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Graphical Abstract
Highlights
Phosphorylation on Y139 of the sheath protein IglB of Francisella.
IglB substitutions Y139A, Y139D or Y139E prevent T6SS formation.
Y139F substitution delays but does not abolish phagosomal escape in macrophages.
Insight into the role of sheath phosphorylation in T6SS biogenesis.
Abstract
The bacterial pathogen Francisella tularensis possesses a noncanonical type VI secretion system (T6SS) that is required for phagosomal escape in infected macrophages. KCl stimulation has been previously used to trigger assembly and secretion of the T6SS in culture. By differential proteomics, we found here that the amounts of the T6SS proteins remained unchanged upon KCl stimulation, suggesting involvement of post-translational modifications in T6SS assembly. A phosphoproteomic analysis indeed identified a unique phosphorylation site on IglB, a key component of the T6SS sheath. Substitutions of Y139 with alanine or phosphomimetics prevented T6SS formation and abolished phagosomal escape whereas substitution with phenylalanine delayed but did not abolish phagosomal escape in J774–1 macrophages. Altogether our data demonstrated that the Y139 site of IglB plays a critical role in T6SS biogenesis, suggesting that sheath phosphorylation could participate to T6SS dynamics.
Data are available via ProteomeXchange with identifier PXD013619; and on MS-Viewer, key lkaqkllxwx
Footnotes
Author contributions: J.Z., M.B., M.C., T.H., I.C.G., and A.C. designed research; J.Z., C.C., H.R., G.P., F.T., and C.L. performed research; J.Z., A.J., M.C., C.L., N.H.K., I.C.G., and A.C. analyzed data; C.C. and T.H. contributed new reagents/analytic tools; N.H.K., I.C.G., and A.C. wrote the paper.
↵* These studies were supported by INSERM, CNRS and Université Paris Descartes Paris Cité Sorbonne. Jason Ziveri was funded by a fellowship from the “Délégation Générale à l'Armement”. Claire Lays was funded by a fellowship from the LABEX ECOFECT (ANR-11-LABX-0048) of Université de Lyon, within the program “Investissement d'Avenir” (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). No author has an actual or perceived conflict of interest with the contents of this article.
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This article contains supplemental material.
- Received April 29, 2019.
- Revision received September 3, 2019.
- © 2019 Ziveri et al.
Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
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