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

Jason Ziveri,Cerina Chhuon,Anne Jamet,Héloïse Rytter,Guénolé Prigent,Fabiola Tros,Monique Barel,Mathieu Coureuil,Claire Lays,Thomas Henry,Nicholas H Keep,Ida Chiara Guerrera,Alain Charbit

doi:10.1074/mcp.ra119.001532

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.

Highlights

Critical Role of a Sheath Phosphorylation Site On the Assembly and Function of an Atypical Type VI Secretion System*□S
The bacterial pathogen Francisella tularensis possesses a noncanonical type VI secretion system (T6SS) that is required for phagosomal escape in infected macrophages
Functional Assays in Primary Cultures—we evaluated the ability of Y139A and Y139F mutants to multiply in primary bone marrow-derived macrophages (BMMs) over a 24 h-period

Summary

Graphical Abstract

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. The bacterial pathogen Francisella tularensis possesses a noncanonical type VI secretion system (T6SS) that is required for phagosomal escape in infected macrophages. 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 identified a unique phosphorylation site on IglB, a key component of the T6SS sheath. 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. In bacteria, polymerization mechanisms are more generally driven by gene expression regulation than by PTMs. T6SS sheath polymerization had never been shown to involve any PTM, we hypothesized that protein phosphorylation might occur in Francisella and contribute to the dynamics of T6SS assembly-disassembly.

EXPERIMENTAL PROCEDURES

The abbreviations used are

RESULTS

DISCUSSION