Activity and Crystal Structure of the Adherent-Invasive Escherichia coli Tle3/Tli3 T6SS Effector/Immunity Complex...

The type VI secretion system (T6SS) delivers enzymatic effectors into target cells to destroy them. Cells of the same strain protect themselves against effectors with immunity proteins that specifically inhibit effectors. Here, we report the identification and characterization of a Tle3 phospholipase effector and its cognate immunity protein Tli3-an outer membrane lipoprotein from adherent-invasive Escherichia coli (AIEC). Enzymatic assays demonstrate that purified Tle3^AIEC has a phospholipase A1, and not A2, activity and that its toxicity is neutralized by the cognate immunity protein Tli3^AIEC. Tli3^AIEC binds Tle3 in a 1:1 stoichiometric ratio. Tle3^AIEC, Tli3^AIEC and the Tle3^AIEC-Tli3^AIEC complex were purified and subjected to crystallization. The Tle3^AIEC-Tli3^AIEC complex structure could not be solved by SeMet phasing, but only by molecular replacement when using an AlphaFold2 prediction model. Tle3^AIEC exhibits an α/β-hydrolase fold decorated by two protruding segments, including a N-terminus loop. Tli3^AIEC displays a new fold of three stacked β-sheets and a protruding loop that inserts in Tle3^AIECcatalytic crevice. We showed, experimentally, that Tle3^AIEC interacts with the VgrG ^AIEC cargo protein and AlphaFold2 prediction of the VgrG^AIEC-Tle3^AIEC complex reveals a strong interaction between the VgrG^AIEC C-terminus adaptor and Tle3^AIEC N-terminal loop.