Structural basis for loading and inhibition of a bacterial T6SS phospholipase effector by the VgrG spike

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. Synopsis Bacterial Type VI secretion system (T6SS) effectors are loaded in the T6SS tube or on the spike complex for delivery into target cells. The current study presents the detailed structure of the T6SS effector Tle1 in complex with the VgrG spike of enteroaggregative Escherichia coli . VgrG trimer binds three Tle1 monomers. VgrG inhibits Tle1 phospholipase activity. N‐terminal secretion sequence of Tle1 interacts with the VgrG C‐terminal TTR domain. The 2.6‐Å resolution cryo‐EM structure of the (VgrG)3‐(Tle1)3 complex reveals a triskelion‐like assembly. Graphical Abstract A cryo‐EM structure reveals interactions between the Type VI secretion system effector Tle1 with the spike protein VgrG from enteroaggregative Escherichia coli .