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 alpha/beta-hydrolase fold decorated by two protruding segments, including a N-terminus loop. Tli3(AIEC) displays a new fold of three stacked beta-sheets and a protruding loop that inserts in Tle3(AIEC)catalytic 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.
Le TTH, Kellenberger C, Boyer M, Santucci P, Flaugnatti N, Cascales E, Roussel A, Canaan S, Journet L, Cambillau C (2023) Activity and Crystal Structure of the Adherent-Invasive Escherichia coli Tle3/Tli3 T6SS Effector/Immunity Complex Determined Using an AlphaFold2 Predicted Model Int J Mol Sci24: 1740
Le TTH, Kellenberger C, Boyer M, Santucci P, Flaugnatti N, Cascales E, Roussel A, Canaan S, Journet L, Cambillau C (2023) Int J Mol Sci24: 1740
