Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of Salmonella pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In Salmonella, these nucleotide alarmones are generated by the synthetase activities of RelA and SpoT proteins. In addition, the (p)ppGpp hydrolase activity of the bifunctional SpoT protein is essential to preserve cell viability. The contribution of SpoT to physiology and pathogenesis has proven elusive in organisms such as Salmonella, because the hydrolytic activity of this RelA and SpoT homologue (RSH) is vital to prevent inhibitory effects of (p)ppGpp produced by a functional RelA. Here, we describe the biochemical and functional characterization of a spoT-Δctd mutant Salmonella strain encoding a SpoT protein that lacks the C-terminal regulatory elements collectively referred to as "ctd." Salmonella expressing the spoT-Δctd variant hydrolyzes (p)ppGpp with similar kinetics to those of wild-type bacteria, but it is defective at synthesizing (p)ppGpp in response to acidic pH. Salmonella spoT-Δctd mutants have virtually normal adaptations to nutritional, nitrosative, and oxidative stresses, but poorly induce metal cation uptake systems and Salmonella pathogenicity island 2 (SPI-2) genes in response to the acidic pH of the phagosome. Importantly, spoT-Δctd mutant Salmonella replicates poorly intracellularly and is attenuated in a murine model of acute salmonellosis. Collectively, these investigations indicate that (p)ppGpp synthesized by SpoT serves a unique function in the adaptation of Salmonella to the intracellular environment of host phagocytes that cannot be compensated by the presence of a functional RelA.IMPORTANCE Pathogenic bacteria experience nutritional challenges during colonization and infection of mammalian hosts. Binding of the alarmone nucleotide guanosine tetraphosphate (ppGpp) to RNA polymerase coordinates metabolic adaptations and virulence gene transcription, increasing the fitness of diverse Gram-positive and Gram-negative bacteria as well as that of actinomycetes. Gammaproteobacteria such as Salmonella synthesize ppGpp by the combined activities of the closely related RelA and SpoT synthetases. Due to its profound inhibitory effects on growth, ppGpp must be removed; in Salmonella, this process is catalyzed by the vital hydrolytic activity of the bifunctional SpoT protein. Because SpoT hydrolase activity is essential in cells expressing a functional RelA, we have a very limited understanding of unique roles these two synthetases may assume during interactions of bacterial pathogens with their hosts. We describe here a SpoT truncation mutant that lacks ppGpp synthetase activity and all C-terminal regulatory domains but retains excellent hydrolase activity. Our studies of this mutant reveal that SpoT uniquely senses the acidification of phagosomes, inducing virulence programs that increase Salmonella fitness in an acute model of infection. Our investigations indicate that the coexistence of RelA/SpoT homologues in a bacterial cell is driven by the need to mount a stringent response to a myriad of physiological and host-specific signatures.

中文翻译：

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Spot 在吞噬体中诱导细胞内沙门氏菌毒力程序。

四磷酸鸟苷 (ppGpp) 和五磷酸鸟苷 (pppGpp) 统称为 (p)ppGpp，调节沙门氏菌发病机制的各个方面，包括营养物质的合成、对炎症介质的抗性和分泌系统的表达。在沙门氏菌中，这些核苷酸警报素是由 RelA 和 SpoT 蛋白的合成酶活性产生的。此外，双功能 Spot 蛋白的 (p)ppGpp 水解酶活性对于保持细胞活力至关重要。SpoT 对生理学和发病机制的贡献在沙门氏菌等生物体中已被证明是难以捉摸的，因为这种 RelA 和 SpoT 同源物 (RSH) 的水解活性对于防止功能性 RelA 产生的 (p)ppGpp 的抑制作用至关重要。这里，我们描述了 spoT-Δctd 突变沙门氏菌菌株的生化和功能表征，该菌株编码缺乏统称为“ctd”的 C 端调节元件的 SpoT 蛋白。表达 spoT-Δctd 变体的沙门氏菌水解 (p)ppGpp 具有与野生型细菌相似的动力学，但它在合成 (p)ppGpp 以响应酸性 pH 值时存在缺陷。沙门氏菌 spoT-Δctd 突变体对营养、亚硝化和氧化应激具有几乎正常的适应能力，但对金属阳离子摄取系统和沙门氏菌致病性岛 2 (SPI-2) 基因的诱导能力较差，以响应吞噬体的酸性 pH 值。重要的是，spoT-Δctd 突变体沙门氏菌在细胞内复制很差，并且在急性沙门氏菌病的小鼠模型中减弱。集体，这些研究表明，由 SpoT 合成的 (p)ppGpp 在沙门氏菌适应宿主吞噬细胞的细胞内环境方面具有独特的功能，而功能性 RelA 的存在无法弥补这一功能。重要性病原菌在定植和感染哺乳动物宿主。警报酮核苷酸四磷酸鸟苷 (ppGpp) 与 RNA 聚合酶的结合可协调代谢适应和毒力基因转录，增加各种革兰氏阳性和革兰氏阴性细菌以及放线菌的适应性。Gammaproteobacteria（如沙门氏菌）通过密切相关的 RelA 和 SpoT 合成酶的联合活性合成 ppGpp。由于其对生长的深刻抑制作用，必须去除 ppGpp；在沙门氏菌中，该过程由双功能 Spot 蛋白的重要水解活性催化。因为 Spot 水解酶活性在表达功能性 RelA 的细胞中是必不可少的，所以我们对这两种合成酶在细菌病原体与其宿主相互作用期间可能承担的独特作用的理解非常有限。我们在这里描述了一个 Spot 截断突变体，它缺乏 ppGpp 合成酶活性和所有 C 末端调节结构域，但保留了出色的水解酶活性。我们对这种突变体的研究表明，SpoT 能够独特地感知吞噬体的酸化，从而诱导在急性感染模型中增加沙门氏菌适应性的毒力程序。