A toxin-antitoxin system provides phage defense via DNA damage and repair

Abstract

Widespread in bacteria and archaea, toxin-antitoxin (TA) systems have been recently demonstrated to function in phage defense. Here we characterize the anti-phage function of a type IV TA system, ShosTA. Using structural and biochemical approaches, we show that ShosT couples phosphoribosyltransferase and pyrophosphatase activities to disrupt purine metabolism, resulting in DNA duplication, cell filamentation and ultimate cell death. ShosA binds DNA and likely recruits other proteins to facilitate DNA homologous recombination to antagonize ShosT's toxicity. We identify Gp0.7 of T7 phage as a trigger for ShosTA system via shutting off the protein synthesis, and the C-terminus-mediated intrinsic instability of ShosA releases the toxicity of the existing ShosT proteins. Collectively, our results provide a novel toxin-antitoxin mechanism for anti-phage immunity and shed light on the triggering of this TA system.

Fig. 1. ShosTA system provides anti-phage defense via toxin-antitoxin mechanism.

a Schematic diagram of ShosTA system. The boundaries for each domain are labeled. b Plating efficiency of various phages infecting the E. coli BL21(DE3) strain containing empty vector or ShosTA system. Data represent plaque-forming units (PFUs) per milliliter of each phage infection. Bar graphs show the average of three replicates with individual data points overlaid. c Phage infection in liquid cultures of the E. coli BL21(DE3) strain containing ShosTA system. The strain containing empty vector is used as the control. Cells are infected at various MOI values. For each MOI, results of three experiments are presented as the average of three replicates with shaded areas indicating SD. Source data are provided as a Source Data file. d Representative plating assay showing ShosA rescue of ShosT toxicity. Plasmids harboring ShosT or ShosTA under arabinose-inducible promoters, respectively, are transformed into E. coli BL21-AI. The arabinose concentrations used for inducing are indicated.

Fig. 2. Structure and activities of ShosT.

a Crystal structure of ShosT. The N- and C-termini of ShosT are indicated. The central β-sheets of the two Rossmann-fold domains are colored orange. b Structure of ShosT hydrolase domain. The secondary structures are labeled. c Superimposition of ShosT hydrolase domain and Bacteroides thetaiotaomicron PPase. d PPase activity of purified ShosT by measuring the absorbance of PPi. Bar graphs represent the mean of four replicates with individual data points overlaid. **P = 0.0045, *P = 0.0286, calculated by two-tailed Student’s t test. Source data are provided as a Source Data file. e Structure of ShosT hydrolase domain complexed with phosphate. The details of the phosphate binding are shown in the enlarged part. The electron density map of phosphate (Fo-Fc omit map, contoured at 3 σ) is shown. f Structure of ShosT PRTase domain. The secondary structures are labeled. g Superimposition of ShosT PRTase domain and human APRT. h Superimposition of ShosT PRTase-PRPP (orange) and PRTase alone (blue). PRPP-binding loops are colored yellow and green in the PRTase-PRPP and PRTase alone structures, respectively. The details of the PRPP binding are shown in the enlarged part. The electron density map of PRPP and the bound Mg²⁺ (Fo-Fc omit map, contoured at 3 σ) is shown. i The enzymatic functions of PRTase and PPase. j Representative plating assay showing the cell toxicity of the indicated ShosT mutants. The arabinose concentrations used for inducing are indicated.

Fig. 3. Structure and activities of ShosA.

a Crystal structure of ShosA C-terminal truncation. The SAM and DprA domains are colored in light orange and orange. b Superimposition of ShosA and Streptococcus pneumoniae DprA. c Structure of ShosA SAM domain. d Structure of ShosA Rossmann-fold domain. e The electrostatic surface potential of ShosA. Blue and red (±5 kT/e) indicate the positively and negatively charged areas of the protein, respectively. f ShosA binds ssDNA in vitro. Electrophoretic mobility shift assay is performed using 5’ Cy3-labeled 54-nt ssDNA. Different concentrations of ShosA proteins (from 0 to 0.8 μM) were incubated with 0.1 μM DNA. Percent of the free DNA is calculated based on the gray scanning analysis. This experiment is repeated three times independently with similar results. g Representative plating assay showing the ShosT toxicity with indicated ShosA mutants. The arabinose concentrations used for inducing are indicated.

Fig. 4. The mechanisms for ShosT-mediated toxicity and ShosA-mediated anti-toxicity.

a Cell growth curves of E. coli BL21(DE3) strain expressing ShosT. Empty vector is used as the control. Results of three experiments are presented as the average of three replicates with shaded areas indicating SD. The time points of the drop of cell growth (blue) and cell collection (purple) are marked. Source data are provided as a Source Data file. b Diagram of the differential metabolite classification (KEGG). c Bubble map of KEGG pathway enrichment (up to top 5). d Fluorescence microscopy of E. coli BL21(DE3) cells expressing the whole system ShosTA, ShosT inactive mutant (D331A/D332A) or wild type ShosT. This experiment is repeated three times independently with similar results. Scale bars: 5 μm. e IP-MS/MS analysis reveals in vivo interactions between ShosA and some recombination-associated proteins. FLAG-tagged ShosA in the context of the full ShosTA system or FLAG-tag alone are used. Bar graphs represent the mean of three replicates with individual data points overlaid. f ShosA rescues E. coli BL21(DE3) cells from the toxicity of ccdB. Left, representative plating assay for indicated plasmids. Right, quantification of the left panel. Bar graphs represent the mean of four replicates with individual data points overlaid. Source data are provided as a Source Data file.

Fig. 5. Phage Gp0.7 triggers the immunity mediated by ShosTA.

a Schematic diagram of the co-expression assay for Gp0.7 and ShosTA. b Co-expression of Gp0.7 and ShosTA results in cell death. Growth curves for E. coli BL21(DE3) cells expressing indicated proteins are shown. Results of three experiments are presented as the average of three replicates with shaded areas indicating SD. c Representative plating assay shows that co-expression of Gp0.7 and ShosTA induces cell death. d The protein levels of ShosT and ShosA upon phage infection measured by western blot. RNAP is used as the loading control. This experiment is repeated three times independently with similar results. e Growth curves for E. coli BL21(DE3) cells treated with chloramphenicol (CAP) for 30 min. Results of three experiments are presented as the average of three replicates with shaded areas indicating SD. The presence of ShosTA results in significant cell death. f Model for the anti-phage mechanism of ShosTA system. Source data are provided as a Source Data file.