Salmonella Reprograms Nucleotide Metabolism in Its Adaptation to Nitrosative Stress

Abstract

The adaptations that protect pathogenic microorganisms against the cytotoxicity of nitric oxide (NO) engendered in the immune response are incompletely understood. We show here that salmonellae experiencing nitrosative stress suffer dramatic losses of the nucleoside triphosphates ATP, GTP, CTP, and UTP while simultaneously generating a massive burst of the alarmone nucleotide guanosine tetraphosphate. RelA proteins associated with ribosomes overwhelmingly synthesize guanosine tetraphosphate in response to NO as a feedback mechanism to transient branched-chain amino acid auxotrophies. Guanosine tetraphosphate activates the transcription of valine biosynthetic genes, thereby reestablishing branched-chain amino acid biosynthesis that enables the translation of the NO-consuming flavohemoglobin Hmp. Guanosine tetraphosphate synthesized by RelA protects salmonellae from the metabolic stress inflicted by reactive nitrogen species generated in the mammalian host response. This research illustrates the importance of nucleotide metabolism in the adaptation of salmonellae to the nutritional stress imposed by NO released in the innate host response.IMPORTANCE Nitric oxide triggers dramatic drops in nucleoside triphosphates, the building blocks that power DNA replication; RNA transcription; translation; cell division; and the biosynthesis of fatty acids, lipopolysaccharide, and peptidoglycan. Concomitantly, this diatomic gas stimulates a burst of guanosine tetraphosphate. Global changes in nucleotide metabolism may contribute to the potent bacteriostatic activity of nitric oxide. In addition to inhibiting numerous growth-dependent processes, guanosine tetraphosphate positively regulates the transcription of branched-chain amino acid biosynthesis genes, thereby facilitating the translation of antinitrosative defenses that mediate recovery from nitrosative stress.

Keywords: Salmonella; adaptive resistance; animal models; cellular redox status; guanosine tetraphosphate; nitric oxide; nitrosative stress; nucleotide metabolism; stringent response.

FIG 1

NO dramatically affects the nucleotide pools of salmonellae. (A) TLC autoradiogram of ³²P-labeled nucleotides extracted from log-phase salmonellae 5 min after exposure to increasing concentrations of spermine NONOate (sNO). (B) Nucleotides were examined 0 to 90 s after treatment with 750 μM sNO. (C) Relative abundance of ATP, GTP, and ppGpp in salmonellae treated with 750 μM sNO (see Fig. S1B for an example of the autoradiograms used in these analyses). n = 4. Data are shown as the mean value ± the standard error of the mean. (D) Model of (p)ppGpp metabolism. RelA synthesizes (p)ppGpp from ATP and GTP or GDP in response to unaminoacylated tRNAs erroneously loaded in the A site of the ribosome. SpoT can both synthesize and hydrolyze (p)ppGpp, whereas GppA hydrolyzes pppGpp to ppGpp. (E) Nucleotides from wild-type (wt) and ΔrelA mutant salmonellae treated for 5 min with 750 μM sNO. (F) Nucleotides in salmonellae treated with 70 μg/ml tetracycline for 3 min before the addition of 0.4 mg/ml serine hydroxamate (SHX) or 33.3 or 300 μM PAPA NONOate (pNO) for 2 min. All of the autoradiograms shown are representative of two to six independent experiments. (G) Nucleotides from salmonellae treated for 5 min with sNO were examined as described for panel A. Extracts in panels A, B, E, and F were separated with 1.25 M KH₂PO₄ buffer, pH 3.4. Specimens in panel G were separated with 0.9 M KH₂PO₄ buffer, pH 3.4. Ctrl, control.

FIG 2

RelA and branched-chain amino acids accelerate the recovery of NO-treated salmonellae. (A) Growth of wild-type (wt) and ΔrelA mutant salmonellae in MOPS glucose minimal medium (Gluc) in the presence or absence of 750 μM spermine NONOate (+sNO). Where indicated, all 20 amino acids (+AA) or branched-chain amino acids (+ILV) were added to the medium. n = 4 to 6. Data are shown as the mean value ± the standard error of the mean. (B) Growth of wild-type and ΔrelA mutant salmonellae challenged with 750 μM sNO in MOPS glucose minimal medium supplemented with branched-chain amino acids (+ILV), isoleucine (+I), leucine (+L), or valine (+V). n = 4. Data are shown as the mean value ± the standard error of the mean. (C) Intracellular concentrations of isoleucine, leucine, and valine were determined by LC-MS in wild-type and ΔrelA mutant salmonellae grown in M9 glucose minimal medium. Where indicated, 750 μM sNO was added to the cultures. n = 4. The data are represented as box-and-whiskers plots. *; P < 0.05 (determined by Mann-Whitney analysis). Ctrl, control.

FIG 3

RelA regulates the expression of branched-chain amino acid biosynthetic loci in salmonellae undergoing nitrosative stress. (A to D) Transcription of ilvB, ilvC, ilvD, and leuA mRNAs in wild-type (wt) and ΔrelA mutant salmonellae grown in MOPS glucose minimal medium was quantified by qRT-PCR. Bacterial cultures were treated with 750 μM spermine NONOate for the times indicated. The results were normalized to internal levels of the ampD housekeeping gene. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (determined by two-way ANOVA). n = 4. Data are shown as the mean value ± the standard error of the mean. (E to H) Western blot analysis of IlvB-3×FLAG, IlvC-3×FLAG, IlvD-3×FLAG, and LeuD-3×FLAG from the Salmonella strains indicated grown in MOPS glucose minimal medium after the addition of 750 μM sNO. Blots are representative of two or three independent experiments. (I) Transcription of ivbL (top) and ilvD (bottom) promoters by RNAP in reaction mixtures containing the concentrations of ppGpp indicated and/or 5 μM DksA. The results are representative of three independent experiments.

FIG 4

RelA aids the translation of the antinitrosative defense Hmp in salmonellae experiencing nitrosative stress. (A) Western blot analysis of Hmp-3×FLAG in wild-type (wt) or ΔrelA mutant salmonellae grown in MOPS glucose minimal medium (Gluc) after the addition of 750 μM spermine NONOate (sNO). Where indicated, the medium was supplemented with all 20 amino acids (+AA) or branched-chain amino acids (+ILV). Blots are representative of five independent experiments. (B) Transcription of hmpA mRNA in salmonellae grown in MOPS glucose minimal medium 60 min after treatment with 750 μM sNO. (C) Growth of wild-type and ΔhmpA mutant salmonellae in M9 glucose minimal medium. Selected samples were treated with 750 μM spermine NONOate (sNO). Where noted, the medium was supplemented with branched-chain amino acids (+ILV). (D) Growth of wild-type and ΔrelA mutant salmonellae in M9 glucose minimal medium. One hour after the addition of 750 μM sNO, bacteria were collected by centrifugation (down arrow) and the pellets were resuspended in either the same medium (up arrow) or fresh M9 medium lacking sNO. n = 4 to 6. Data are shown as the mean value ± the standard error of the mean. ctrl, control.

FIG 5

Importance of relA and ilvD in the pathogenesis of salmonellae. (A to C) C3H/HeN mice were infected p.o. with 5 × 10⁶ CFU of the Salmonella strains indicated. Murine survival was monitored for 28 days. The drinking water in panel B was treated with a 2.5% solution of the iNOS inhibitor aminoguanidine (AG). n = 5 to 10 mice. Log rank analysis results: wild type versus ΔrelA mutant, P < 0.01; wild type versus ΔilvD mutant, P < 0.01; wild type versus ΔlivD-F ΔbrnQ mutant, P = 0.141; wild type versus ΔrelA ΔlivD-F ΔbrnQ mutant, P < 0.001. No statistically significant difference between the wild type and the ΔrelA mutant in AG-treated C3H/HeN mice was found (P = 0.937).

FIG 6

Integrated model of the role of nucleotide metabolism and branched-chain amino acids in the adaptation of salmonellae to nitrosative stress. Early signaling events in response to nitrosative stress. NO inhibits respiration and glycolysis. The depletion of ATP that follows the NO-mediated inhibition of substrate level and oxidative phosphorylation leads to drops in GTP, CTP, and UTP. NO also inhibits valine biosynthesis. In addition, drops in valine stimulate the accumulation of unaminoacylated tRNA^Val, stimulating the synthesis of ppGpp by RelA associated with ribosomes. Depletion of NTPs and valine and synthesis of ppGpp inhibit translation and bacterial growth. Late adaptive responses by which ppGpp aids in the recovery of salmonellae from nitrosative stress are shown on the right. The ppGpp produced by RelA activates the transcription of branched-chain amino acid (BCAA) biosynthetic genes, restoring valine levels that are needed for the translation of the NO-consuming flavohemoprotein Hmp.