Structure-Function Analysis of the TssL Cytoplasmic Domain Reveals a New Interaction between the Type VI Secretion Baseplate and Membrane Complexes

- Systematic interaction studies reveal contact between the T6SS TssL and TssE subunits.
- The cytoplasmic domain of TssL delimitates a cleft that interacts with TssM.
- Two loops with distinct conformations mediate interactions with TssE and TssK.
- TssL interactions with TssM, TssE, and TssK are required for function of the T6SS.

The type VI secretion system (T6SS) is a multiprotein complex that delivers toxin effectors in both prokaryotic and eukaryotic cells. It is constituted of a long cytoplasmic structure-the tail-made of stacked Hcp hexamers and wrapped by a contractile sheath. Contraction of the sheath propels the inner tube capped by the VgrG spike protein toward the target cell. This tubular structure is built onto an assembly platform-the baseplate-that is composed of the TssEFGK-VgrG subunits. During the assembly process, the baseplate is recruited to a trans-envelope complex comprising the TssJ outer membrane lipoprotein and the TssL and TssM inner membrane proteins. This membrane complex serves as a docking station for the baseplate/tail and as a channel for the passage of the inner tube during sheath contraction. The baseplate is recruited to the membrane complex through multiple contacts including interactions of TssG and TssK with the cytoplasmic loop of TssM and of TssK with the cytoplasmic domain of TssL, TssLCyto. Here, we show that TssLCyto interacts also with the TssE baseplate subunit. Based on the available TssLCyto structures, we targeted conserved regions and specific features of TssLCyto in enteroaggregative Escherichia coli. By using bacterial two-hybrid analysis and co-immunoprecipitation, we further show that the disordered L3-L4 loop is necessary to interact with TssK and that the L6-L7 loop mediates the interaction with TssE, whereas the TssM cytoplasmic loop binds the conserved groove of TssLCyto. Finally, competition assays demonstrated that these interactions are physiologically important for T6SS function.

Graphical Abstract94