Structural basis for loading and inhibition of a bacterial T6 SS phospholipase effector by the VgrG spike

Abstract

The bacterial type VI secretion system (T6SS) is a macromolecular machine that injects effectors into prokaryotic and eukaryotic cells. The mode of action of the T6SS is similar to contractile phages: the contraction of a sheath structure pushes a tube topped by a spike into target cells. Effectors are loaded onto the spike or confined into the tube. In enteroaggregative Escherichia coli, the Tle1 phospholipase binds the C-terminal extension of the VgrG trimeric spike. Here, we purify the VgrG-Tle1 complex and show that a VgrG trimer binds three Tle1 monomers and inhibits their activity. Using covalent cross-linking coupled to high-resolution mass spectrometry, we provide information on the sites of contact and further identify the requirement for a Tle1 N-terminal secretion sequence in complex formation. Finally, we report the 2.6-Å-resolution cryo-electron microscopy tri-dimensional structure of the (VgrG) 3 -(Tle1) 3 complex revealing how the effector binds its cargo, and how VgrG inhibits Tle1 phospholipase activity. The inhibition of Tle1 phospholipase activity once bound to VgrG suggests that Tle1 dissociation from VgrG is required upon delivery.

Activity, delivery, and diversity of Type VI secretion effectors

“…Bacterial TTR-like domains are globular structures composed of two small β-sheets structurally resembling the eukaryotic TTR β-sandwich structure (Flaugnatti et al, 2020). In enteroaggregative E. coli VgrG EAEC , the TTR is fused to the VgrG gp5 needle-like domain through highly flexible loops (Flaugnatti et al, 2020). TTR interacts with the flexible N-terminal extension of the Tle1 effector which inserts its β-sheets to form a β-barrel with TTR (Flaugnatti et al, 2020).…”

“…DUF2345, as well as long α-helices aligned against the length of the needle, make numerous contacts with loaded effector. Together with the TTR domain, they can be considered an internal adaptor domain for the direct recruitment of the effectors (Flaugnatti et al, 2020). DUF2345 domains can perform additional functions.…”

“…Moreover, the same VgrG spike can accommodate different effectors using different PAAR or adaptor proteins, and some effectors could be recruited to several different assemblies (Burkinshaw et al, 2018;Cianfanelli et al, 2016a;Whitney et al, 2014;Wood et al, 2019b). Processes that could coordinate the secretion of different effectors and the bases for the selection of the effectors are so far unclear.The first high-resolution Cryo-EM structures of T6SS secretion spike/effector complexes revealed networks of highly specific interactions (Flaugnatti et al, 2020;Quentin et al, 2018). These examples represent only a few out of many possible secretion complex architectures.…”

Combining high throughput and high quality for cryo-electron microscopy data collection

“…It is also of primary importance to carefully select the acquisition parameters (pixel size and defocus range) according to the expected/desired resolution in order to get the maximum out of the data-collection session. We now routinely collect SPA data sets using active beam-tilt compensation and fringe-free imaging at a pixel size of $0.8 Å (Wandzik et al, 2020;Qi, Di Minin et al, 2019;Qi, Sorrentino et al, 2019;Cannac et al, 2020;Flaugnatti et al, 2020;Zivanov et al, 2018;Muir et al, 2020;Oosterheert et al, 2018) or even smaller (Weis et al, 2019). The ability to collect more/better data in a shorter time allows increasingly dynamic, flexible and heterogeneous complexes to be imaged (time-resolved studies, mechanistic studies etc.…”

Interbacterial competition and anti-predatory behavior of environmentalVibrio choleraestrains

“…It is composed of a membrane-spanning portion, a tube structure made of stacks of Hcp hexamer rings, and a tube-surrounding sheath. The sheath is composed of the two proteins VipA and VipB which, upon contraction, propels the inner tube out of the cell together with its effector-decorated tip proteins (VgrG and PAAR) (Zoued et al, 2014;Cherrak et al, 2019;Flaugnatti et al, 2020).…”