Research Article: Killing with proficiency: Integrated post-translational regulation of an offensive Type VI secretion system

Date Published: July 27, 2018

Publisher: Public Library of Science

Author(s): Adam Ostrowski, Francesca R. Cianfanelli, Michael Porter, Giuseppina Mariano, Julien Peltier, Jun Jie Wong, Jason R. Swedlow, Matthias Trost, Sarah J. Coulthurst, Melanie Blokesch.

http://doi.org/10.1371/journal.ppat.1007230

Abstract

The Type VI secretion system (T6SS) is widely used by bacterial pathogens as an effective weapon against bacterial competitors and is also deployed against host eukaryotic cells in some cases. It is a contractile nanomachine which delivers toxic effector proteins directly into target cells by dynamic cycles of assembly and firing. Bacterial cells adopt distinct post-translational regulatory strategies for deployment of the T6SS. ‘Defensive’ T6SSs assemble and fire in response to incoming attacks from aggressive neighbouring cells, and can utilise the Threonine Protein Phosphorylation (TPP) regulatory pathway to achieve this control. However, many T6SSs are ‘offensive’, firing at all-comers without the need for incoming attack or other cell contact-dependent signal. Post-translational control of the offensive mode has been less well defined but can utilise components of the same TPP pathway. Here, we used the anti-bacterial T6SS of Serratia marcescens to elucidate post-translational regulation of offensive T6SS deployment, using single-cell microscopy and genetic analyses. We show that the integration of the TPP pathway with the negative regulator TagF to control core T6SS machine assembly is conserved between offensive and defensive T6SSs. Signal-dependent PpkA-mediated phosphorylation of Fha is required to overcome inhibition of membrane complex assembly by TagF, whilst PppA-mediated dephosphorylation promotes spatial reorientation and efficient killing. In contrast, the upstream input of the TPP pathway defines regulatory strategy, with a new periplasmic regulator, RtkS, shown to interact with the PpkA kinase in S. marcescens. We propose a model whereby the opposing actions of the TPP pathway and TagF impose a delay on T6SS re-assembly after firing, providing an opportunity for spatial re-orientation of the T6SS in order to maximise the efficiency of competitor cell targeting. Our findings provide a better understanding of how bacterial cells deploy competitive weapons effectively, with implications for the structure and dynamics of varied polymicrobial communities.

Partial Text

Bacteria constantly face a challenging external environment, with their survival dependent on the ability to adapt to abiotic conditions, overcome host defences or successfully compete against rival bacterial cells. Critical to all of these is the use of sophisticated protein secretion systems to translocate specific proteins across the bacterial envelope to the cell exterior, extracellular milieu or directly into other cells [1]. The Type VI secretion system (T6SS) is widespread in Gram-negative bacteria and is able to deliver toxic proteins, known as effectors, directly into target cells. Whilst some T6SSs are deployed by pathogenic bacteria against host eukaryotic organisms as direct virulence factors, it is now believed that the majority of T6SSs are involved in inter-bacterial competition and represent a crucial factor in conferring a fitness advantage in a variety of polymicrobial niches [2–4]. Many important pathogens, in addition to commensals and environmental bacteria, use anti-bacterial T6SSs to deliver anti-bacterial toxins into rival bacterial cells, whilst protecting themselves and their siblings from intoxication through possession of cognate immunity proteins able to neutralise each T6SS-delivered effector. T6SS-dependent anti-bacterial effectors include families of peptidoglycan hydrolases, phospholipases and DNases, in addition to examples of pore forming toxins, NAD-glycohydrolases and others of currently unknown function [2, 4–6]. The T6SS can promote very efficient killing of competitors and its importance in maintaining and disrupting complex communities such as the human gut microbiota is becoming increasingly appreciated [7, 8].

Whilst anti-bacterial T6SSs are widespread and effective weapons during inter-bacterial competition, the broad strategy by which they are deployed can vary dramatically, even between similar systems. Some systems, such as P. aeruginosa H1-T6SS and EAEC SciX T6SS, are described as ‘defensive’ since they assemble and fire in response to an incoming attack from a neighbouring cell, whereas others are considered ‘offensive’ since they fire without any requirement for incoming attack or other cell contact-based signal, including the T6SSs of V. cholerae and S. marcescens [16–18]. The best characterised defensive system is P. aeruginosa H1-T6SS, where incoming attack is sensed by the TagQRST proteins, causing activation of the PpkA kinase, phosphorylation of Fha and assembly of an actively-firing T6SS at the site of the assault. This TPP regulatory system results in a single cell ‘dueling’ phenotype where neighbouring cells repeatedly assemble adjacent, retaliatory T6SS foci at the interface between them, and an inability of wild type P. aeruginosa to kill non-aggressive competitors such as E. coli K12 or secrete proteins in liquid media [18, 21]. On the other hand, the S. marcescens Db10 T6SS represents a typical example of an offensive system, killing non-attacking competitors efficiently and readily firing in liquid or in isolation from other cells [17, 26, 27]. Interestingly, however, the P. aeruginosa and S. marcescens T6SSs share a similar TPP pathway and TagF, and both require PpkA-mediated phosphorylation of Fha for T6SS activity, despite the opposite final regulatory outcome [21, 27]. This study has now demonstrated that the interaction of the TPP pathway with the core machinery is conserved between the offensive and defensive systems, whilst a novel input protein, RtkS, forms the top of the offensive pathway.

 

Source:

http://doi.org/10.1371/journal.ppat.1007230