Identification, structure, and function of a novel type VI secretion peptidoglycan glycoside hydrolase effector-immunity pair

Abstract

Bacteria employ type VI secretion systems (T6SSs) to facilitate interactions with prokaryotic and eukaryotic cells. Despite the widespread identification of T6SSs among Gram-negative bacteria, the number of experimentally validated substrate effector proteins mediating these interactions remains small. Here, employing an informatics approach, we define novel families of T6S peptidoglycan glycoside hydrolase effectors. Consistent with the known intercellular self-intoxication exhibited by the T6S pathway, we observe that each effector gene is located adjacent to a hypothetical open reading frame encoding a putative periplasmically localized immunity determinant. To validate our sequence-based approach, we functionally investigate a representative family member from the soil-dwelling bacterium Pseudomonas protegens. We demonstrate that this protein is secreted in a T6SS-dependent manner and that it confers a fitness advantage in growth competition assays with Pseudomonas putida. In addition, we determined the 1.4 Å x-ray crystal structure of this effector in complex with its cognate immunity protein. The structure reveals the effector shares highest overall structural similarity to a glycoside hydrolase family associated with peptidoglycan N-acetylglucosaminidase activity, suggesting that T6S peptidoglycan glycoside hydrolase effector families may comprise significant enzymatic diversity. Our structural analyses also demonstrate that self-intoxication is prevented by the immunity protein through direct occlusion of the effector active site. This work significantly expands our current understanding of T6S effector diversity.

Keywords: Effector-Immunity Pair; Glycoside Hydrolases; Interbacterial Interactions; Microbiology; Peptidoglycan; Protein Crystallization; Protein Secretion; Type VI Secretion.

FIGURE 1.

Overview of the tge1–3 families. Genomic arrangement of tge1 (A), tge2 (B), and tge3 (C) families in representative organisms. Shading is used to indicate effector (light) and immunity (dark) open reading frames. Accession numbers and sequence alignments of Tge2 and Tge3 effector and immunity proteins are available in supplemental Figs. S1 and S2.

FIGURE 2.

Tge2^PP-Tgi2^PP are an effector-immunity pair secreted by the T6SS. A, growth of E. coli on solid media harboring inducible plasmids expressing the indicated proteins. Empty vector controls are indicated by a dash. Error bars represent ± S.D. (n = 3). B, optical density of E. coli in liquid media harboring a vector control or inducible plasmids expressing the indicated proteins. The arrow indicates the time at which protein expression was induced. Error bars represent ± S.D. (n = 3). C, representative phase contrast micrographs of E. coli expressing peri-Tge2^PP or peri-Tge2^PP E69Q. Images were acquired 45 min after IPTG induction. Scale bar, 2 μm. D, Western blot analysis of E. coli expressing vector control, Tge2^PP and Tge2^PP E69Q. RNA polymerase (RNAP) was used as a loading control. E, growth of E. coli on solid media harboring inducible plasmids expressing the indicated proteins. Empty vector controls are indicated by a dash. Error bars represent ± S.D. (n = 3). F, ITC analysis showing the interaction between Tge2^PP and Tgi2^PP. The top panel displays the heats of injection, whereas the bottom panel shows the normalized integration data as a function of the molar syringe and cell concentrations. G, Tge2^PP is secreted in a T6-dependent manner. Western blot analysis of the cell and supernatant (sup) fractions of the indicated P. protegens strains expressing Tge2^PP fused to a C-terminal vesicular stomatitis viral glycoprotein (VSV-G) tag. H, Tge2^PP provides a competitive growth advantage under cell contact-promoting conditions. Competitive outcome of the indicated P. protegens strains against P. putida. Asterisks denote competition outcomes significantly different than wild-type P. protegens (n = 4; *, p < 0.05). Error bars indicate ± S.D.

FIGURE 3.

Overall structure of Tge2^PP in complex with Tgi2^PP. A, Tge2^PP adopts a lysozyme-like fold. Ribbon (left) and surface (right) representations of Tge2^PP shown at two orthogonal orientations. Secondary structure elements and the catalytic acid, Glu⁶⁹, are labeled. Tgi2^PP is omitted for clarity. B, structural overlay of Tge2^PP (blue), Auto (orange), and FlgJ (gray) shown as schematic representations (left panel). Superposition of the Tge2^PP, Auto and FlgJ active site residues are shown as stick representations (right panel). The labels indicate the residue numbering of Tge2^PP followed by Auto and FlgJ. C, structure of the Tge2^PP·Tgi2^PP complex. Tge2^PP and Tgi2^PP are shown as surface and ribbon representations, respectively. Secondary structure elements of Tgi2^PP are labeled, and Glu⁶⁹ of Tge2^PP is colored yellow. D, a protruding loop of Tgi2^PP occludes the active site of Tge2^PP. Shown is a close-up view of the β2-β3 loop of Tgi2^PP interacting with the active site of Tge2^PP. Invariant residues Gly⁹⁴ and Ala⁹⁵ of Tgi2^PP interact with His⁸⁸ and Tyr¹⁴⁷ in the active site cleft of Tge2^PP.

FIGURE 4.

Structurally characterized lysozyme inhibitors display distinct folds but act via a common mechanism. Comparison of the Tge2^PP·Tgi2^PP complex to vertebrate lysozymes in complex with proteinaceous bacterial lysozyme inhibitors. Shown are the Tge2^PP·Tgi2^PP complex (A), E. coli periplasmic lysozyme inhibitor of G-type lysozyme (PliG^EC) in complex with salmon G-type lysozyme (SalG) (B), E. coli proteinaceous inhibitor of vertebrate lysozyme (Ivy^EC) in complex with hen egg white lysozyme (HEWL) (C), and membrane-bound lysozyme inhibitor of C-type lysozyme (MliC) in complex with hen egg white lysozyme (D) (–47). For each structural model, the lysozyme-like protein and its associated inhibitor are shown as surface and ribbon representations, respectively.