A glycine zipper motif governs translocation of type VI secretion toxic effectors across the cytoplasmic membrane of target cells

Type VI secretion systems (T6SSs) can deliver diverse toxic effectors into eukaryotic and bacterial cells. Although much is known about the regulation and assembly of T6SS, the translocation mechanism of effectors into the periplasm and/or cytoplasm of target cells remains elusive. Here we used the Agrobacterium tumefaciens DNase effector Tde1 to unravel the mechanism of translocation from attacker to prey. We demonstrate that Tde1 loading onto the secretion machinery is mediated via binding to its adaptor through the N-terminus, which harbours continuous copies of GxxxG motifs resembling the glycine zipper structure found in proteins involved in the membrane channel formation. By amino acid substitutions on a conserved glycine zipper motif, we showed G39xxxG43 motif governs the delivery into target cells by permeabilizing the cytoplasmic membrane. The findings demonstrate effector itself mediates target cell entry. Considering the widespread presence of GxxxG motifs in bacterial effectors and pore-forming toxins, we proposed that glycine zipper mediated permeabilization is a conserved mechanism used by bacterial effectors for translocation across target cell membranes.