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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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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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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DOI: https://doi.org/10.1038/ncb1605
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