A Salmonella Toxin Promotes Persister Formation through Acetylation of tRNA

Abstract

The recalcitrance of many bacterial infections to antibiotic treatment is thought to be due to the presence of persisters that are non-growing, antibiotic-insensitive cells. Eventually, persisters resume growth, accounting for relapses of infection. Salmonella is an important pathogen that causes disease through its ability to survive inside macrophages. After macrophage phagocytosis, a significant proportion of the Salmonella population forms non-growing persisters through the action of toxin-antitoxin modules. Here we reveal that one such toxin, TacT, is an acetyltransferase that blocks the primary amine group of amino acids on charged tRNA molecules, thereby inhibiting translation and promoting persister formation. Furthermore, we report the crystal structure of TacT and note unique structural features, including two positively charged surface patches that are essential for toxicity. Finally, we identify a detoxifying mechanism in Salmonella wherein peptidyl-tRNA hydrolase counteracts TacT-dependent growth arrest, explaining how bacterial persisters can resume growth.

Graphical abstract

Figure 1

A GNAT-Related Toxin of Salmonella Extends Lag Phase by Halting Protein Synthesis (A) Growth curves of S. Typhimurium 12023 Δta8 carrying pNDM220 (−T8) or pNDM220::t8 (+T8) in fresh rich medium. Expression of t8 was induced upon addition of IPTG during mid-exponential (left panel) or lag (right panel) phase. (B) Growth curves of S. Typhimurium 12023 Δta8 carrying pNDM220 (−T8), pNDM220::t8 (+T8), pBAD33::a8 (+A8), or pNDM220::t8 and pBAD33::a8 (+T8+A8). All cultures were supplemented with arabinose and IPTG in fresh rich medium during lag phase. (C) Levels of incorporation of radiolabeled methionine (left panel), thymidine (middle panel), or uracil (right panel) during pulse-chase assays in lag phase cultures of S. Typhimurium 12023 Δta8 carrying pNDM220 (−T8) or pNDM220::t8 (+T8), or treated with bacteriostatic concentrations of chloramphenicol, ciprofloxacin, or rifampicin. All cultures were supplemented with IPTG at t0. All measures were normalized to those of the control samples at 5 min. Data represent the mean ± SEM (n ≥ 3) and were analyzed using a Student’s t test (ns, non-significant; ^∗p < 0.05; ^∗∗∗p < 0.005) (see also Figure S1).

Figure 2

T8 Is a Unique Type of Acetyltransferase (A) Growth curves of S. Typhimurium 12023 Δta8 expressing from pNDM220, the wild-type toxin (+T8), or point mutant toxins (+T8^A93P, +T8^Y140F), or carrying the empty vector (−T8). All cultures were supplemented with IPTG in fresh rich medium during lag phase. Data represent the mean ± SEM (n ≥ 3) and were analyzed using a Student’s t test (ns, non-significant). (B) Cartoon representation of the dimeric structure of T8^Y140F (top panel). Chain A is colored from blue at the N terminus to red at the C terminus, and Chain B is colored light gray. Ac-CoA molecules are shown as stick representations. Sequence of T8 (lower panel) with secondary structure elements illustrated and colored as in the top panel. Black dots indicate Ac-CoA binding residues and gray diamonds indicate residues involved in dimer formation (PDB: 5FVJ). (C) Superimposition of T8^Y140F (gray) with three other GNATs identified as being close structural homologs by the DALI server (Holm and Rosenström, 2010)—PDB: 2R7H (yellow), 2CNM (orange), and 4PV6 (purple), members of the N-acetyltransferase superfamily. Unique features of T8 are labeled and highlighted in black circles (see also Figures S2 and S3).

Figure 3

TacT Acetylates tRNA in Cell-Free Expression Assay (A) Cell-free expression assays leading to the production of the control protein DHFR (red asterisk) from template DNA without T8 (lanes 1 and 3) or with T8 (lanes 2 and 4) added from the onset of the assay. [¹⁴C]Ac-CoA was added to lanes 3 and 4. All samples were analyzed by SDS-PAGE. Production of DHFR was revealed by Coomassie staining (top panel) and acetylation by autoradiography (middle panel). tRNA molecules were detected by northern blotting using a radiolabeled probe specific of tRNA^Ala (lower panel). (B) Acetylation of tRNA molecules during cell-free expression was tracked by addition of [¹⁴C]Ac-CoA in all samples. Samples were only supplemented with template DNA to fire expression after 60 min of incubation (lanes 5 and 6) of all components of transcription and translation machineries without T8 (lanes 1, 3, and 5) or with T8 (lanes 2, 4, and 6) added to the samples from the onset of the assay. After 0, 60, or 120 min of incubation, production of DHFR was detected by SDS-PAGE followed by Coomassie staining (top panel). Concomitantly, extracted tRNA molecules were separated on acid-urea polyacrylamide gel and revealed by methylene blue staining (middle panel) followed by autoradiography (lower panel). Red asterisks mark the position of the DHFR in all panels containing Coomassie-stained gels (see also Figure S4).

Figure 4

Positive Patches at the Surface of TacT Dimers Are Essential for Toxicity (A) Electrostatic potential of the surface of the TacT dimer showing positive potential in blue, negative in red, and residues mentioned in the text labeled. Top panel shows the overall dimer structure (a 45° rotation about the x axis from Figure 2B). Bottom panel shows a walleye stereo image with a charged tRNA acceptor stem (yellow sticks) modeled in the active site. (B) Effect of amino acid substitutions on TacT toxicity. Growth curves of S. Typhimurium 12023 ΔtacAT expressing from pNDM220, the wild-type toxin (+TacT), or point mutant toxins, or carrying the empty vector (−TacT). All cultures were supplemented with IPTG in fresh rich medium during lag phase. Kinetics illustrate the effect of toxins bearing substitutions in the positively charged groove (top panel: R91E, K33E, K36E, R91E/K33E), or in the second positive patch of the second TacT monomer (lower panel: R77E, R78E, R158E, K146E). Data represent the mean ± SEM (n ≥ 3) and were analyzed using a Student’s t test (^∗∗∗p < 0.005) (see also Figure S5).

Figure 5

TacT Acetylates the Amino Acid Charged onto Salmonella tRNAs (A) Proposed model of action of TacT. TacT inhibits translation by acetylation of the primary amine group of amino acids charged onto tRNA molecules. The negatively charged tRNA molecule binds to TacT through its positive surface residues such that the primary amine group of the amino acid carried by the tRNA sits in the active site of the acetylase. Upon acetylation, the alpha amine group engaged with the acetyl moiety is blocked and cannot react for peptide bond formation with the carboxyl group of the amino acid loaded on the tRNA in the ribosomal P site. (B) tRNA molecules were extracted from S. Typhimurium 12023 ΔtacAT and preserved (lanes 1 and 2) or alkali treated in vitro (lane 3) before subsequent incubation with purified TacT (lanes 1 and 3) or purified TacT^Y140F (lane 2). (C) Acetylation of tRNA molecules extracted from Salmonella by purified TacT. tRNAs were extracted from S. Typhimurium 12023 ΔtacAT (lanes 1 and 2) or S. Typhimurium 12023 ΔtacAT carrying pNDM220::t8 (+TacT) (lanes 3 and 4), then incubated in vitro with purified TacT. All samples were supplemented with [¹⁴C]Ac-CoA. Treated tRNA molecules were separated on acid-urea polyacrylamide gel and revealed by methylene blue staining (top panels), and TacT-dependent acetylation was revealed by autoradiography (lower panels).

Figure 6

Pth Counteracts TacT Toxicity and TacT-Dependent Persister Formation (A) Growth curves of S. Typhimurium 12023 Δta8ΔcobB carrying pNDM220 (ΔcobB − TacT) or pNDM220::t8 (ΔcobB +TacT) in fresh rich medium. Expression of tacT was induced upon addition of IPTG during mid-exponential (left panel) or lag (right panel) phase. Growth curves from Figure 1A, right panel, were reported in gray for comparison purposes. Data represent the mean ± SEM (n ≥ 3) and were analyzed using a Student’s t test (^∗p < 0.05; ^∗∗p < 0.01). (B) Growth curves of S. Typhimurium 12023 ΔtacAT carrying pNDM220 (−TacT), pNDM220::t8 (+TacT), pBAD33::pth (+Pth), or pNDM220::t8 and pBAD33::pth (+TacT +Pth). All cultures were supplemented with arabinose and IPTG in fresh rich medium during lag phase. Data represent the mean ± SEM (n ≥ 3) and were analyzed using a Student’s t test (ns, non-significant). (C) Pth hydrolyses the ester bond between the nascent peptide and tRNA upon release of free peptidyl-tRNA molecules. Pth does not deacylate charged tRNA, but recognizes and cleaves acetylated aminoacyl-tRNA molecules as dipeptidyl-tRNA. (D) Exposure of tRNA molecules acetylated by TacT to Pth treatment in vitro. Cell-free expression assays leading to the production of the control protein DHFR were supplemented with TacT (lanes 3 and 4) from the onset of the assay, then tRNA molecules were extracted and TacT-dependent acetylation was assessed by autoradiography, before (lane 3) or after (lane 4) samples were treated with Pth (lower panel). (E) Exposure of tRNA molecules acetylated by TacT to Proteinase K treatment in vitro. tRNA molecules were extracted from S. Typhimurium 12023 ΔtacAT and incubated in vitro with purified TacT (lanes 2 and 3). Proteinase K was subsequently added to one sample (lane 3). (D and E) All samples were supplemented with [¹⁴C]Ac-CoA. Treated tRNA molecules were separated on acid-urea polyacrylamide gel and revealed by methylene blue staining (top panels), and TacT-dependent acetylation was revealed by autoradiography (lower panels). (F) Proportion of bacteria surviving 4 hr exposure to bactericidal concentrations of cefotaxime in cultures of S. Typhimurium 12023 ΔtacAT, ΔtacAT pNDM220::tacT, pNDM220::tacT and pBAD33::tacA, pNDM220::tacT and pBAD33::pth, or wild-type. Arabinose and IPTG were added to all cultures in fresh medium during lag phase, then antibiotic treatment started 1 hr later. Data represent the mean ± SEM (n ≥ 3) and were analyzed using a Student’s t test (ns, non-significant; ^∗∗∗p < 0.001) (see also Figures S6 and S7).

Figure 7

Model of TacT-Dependent Persister Formation and Resumption of Growth TacT, an acetyltransferase, inhibits translation by modification of the primary amine group of amino acids charged onto tRNA molecules. Upon acetylation (red arrow), the alpha amine group is blocked and cannot form a peptide bond with the nascent peptide, and bacteria stop growing. TacT activity on translation is counteracted by the action of Pth, which detoxifies the corrupted tRNAs (green arrow). When the effect of Pth is greater than that of TacT and detoxification is complete, translation resumes (blue arrow) and persisters regrow.