Family of Tle3 phospholipase effector .The type VI lipase effectors Tle1-Tle5 secreted by the bacterial type VI secretion system (T6SS) have been identified as antibacterial effectors that hydrolyze membrane phospholipids. The Tle1-4 display phospholipase A1 and A2 (PLA1) (PLA2) activities. Tle1 belongs to Duf_2235 family (now T6SS-TLE1), TLE2 to Lipase_3, TLE3 to alpha/ beta hydrolase (now T6SS-TLE3) and Tle4 belongs to PGAP1 (Tle5 family, such as Pseudomonas aeruginosa PldA and PldB, are phospholipases D and are not alpha/beta hydrolases). Tle3 phospholipase effector from adherent-invasive Escherichia coli (AIEC) Tle3AIEC possesses phospholipase A1 (PLA1) activity (specific activity (SA) = 7.65 nmole.min-1.mg-1) but no PLA2 activity. The T6SS-TLE3 is decorated by two protruding segments, including a N-terminus loop containing part of the oxyanion hole residues. The long C-terminus contains the catalytic site histidine and in some members is related to the DUF3274 family (most members of this family are not linked to alpha/beta hydrolases). In T6SS, protection against its own toxic effector is ensured in bacteria by the production of immunity proteins that specifically bind and inhibit Tle3. Tli (Type VI lipase immunity) binds tightly to Tle3. There is also a strong interaction between the VgrGAIEC C-terminus adaptor and Tle3AIEC N-terminal loop.
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.
Membranes allow the compartmentalization of biochemical processes and are therefore fundamental to life. The conservation of the cellular membrane, combined with its accessibility to secreted proteins, has made it a common target of factors mediating antagonistic interactions between diverse organisms. Here we report the discovery of a diverse superfamily of bacterial phospholipase enzymes. Within this superfamily, we defined enzymes with phospholipase A1 and A2 activity, which are common in host-cell-targeting bacterial toxins and the venoms of certain insects and reptiles. However, we find that the fundamental role of the superfamily is to mediate antagonistic bacterial interactions as effectors of the type VI secretion system (T6SS) translocation apparatus; accordingly, we name these proteins type VI lipase effectors. Our analyses indicate that PldA of Pseudomonas aeruginosa, a eukaryotic-like phospholipase D, is a member of the type VI lipase effector superfamily and the founding substrate of the haemolysin co-regulated protein secretion island II T6SS (H2-T6SS). Although previous studies have specifically implicated PldA and the H2-T6SS in pathogenesis, we uncovered a specific role for the effector and its secretory machinery in intra- and interspecies bacterial interactions. Furthermore, we find that this effector achieves its antibacterial activity by degrading phosphatidylethanolamine, the major component of bacterial membranes. The surprising finding that virulence-associated phospholipases can serve as specific antibacterial effectors suggests that interbacterial interactions are a relevant factor driving the continuing evolution of pathogenesis.
.The type VI lipase effectors Tle1-Tle5 secreted by the bacterial type VI secretion system (T6SS) have been identified as antibacterial effectors that hydrolyze membrane phospholipids. The Tle1-4 display phospholipase A1 and A2 (PLA1) (PLA2) activities. Tle1 belongs to Duf_2235 family (now T6SS-TLE1), TLE2 to Lipase_3, TLE3 to alpha/ beta hydrolase (now T6SS-TLE3) and Tle4 belongs to PGAP1 (Tle5 family, such as Pseudomonas aeruginosa PldA and PldB, are phospholipases D and are not alpha/beta hydrolases).