Critical Role of a Sheath Phosphorylation Site On the Assembly and Function of an Atypical Type VI Secretion System

• Published in: Molecular & cellular proteomics Vol. 18; no. 12; pp. 2418 - 2432
• Main Authors: Ziveri, Jason, Chhuon, Cerina, Jamet, Anne, Rytter, Héloïse, Prigent, Guénolé, Tros, Fabiola, Barel, Monique, Coureuil, Mathieu, Lays, Claire, Henry, Thomas, Keep, Nicholas H., Guerrera, Ida Chiara, Charbit, Alain
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2019; American Society for Biochemistry and Molecular Biology; The American Society for Biochemistry and Molecular Biology
• Subjects: Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Cell Line; Electronic Data Processing; Francisella; Francisella tularensis - genetics; Francisella tularensis - metabolism; Francisella tularensis - ultrastructure; Gas Chromatography-Mass Spectrometry; Humans; IglB; Life Sciences; Macrophages - microbiology; Microbiology; Microbiology and Parasitology; Molecular Structure; Mutagenesis, Site-Directed; Pathogens; Phosphoproteome; Phosphorylation; Potassium Chloride - pharmacology; Protein Processing, Post-Translational; Proteomics; Tandem Mass Spectrometry; Type 6 secretion system; Type VI Secretion Systems - chemistry; Type VI Secretion Systems - drug effects; Type VI Secretion Systems - genetics; Type VI Secretion Systems - metabolism; Pathogens; Phosphorylation; Microbiology; Francisella; IglB; Bacteria; Phosphoproteome; Type 6 secretion system
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.RA119.001532

KCl stimulation triggers assembly of the Francisella T6SS in culture. Differential whole cell proteomics reveals that the amounts of the T6SS proteins remain unchanged upon KCl stimulation. A phosphoproteomic analysis identifies a phosphorylation on the T6SS sheath, at site Y139 of IglB. Our data demonstrate that site Y139 of IglB plays a critical role in T6SS biogenesis, providing novel mechanistic insight into the role of sheath phosphorylation in T6SS biogenesis. [Display omitted] 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. 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