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

Abstract

The bacterial pathogen Francisella tularensis possesses a non-canonical 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. Proteomic and Phosphoproteomic analyses Reagents and chemicals. For protein digestion, dithiothreitol, iodoacetamide and ammonium bicarbonate were purchased from Sigma-Aldrich (St Louis, MO, USA). For phosphopeptide enrichment and LC-MS/MS analysis, trifluoroacetic acid (TFA), formic acid, acetonitrile and HPLC-grade water were purchased from Fisher Scientific (Pittsburgh, PA, USA) at the highest purity grade. Protein digestion. For proteomic analysis, F. novicida was analyzed in three independent biological replicates. Protein concentration was determined by DC assay (Biorad, CA, USA) according to the manufacturer's instructions. An estimated 1.2 mg of proteins for each

The pentose phosphate pathway constitutes a major metabolic hub in pathogenic Francisella

“…Constructions of GFP-expressing strains. Plasmid pKK-pGro-GFP [70] was introduced by chemical transformation into wild-type F. novicida (designated WT-GFP) and in the ΔtktA, ΔrpiA, Δrpe and ΔtalA mutants, to generate strains that constitutively expressing GFP.…”

Importance of protein Ser/Thr/Tyr phosphorylation for bacterial pathogenesis

“…Finally, the phosphoproteome of a subspecies of Francisella tularensis (the causative agent of 'rabbit fever') was profiled in response to KCl, which induces T6SS formation required for immune evasion. Together with functional experiments, this study was able to establish Tyr phosphorylation of a T6SS sheath component to be essential for T6SS biogenesis [158]. Quantitative phosphoproteomics can also shed light on mechanisms of antibacterial drug resistance.…”

Dissociation between the critical role of ClpB of Francisella tularensis for the heat shock response and the DnaK interaction and its important role for efficient type VI secretion and bacterial virulence

“…It was proposed that these parts are buried in the extended protein, whereas, once the sheath contracts, the N-terminal ClpV-binding domain is exposed, thereby facilitating sheath disassembly [47]. Although no direct evidence of a ClpB-IglB interaction is available and we failed to purify soluble IglA-B to provide such evidence, co-localization of IglA and ClpB has been observed by live-cell microscopy [6,48], similar to that observed for ClpV and VipB [13,14]. Thus, if there is an interaction, then this must be distinct from that of ClpV-VipB, since there are no regions in Francisella ClpB and IglB with similarity to the interacting regions of ClpV and VipB…”