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms. Mol. Cell 6, 1169–1182 (2000).

    Article  CAS  Google Scholar 

  24. Li, J. et al. Structural and functional versatility of the FHA domain in DNA-damage signaling by the tumor suppressor kinase Chk2. Mol. Cell 9, 1045–1054 (2002).

    Article  CAS  Google Scholar 

  25. Smith, E. E. et al. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc. Natl Acad. Sci. USA 103, 8487–8492 (2006).

    Article  CAS  Google Scholar 

  26. Ponting, C. P., Aravind, L., Schultz, J., Bork, P. & Koonin, E. V. Eukaryotic signalling domain homologues in archaea and bacteria. J. Mol. Biol. 289, 729–745 (1999).

    Article  CAS  Google Scholar 

  27. Whittaker, C. A. & Hynes, R. O. Distribution and evolution of von Willebrand/integrin A domains: widely dispersed domains with roles in cell adhesion and elsewhere. Mol. Biol. Cell 13, 3369–3387 (2002).

    Article  CAS  Google Scholar 

  28. Salunkhe, P. et al. A cystic fibrosis epidemic strain of Pseudomonas aeruginosa displays enhanced virulence and antimicrobial resistance. J. Bacteriol. 187, 4908–4920 (2005).

    Article  CAS  Google Scholar 

  29. Rietsch, A., Vallet-Gely, I., Dove, S. L. & Mekalanos, J. J. ExsE, a secreted regulator of type III secretion genes in Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 102, 8006–8011 (2005).

    Article  CAS  Google Scholar 

  30. Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnol. 22, 1567–1572 (2004).

    Article  CAS  Google Scholar 

  31. Shen, A. & Higgins, D. E. The MogR transcriptional repressor regulates nonhierarchal expression of flagellar motility genes and virulence in Listeria monocytogenes. PLoS Pathog. 2, e30 (2006).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors wish to thank: C. Petzold, B. Smart and R. Tomaino for assistance with mass spectrometry; E. Cameron, A. Rietsch, S. Dove, A. Goodman, J. Thompson, S. Lory, M. Schelle, B. Carlson, A. Lauring and E. Ballister, researchers at the Molecular Foundry, and members of the Lory, Bertozzi and Mekalanos laboratories for helpful discussions and comments; A. Shen for providing MogR; and C. Winstanley for sharing P. aeruginosa clinical isolates. This study was supported by grants to J.J.M. from the National Institutes of Health (NIH; AI26289) and a supplement to Argonne National Lab U54 GM074942 for functional studies based on high resolution structures obtained in the Protein Structure Initiative and the U.S. Department of Energy, OBER under Contract W-31-109-ENG-38. J.D.M. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-1873-05).

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Correspondence to John J. Mekalanos.

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Supplementary Figures S1, S2, S3, S4, S5 and Supplementary Table T1 (PDF 292 kb)

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Mougous, J., Gifford, C., Ramsdell, T. et al. Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa. Nat Cell Biol 9, 797–803 (2007). https://doi.org/10.1038/ncb1605

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