Structure of the Type VI Secretion System Contractile Sheath

Abstract

Summary Bacteria use rapid contraction of a long sheath of the Type VI secretion system (T6SS) to deliver effectors into a target cell. Here we present an atomic resolution structure of a native contracted Vibrio cholerae sheath determined by cryo-electron microscopy. The sheath subunits, composed of tightly interacting proteins VipA and VipB, assemble into a six-start helix. The helix is stabilized by a core domain assembled from four β-strands donated by one VipA and two VipB molecules. The fold of inner and middle layers is conserved between T6SS and phage sheaths. However, the structure of the outer layer is distinct and suggests a mechanism of interaction of the bacterial sheath with an accessory ATPase, ClpV, that facilitates multiple rounds of effector delivery. Our results provide a mechanistic insight into assembly of contractile nanomachines that bacteria and phages use to translocate macromolecules across membranes.

Cryo‐ EM reconstruction of Type VI secretion system baseplate and sheath distal end

“…We isolated the VipA‐N3 sheath variant (3‐amino acid insertion into the N‐terminal VipA linker, tagged with msfGFP) from cells and purified it by ultracentrifugation as described previously (Kudryashev et al , 2015). Negative stain electron microscopy showed sheath assemblies of various lengths in apparently extended conformations (Fig 1A).…”

“…However, the subunits of the non‐contractile VipA‐N3 mutant sheath are connected to the previous sheath ring only through VipB linker and the elongated VipA linker connects to a neighboring subunit on the same sheath ring (Wang et al , 2017). This also means that the first sheath ring can only be connected to the baseplate by six linkers and not by twelve as in the wild‐type assembly or in the T4 phage (Kudryashev et al , 2015; Taylor et al , 2016). Even though our reconstruction lacks the resolution needed to resolve details of sheath–baseplate connection, there is a density originating from a sheath protomer likely corresponding to the VipB C‐terminal linker, which is most likely connected to TssE in the wedge (Fig EV4).…”

“…Evolutionarily related Type VI secretion system (T6SS) is used by bacteria to deliver proteins into both bacterial and eukaryotic cells (Mougous et al , 2006; Pukatzki et al , 2006; Hood et al , 2010; MacIntyre et al , 2010; Durand et al , 2014; Ho et al , 2014; Alcoforado Diniz et al , 2015; Hachani et al , 2016). The current model of T6SS biogenesis and mode of action is largely based on well‐understood phage assembly (Leiman et al , 2009; Lossi et al , 2011, 2013; Ho et al , 2014; Zoued et al , 2014; Clemens et al , 2015; Kudryashev et al , 2015; Cianfanelli et al , 2016). However, many important features are unknown mostly due to the lack of high‐resolution structural information of T6SS.…”

“…The V. cholerae sheath subunits are interconnected by a mesh of VipA N‐terminal and VipB C‐terminal linkers (Kudryashev et al , 2015) similarly to T6SS sheath of Francisella novicida and R‐type pyocin sheath (Clemens et al , 2015; Ge et al , 2015). The sheath was shown to assemble across the entire width of a cell, and this allowed to use live‐cell fluorescence microscopy to study its dynamics and subcellular localization (Basler et al , 2012; Brunet et al , 2013; Gerc et al , 2015; Zoued et al , 2016; Vettiger et al , 2017).…”

VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor–effector complex

“…Functional and structural studies have shown that the T6SS nanomachine shares striking similarities with the bacteriophage tail structure (14)(15)(16)(17)(18)(19)(20)(21)(22). Accumulating evidence suggests that the baseplate complex is recruited into the membrane-associated protein complex and initiates the polymerization of a TssB-TssC (VipAVipB) contractile sheath.…”