Salmonella antibacterial Rhs polymorphic toxin inhibits translation through ADP-ribosylation of EF-Tu P-loop

Abstract

Rearrangement hot spot (Rhs) proteins are members of the broad family of polymorphic toxins. Polymorphic toxins are modular proteins composed of an N-terminal region that specifies their mode of secretion into the medium or into the target cell, a central delivery module, and a C-terminal domain that has toxic activity. Here, we structurally and functionally characterize the C-terminal toxic domain of the antibacterial Rhsmain protein, TreTu, which is delivered by the type VI secretion system of Salmonella enterica Typhimurium. We show that this domain adopts an ADP-ribosyltransferase fold and inhibits protein synthesis by transferring an ADP-ribose group from NAD+ to the elongation factor Tu (EF-Tu). This modification is specifically placed on the side chain of the conserved D21 residue located on the P-loop of the EF-Tu G-domain. Finally, we demonstrate that the TriTu immunity protein neutralizes TreTu activity by acting like a lid that closes the catalytic site and traps the NAD+.

Graphical Abstract

Salmonella enterica Typhimurium eliminates bacterial rivals by delivering the Tre^Tu type VI secretion Rhs polymorphic toxin that arrests protein synthesis through ADP-ribosylation of EF-Tu.

Figure 1.

Salmonella enterica Rhs^main Tre^Tu C-terminal domain has antibacterial cytoplasmic-acting activity and is neutralized by Tri^Tu. (A) Schematic representation of the S. enterica Typhimurium LT2 T6SS gene cluster. Genes encoding core and accessory T6SS components are depicted as black arrows. Genes encoding effector protein and domain, and immunity proteins are colored in red and green, respectively. The structural parts of rhs^main and rhs^orphan genes are stripped in black and red. (B) The schematic representation of the rhs^main. PAAR domain is colored in blue, transmembrane domains in gray, RHS repeats in brown and Tre^Tu toxin domain in red. (C) Toxicity assay in the heterologous host E. coli. Cultures of E. coli cells bearing the empty or Tre^Tu toxin-encoded pBAD33rbs and empty or Tri^Tu immunity-encoded pKK22.3 plasmids were serially diluted and spotted on LB agar plates supplemented with 0.2% arabinose and 1 mM IPTG to induce expression from pBAD33 and pKK vectors, respectively. (D) Gel filtration analyses using Superdex 75 10/30 column. The Tre^Tu–Tri^Tu complex was purified via the 6× His tag fused to Tre^Tu. After TEV protease cleavage, the complex was subjected to size-exclusion chromatography, revealing a unique peak (black peak and black star). In the other assay, the complex bound on the metal-affinity resin was subjected to denaturation by 8 M urea. The two fractions (elution containing Tre^Tu and urea wash containing Tri^Tu) were then refolded and subjected to size-exclusion chromatography (red and green peaks and stars, respectively). The elution profiles of molecular mass standards indicated on top (in kDa) are shown in dotted lines. (E) SDS-PAGE and Coomassie blue staining of the various purified samples: His-TEV-Tre^Tu/Tri^Tu complex (lane 1), His-TEV-Tre^Tu (lane 2), TEV-digested Tre^Tu/Tri^Tu complex (lane 3), and denaturated/refolded Tre^Tu (lane 4) and Tri^Tu (lane 5).

Figure 2.

Tre^Tu structure. (A) Ribbon representation of the S. enterica Tre^Tu domain. The β -strands, including the pseudo-β-strand β4, are numbered. (B) Secondary structures of Tre^Tu. The putative residues involved in NAD⁺ binding and catalysis are colored by element. (C). Magnification of the enzymatic pocket highlighting the residues involved in NAD⁺ binding and catalysis. (D and E) Toxicity assay in the heterologous host E. coli. Cultures of E. coli cells producing the Tre^Tu toxin and its variants from the pBAD33rbs vector were serially diluted and spotted on LB-agar plates supplemented with 1% glucose (repression conditions, left panel) or 0.2% L-arabinose (induction conditions, right panel). Effect of mutations in the putative catalytic triad are shown in (D), and further residues suggested to be involved in NAD⁺ binding and catalysis are shown in (E).

Figure 3.

Tre^Tu ADP-ribosylates the EF-Tu. (A) Time-course ADP-ribosylation assays of cellular extracts of E. coli in presence of the Tre^Tu toxin and biotin-labeled NAD⁺. (B) Effect of Tre^Tu toxin on coupled in vitro transcription-translation assays. Synthesis of GFP-strepII protein was followed in the presence of Tre^Tu toxin, NAD⁺ and Tri^Tu immunity protein. Synthesized GFP-strepII was then recognized by western blotting with anti-strep antibodies (C) ADP-ribosylation of purified his-EF-Tu by the Tre^Tu toxin in presence of biotin-labeled NAD⁺ and of the Tri^Tu immunity protein. ‘Aft’ signifies immunity protein added after the reaction and allowing the reaction to continue.

Figure 4.

Tre^Tu ADP-ribosylates EF-Tu at residue D21. (A) Deconvoluted mass spectra of EF-Tu in presence of NAD⁺ and GTP before (top panel) and after (bottom panel) incubation with Tre^Tu toxin. The two major forms correspond to EF-Tu without the initiator methionine (purple) and without the initiator methionine plus an hexose (cyan), respectively. (B) Fragmentation spectra of the TIGHVD(ADP-ribose)HGKTTL peptide obtained after HCD activation. b/y ions are labeled respectively in dark blue and dark green. Upon HCD activation, ADP-ribose fragmentation releases the ADP moiety which forms three distinctive ions corresponding from left to right to adenosine, adenosine-phosphate and adenosine diphosphate ions. The ribose-phosphate moiety remains on the peptide fragments, labeled with a star (b*, y* respectively in light blue and light green). Parent peptide can be observed free of modification or with ribose-phosphate addition due to the sensibility of the modification to the activation method. Both forms produce also lots of water loss (labeled ‘-H2O’). (C) EF-Tu structure (blue, PDB:1ob2) highlighting the exposition of residue D21 side chain, which is located on the P-loop of the G domain (domain I). (D) In the translating ribosome complex (PDB:4v5l), the side chain of D21 is enclosed in a 23S RNA pocket formed by nucleobases G2655, C2658 and G2659. (E) ADP-ribosylation of the wild-type EF-Tu and its D21A, D21E and D21N substitution variants. About 20 μM of purified his-EF-Tu was incubated with GTP, 0.1 μM of biotinylated NAD⁺ and 1 μM of Tre^Tu toxin for 15 min. Proteins were stained by Ponceau rouge after SDS-PAGE and transfer on nitrocellulose membrane (upper panel). The ADP-ribosylation was detected by streptavidin-AP conjugate (lower panel).

Figure 5.

Tre^Tu toxin ADP-ribosylates GTP-bound EF-Tu. (A) Effect of GTP and/or tRNA or EF-Ts addition on EF-Tu ADP ribosylation by Tre^Tu. The composition of each reaction is indicated above the panels. Final concentrations of 20 μM of EF-Tu, 0.1 μM of 6-biotin-17-NAD, 1 μM of Tre^Tu toxin, 1 mM of GTP, 100 μM of tRNA or 50 μM of EF-Ts were used for the reactions. (B) Effect of the different forms of guanosine phosphate on EF-Tu ADP-ribosylation. Nucleotide-free his-EF-Tu was incubated with the indicated nucleotide and the reactions were performed as previously described. Proteins were stained by Ponceau rouge after SDS-PAGE and transfer on nitrocellulose membrane (upper panels). ADP-ribosylation was detected by streptavidin-AP conjugate (lower panels).

Figure 6.

Tri^Tu immunity protein neutralizes the Tre^Tu toxin blocking its NAD⁺ pocket and C-terminal α-helix. (A,B) Side (A) and top (B) views of the Tre^Tu-Tri^Tu complex crystal structure. Tre^Tu is shown in red while Tri^Tu is shown in green. (C) Magnification of the NAD⁺ binding pocket, highlighting the NAD⁺ molecule (yellow) and the side chains of the putative active site. (D) Magnification of the Tre^Tu C-terminal helix, highlighting the Tre^Tu (red) and Tri^Tu (green) residues (colored by element) at the contact site. (E) Effect of Tre^Tu C-terminal α -helix substitutions on toxicity in the heterologous host E. coli. Cultures of E. coli cells producing the Tre^Tu toxin and its variants from the pBAD33rbs vector were serially diluted and spotted on LB-agar plates supplemented with 1% glucose (repression conditions, left panel) or 0.2% L-arabinose (induction conditions, right panel).