SalmonellaReprograms 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. 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.

The alarmone (p)ppGpp is part of the heat shock response ofBacillus subtilis

“…Interestingly, accumulation of (p)ppGpp upon heat or oxidative stress and its importance for stress resistance has also been reported in other Firmicutes and also Proteobacteria that differ widely in terms of (p)ppGpp signaling [56–58,74,75]. Accumulation of (p)ppGpp was shown to protect cells from salt or osmotic stress [76, 77].…”

“…1E, 2A-D). Activation of the SR by Rel-mediated (p)ppGpp synthesis during heat and oxidative stress has been reported for diverse taxa, suggesting that the underlying mechanisms could be conserved [56–58]. However, little is known about the mechanism of Rel activation upon environmental stress.…”

“…1F, S1E). Therefore, the underlying activation mechanisms during environmental stress likely shares some similarities to the well-studied SR-activation upon amino acid deprivation and may also involve the sensing of uncharged tRNA on the ribosome [17,18,56,58].…”

“…Proteotoxic and oxidative stress results in the inactivation of labile enzymes and may thereby impair uptake or biosynthesis of certain amino acids and/or modulate the activity of aminoacyl-tRNA synthetases, resulting in an accumulation of uncharged tRNA which can serve as a signal to activate Rel [58, 60]. In addition, tRNAs and proteins of the translational machinery are prone to oxidation or modification upon stress, leading to translation stalling, which can also elicit the SR [61].…”

“…Accumulation of (p)ppGpp was shown to protect cells from salt or osmotic stress [76, 77]. Conversely, the lack of (p)ppGpp is known to renders cells sensitive to heat or oxidative stress [58,78,79], suggesting that activation of the SR, allowing the fast down regulation of translation, is an important and conserved part of the response to environmental stress in bacteria. It is interesting to note that SR was also implicated in B. subtilis competence development, facilitating a cellular state (also referred to as the K-state) where cells cease to divide, and most transcription and translation is strongly down-regulated.…”