Prophage terminase with tRNase activity sensitizes Salmonella enterica to oxidative stress

Abstract

Phage viruses shape the evolution and virulence of their bacterial hosts. The Salmonella enterica genome encodes several stress-inducible prophages. The Gifsy-1 prophage terminase protein, whose canonical function is to process phage DNA for packaging in the virus head, unexpectedly acts as a transfer ribonuclease (tRNase) under oxidative stress, cleaving the anticodon loop of tRNA^Leu. The ensuing RNA fragmentation compromises bacterial translation, intracellular survival, and recovery from oxidative stress in the vertebrate host. S. enterica adapts to this transfer RNA (tRNA) fragmentation by transcribing the RNA repair Rtc system. The counterintuitive translational arrest provided by tRNA cleavage may subvert prophage mobilization and give the host an opportunity for repair as a way of maintaining bacterial genome integrity and ultimately survival in animals.

Fig. 1.. Transcriptional adaptations to the repression of de novo translation in S. enterica undergoing oxidative stress.

(A and D) Immunoblots with Ponceau S–stained controls and (B and E) a corresponding densitometric analysis examining puromycin incorporation by S. enterica 30 min after H₂O₂ treatment. Densitometry was normalized against Ponceau S–stained lanes with ImageJ. Data are shown as mean ± SD (n = 3 independent observations); ****P ≤ 0.0001 as determined by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test. (C) Killing of S. enterica by H₂O₂ in PBS after 30 min of treatment. Data are shown as mean ± SD (n = 4); ****P ≤ 0.0001 and ***P ≤ 0.001 as determined by one-way ANOVA with Dunnet’s multiple comparison test. (F) Differentially expressed genes in S. enterica after 30 min of 400 μM H₂O₂ treatment as assessed by RNA-seq (30). Each circle represents the average log₂-fold change of four biological replicates. Genes with a log₂-fold change >0.8 or <−0.8 are depicted on the top and bottom, respectively. Orange and green boxes represent pathogenicity islands, and the yellow box represents S. enterica plasmid genes.

Fig. 2.. Repair of tRNA by the RNA repair Rtc system protects S. enterica from the phagocyte NADPH oxidase.

Densitometry (A and B) of the puromycin⁺ proteome in WT and rtc mutants. Specimens in (B) were treated with 400 μM H₂O₂. Data are shown as mean ± SD (n = 4). (C) tRNA fragmentation was visualized by Northern blotting in S. enterica after 5 mM H₂O₂ treatment. (D) Densitometry of tRNA fragments 2 hours after H₂O₂ treatment, corresponding to data in (C). Data are shown as mean ± SD (n = 3). (E) Position of tRNA^LeuPQTV cleavage in H₂O₂-treated S. enterica as assessed by sequencing and 3′ RACE. (F) Killing of S. enterica after 2 hours of treatment with 400 μM H₂O₂ in phosphate-buffered saline (PBS). Data are shown as mean ± SD (n = 10). (G) Intracellular survival of S. enterica in bone marrow–derived macrophages from C57BL/6 (B6) and Cybb^−/− mice. Data are shown as mean ± SD (n = 6). (H) Competitive index of S. enterica in livers and spleens of C57BL/6 (B6) and Cybb^−/− mice 3 days after i.p. inoculation with equal numbers of WT and ΔrtcBA S. enterica (n = 6 to 7). (I) Histopathology of paraffin-embedded, hematoxylin and eosin (H&E)–stained liver tissues isolated 3 days after infection. Scale bars, 50 μm. The panel on the right shows the average number of microabscesses and necrotic foci per 200X field of liver tissue stained with H&E (n = 6 to 7). *P ≤ 0.05, **P ≤ 0.01, ***P < 0.001, and ****P < 0.0001 as determined by Student’s t test [(A), (D), and (F)], Mann-Whitney test (G), one-way ANOVA with Dunnet’s multiple comparison test (B), or two-way ANOVA (H).

Fig. 3.. The SOS response activates prophage-dependent tRNA cleavage in S. enterica undergoing oxidative stress.

(A) Differentially expressed genes (DEGs) in WT S. enterica after treatment with 400 μM H₂O₂. DEGs were identified by DESeq2 and edgeR with tagwise dispersion and FDR-corrected P values. (B, D, and E) tRNA^LeuPQTV fragments were visualized by Northern blotting in log-phase S. enterica after treatment with 5 mM H₂O₂. Densitometric quantification of 5′ /intact tRNA is presented below each lane. (C and F) rtcA transcription in log-phase S. enterica 1 hour after treatment with 400 μM H₂O₂ or PBS. Cycle threshold values were normalized to the rpoD housekeeping gene. Data are shown as mean ± SD [(C), n = 4; (F), n = 3]. **P < 0.01 as determined by unpaired Mann-Whitney test. Transcription in (F) is expressed relative to WT controls. (G) Densitometry of the de novo translated proteome as assessed by puromycin incorporation in the indicated S. enterica strains grown in MOPS-GLC media and treated with 400 μM H₂O₂ for 2 hours. Data are shown as mean ± SD (n = 3); *P ≤ 0.05 as determined by Student’s t-test. (H) Antimicrobial activity of H₂O₂ on S. enterica 2 hours after treatment. Data are shown as mean ± SD (n = 6); ****P ≤ 0.0001 as determined by one-way ANOVA with Dunnet’s multiple comparison test. (I) Competitive index of S. enterica in livers and spleens of C57BL/6 (B6) and Cybb^−/− mice 3 days after i.p. inoculation of equal numbers of WT and Gifsy-1–deficient S. enterica (n = 5 to 8). ***P < 0.001 as determined by Mann-Whitney test.

Fig. 4.. Gifsy-1 terminase cleaves tRNA in S. enterica during oxidative stress.

(A) Genomic organization of Gifsy-1 region in S. enterica 14028s. (B, D, and E) tRNA^LeuPQTV fragments were visualized by Northern blotting in S. enterica grown to log phase in LB broth and treated with 5 mM H₂O₂. Strains in (E) expressing pBAD or pBAD-gpA were treated with 500 μM H₂O₂. (C) Phylogenetic tree of full-length S. enterica GpA and known ribonucleases. (F and I) Total RNA extracted from log-phase S. enterica was treated with recombinant GpA proteins. Recombinant GpA variants in (I), and where indicated in (F), were treated with H₂O₂. tRNA^LeuPQTV fragments were visualized with Northern blot. Densitometric quantification of 5′ fragment and intact tRNA is presented below each lane. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; E, Glu; G, Gly; H, His; K, Lys; R, Arg. In the mutants, other amino acids were substituted at certain locations; for example, H432A indicates that histidine at position 432 was replaced with alanine. (G) Site of cleavage of tRNA^LeuPQTV by H₂O₂-treated recombinant GpA protein was determined by 3′ RACE and sequencing. (H) AlphaFold representation of the C-terminal nuclease domain of GpA. Walker A motif is in green, whereas α helices and β sheets of the predicted nuclease site are in pink and cyan, respectively. Residues mutated in (I) are shown in red. (J) Survival of S. enterica grown overnight in LB broth and treated for 2 hours with 400 μM H₂O₂ in PBS. Data are shown as the mean ± SD (n = 16 to 26). (K) Competitive index of S. enterica in livers of C57BL/6 (B6) and Cybb^−/− mice 3 days after i.p. inoculation of equal numbers of WT and ΔgpA S. enterica (n = 5 to 7). **P < 0.01 and ****P ≤ 0.0001 as determined by Mann-Whitney test [(J) and (K)].