The type VI secretion system (T6SS) is a phage-derived contractile nanomachine primarily involved in interbacterial competition. Its pivotal component, TssA, is indispensable for the assembly of the T6SS sheath structure, the contraction of which propels a payload of effector proteins into neighboring cells. Despite their key function, TssA proteins exhibit unexpected diversity and exist in two major forms, a short form (TssAS) and a long form (TssAL). While TssAL proteins interact with a partner, called TagA, to anchor the distal end of the extended sheath, the mechanism for the stabilization of TssAS-containing T6SSs remains unknown. Here we discover a class of structural components that interact with short TssA proteins and contribute to T6SS assembly by stabilizing the polymerizing sheath from the baseplate. We demonstrate that the presence of these components is important for full sheath extension and optimal firing. Moreover, we show that the pairing of each form of TssA with a different class of sheath stabilization proteins results in T6SS apparatuses that either reside in the cell for some time or fire immediately after sheath extension. We propose that this diversity in firing dynamics could contribute to the specialization of the T6SS to suit bacterial lifestyles in diverse environmental niches.

The bacterial pathogen Pseudomonas aeruginosa uses three type VI secretion systems (T6SSs) to drive a multitude of effector proteins into eukaryotic or prokaryotic target cells. The T6SS is a supramolecular nanomachine, involving a set of 13 core proteins, which resembles the contractile tail of bacteriophages and whose tip is considered as a puncturing device helping to cross membranes. Effectors can attach directly to the T6SS spike which is composed of a VgrG (valine-glycine-rich proteins) trimer, of which P. aeruginosa produces several. We have previously shown that the master regulator RsmA controls the expression of all three T6SS gene clusters (H1-, H2- and H3-T6SS) and a range of remote vgrG and effector genes. We also demonstrated that specific interactions between VgrGs and various T6SS effectors are prerequisite for effector delivery in a process we called “à la carte delivery.” Here, we provide an in-depth description on how the two H2-T6SS-dependent effectors PldA and PldB are delivered via their cognate VgrGs, VgrG4b and VgrG5, respectively. We show that specific recognition of the VgrG C terminus is required and effector specificity can be swapped by exchanging these C-terminal domains. Importantly, we established that effector recognition by a cognate VgrG is not always sufficient to achieve successful secretion, but it is crucial to provide effector stability. This study highlights the complexity of effector adaptation to the T6SS nanomachine and shows how the VgrG tip can possibly be manipulated to achieve effector delivery.

Pseudomonas aeruginosa encodes three type VI secretion systems, each comprising a dozen distinct proteins, which deliver toxins upon T6SS sheath contraction. The least conserved T6SS component, TssA, has variations in size (short: TssA1 and TssA3, long: TssA2) which influence domain organisation and structure. Here we show that the TssA Nt1 domain interacts directly with the sheath in a specific manner, while the C-terminus is essential for oligomerisation. We built chimeric TssA proteins by swapping C-termini and showed that these can be functional even when made of domains from different TssA sub-groups. Functional specificity requires the Nt1 domain, while the origin of the C-terminal domain is more permissive for T6SS function. We identify two regions, loop and hairpin, that contribute to sheath binding. We propose a docking mechanism of TssA proteins with the sheath, and a model for how sheath assembly is coordinated by TssA proteins from this position.

20 21 2 These authors have contributed equally to this work. 22 23 24 KEYWORDS 25 26 Type VI secretion system, interbacterial competition, contact-dependent killing, toxin 27 delivery, TssA, TagB, TagA, sheath stabilization, sheath contraction, Pseudomonas. 28 29 30 ABSTRACT 31 32The type VI secretion system (T6SS) is a phage-derived contractile nanomachine primarily 33 involved in interbacterial competition. Its pivotal component, TssA, is indispensable for the 34 assembly of the T6SS sheath structure, the contraction of which propels a payload of effector 35proteins into neighboring cells. Despite their key function, TssA proteins exhibit unexpected 36 diversity and exist in two major forms, a short (TssAS) and a long (TssAL) TssA. Whilst 37TssAL proteins interact with a partner, called TagA, to anchor the distal end of the extended 38sheath, the mechanism for the stabilization of TssAS-containing T6SSs remains unknown. 39Here we discover a novel class of structural components that interact with short TssA 40proteins and contribute to T6SS assembly by stabilizing the polymerizing sheath from the 41 baseplate. We demonstrate that the presence of these components is important for full sheath 42 extension and optimal firing. Moreover, we show that the pairing of each form of TssA with 43 a different class of sheath stabilization proteins results in T6SS apparatuses that either reside 44in the cell for a while or fire immediately after sheath extension, thus giving rise to different 45 aggression behaviors. We propose that this functional diversity could contribute to the 46 specialization of the T6SS to suit bacterial lifestyles in diverse environmental niches. 47 48 49Bacteria live in complex polymicrobial communities that are shaped by interspecies 50 cooperation and competition. As resources are limited, antagonistic strategies are a major 51 driver of survival and success for bacterial populations. One of the most elaborate bacterial 52weapons is the type VI secretion system (T6SS), which not only promotes inter-bacterial and 53inter-kingdom competition (1-3), but is also involved in the interaction of bacteria with their 54