Baseplate Component TssK and Spatio-Temporal Assembly of T6SS in Pseudomonas aeruginosa.

David Liebl,Viviana Job,Mylène Robert-Genthon,Valentina Cogoni,Ina Attrée

doi:10.3389/fmicb.2019.01615

Abstract

The Gram-negative bacteria use the contractile multi-molecular structure, called the Type VI Secretion System (T6SS) to inject toxic products into eukaryotic and prokaryotic cells. In this study, we use fluorescent protein fusions and time-lapse microscopy imaging to study the assembly dynamics of the baseplate protein TssK in Pseudomonas aeruginosa T6SS. TssK formed transient higher-order structures that correlated with dynamics of sheath component TssB. Assembly of peri-membrane TssK structures occurred de novo upon contact with competing bacteria. We show that this assembly required presence of TagQ-TagR envelope sensors, activity of PpkA kinase and anchoring to the inner membrane via TssM. Disassembly and repositioning of TssK component was dependent on PppA phosphatase and indispensable for repositioning and deployment of the entire contractile apparatus toward a new target cell. We also show that TssE is necessary for correct elongation and stability of TssB-sheath, but not for TssK assembly. Therefore, in P. aeruginosa, assembly of the TssK-containing structure relays on the post-translational regulatory envelope module and acts as spatio-temporal marker for further recruitment and subsequent assembly of the contractile apparatus.

Highlights

Gram-negative bacteria deploy a number of complex envelope-embedded multi-protein nanomachines adjusted exclusively for protein export across bacterial membrane and interaction with eukaryotic and prokaryotic species (Filloux et al, 2008; Hayes et al, 2010)
Our results show dynamic assembly of the T6SS baseplate structure that depends on P. aeruginosa post-translational signaling cascade, as well as the key functions the baseplate provides for assembly and relocation of the T6SS nanomachinery to a site of contact with competing bacteria
In a subpopulation of bacteria, TssKsfGFP assembled into discernible foci localized to the bacterial periphery, reminiscent to TssK foci observed in Escherichia coli (Brunet et al, 2015; Cherrak et al, 2018), whereas TssB-GFP assembled into elongated rod-like structures with substantially higher fluorescence intensity relative to TssK foci (Figure 1A)

Summary

Introduction

Gram-negative bacteria deploy a number of complex envelope-embedded multi-protein nanomachines adjusted exclusively for protein export across bacterial membrane and interaction with eukaryotic and prokaryotic species (Filloux et al, 2008; Hayes et al, 2010). One of these is Type VI secretion system (T6SS), originally identified in Vibrio cholerae using Dictyostelium host (Pukatzki et al, 2006). Upon activation of the system, certain components such as Hcp and VgrG, could be exchanged between neighboring bacteria to launch rapid response to external stimuli (Vettiger and Basler, 2016)

Objectives

Methods

Results

Conclusion