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

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.

Distribution, Function and Regulation of Type 6 Secretion Systems of Xanthomonadales

“…T6SS clusters from group 3 contain tagF-pppA-ppkA-fha genes, suggesting that activation of T6SS assembly and firing is dependent on the activation of the kinase PpkA, as originally described in P. aeruginosa (Mougous et al, 2007; Casabona et al, 2013). Recent work has demonstrated that PpkA acts by counteracting the inhibitory effect of TagF on T6SS activity, and the distinction between defensive and offensive T6SSs is mainly determined by the upstream input from protein(s) responsible for activation of PpkA (Lin et al, 2018; Ostrowski et al, 2018). In the defensive T6SS of P. aeruginosa , an incoming attack is sensed in the periplasm by TagQRST, while in the offensive Serratia marcensens T6SS, the signal is sensed by RtkS (Ostrowski et al, 2018).…”

“…Recent work has demonstrated that PpkA acts by counteracting the inhibitory effect of TagF on T6SS activity, and the distinction between defensive and offensive T6SSs is mainly determined by the upstream input from protein(s) responsible for activation of PpkA (Lin et al, 2018; Ostrowski et al, 2018). In the defensive T6SS of P. aeruginosa , an incoming attack is sensed in the periplasm by TagQRST, while in the offensive Serratia marcensens T6SS, the signal is sensed by RtkS (Ostrowski et al, 2018). The offensive/defensive model has been described in only a few antibacterial T6SS and the mechanism of post-translational activation of anti-eukaryotic machines is possibly different.…”

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

“…During this process, involved in the defense of bacteria to a neighboring attack, a membrane-bound threonine kinase phosphorylates a forkheadassociated domain-containing Protein (FhA or TagH), which promotes the assembly of an active conformation of the T6SS at the site of the attack (52)(53)(54). Recently, the offensive antibacterial T6SS of Serratia marcescens has also been shown be controlled by the opposing actions of the TPP pathway and TagF on assembly of the core machinery (55). The TPP represents the only currently identified post-translational mechanism regulating T6SS biogenesis.…”

The evolution of tit-for-tat in bacteria via the type VI secretion system

“…There is remarkable variation in the regulation and use of T6SS weaponry across species. Bacteria activate and deploy the T6SS across a range of environmental contexts [16][17][18][19] and against both prokaryotic and eukaryotic targets [20][21][22] . The specific pattern of firing by cells also varies: whereas placement of T6SS assembly appears to be random in some species, such as Vibrio cholerae, Serratia marcescens, and Acinetobacter baylyi [23][24][25] , other bacteria are known to fire from specific locations on their cell membranes.…”