Francisella tularensis is a facultative intracellular pathogen that causes the zoonoticdisease tularemia in human and animal hosts. This bacterium possesses a non-canonical type VI secretion systems (T6SS) required for phagosomal escape and access to its replicative niche in the cytosol of infected macrophages. KCl stimulation has been previously used to trigger assembly and secretion of the Francisella T6SS in culture. We found that the amounts of essentially all the TSS6 proteins remained unchanged upon KCl stimulation. We therefore hypothesized that a post-translational modification might be involved in T6SS assembly. A whole cell phosphoproteomic analysis allowed us to identify a unique phosphorylation site on IglB, the TssC homologue and key component of the T6SS sheath. Importantly, the phosphorylated form of IglB was not present in the contracted sheath and 3D modeling indicated that the charge repulsion provoked by addition of a phosphogroup on tyrosine 139 was likely to weaken the stability of the sheath structure. Substitutions of the phosphorylatable residue of IglB (tyrosine 139) with alanine or with phosphomimetics prevented T6SS formation and totally impaired phagosomal escape. In contrast, the substitution with the non-phosphorylatable aromatic analog phenylalanine impaired but did not prevent phagosomal escape and cytosolic bacterial multiplication in J774-1 macrophages. Altogether these data suggest that phosphorylation of the sheath participates to T6SS disassembly. Post-translational modifications of the sheath may represent a previously unrecognized mechanism to finely modulate the dynamics of T6SS assembly disassembly.

A post-translational modification of the sheath 1 modulates Francisella type VI secretion system assembly and function