In-depth characterization of the metabolic effect of the bacterial alamorne ppGpp

细菌阿拉莫恩 ppGpp 代谢效应的深入表征

• 批准号: 10578783
• 负责人: Boyuan Wang
• 金额: $ 24.9万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10578783
• 关键词: Amino Acids; Antibiotic Therapy; Antibiotics; Attenuated; Bacteria; Binding; Binding Proteins; Binding Sites; Biochemical; Biochemical Pathway; Biological Assay; Biology; Biophysics; Cell Wall; Cell physiology; Cells; Chemistry; Data; Drug Design; Enzymes; Escherichia coli; Fatty Acids; Filtration; Gene Expression; Genetic Transcription; Glutamate Dehydrogenase; Growth; Guanosine; Guanosine Triphosphate Phosphohydrolases; Isocitrate Dehydrogenase; Libraries; Ligands; Mass Spectrum Analysis; Mediating; Metabolic; Metabolic Pathway; Metabolism; Metabolite Interaction; Methods; Molecular; Nucleotides; Organism; Pathway interactions; Pharmaceutical Preparations; Physiological; Point Mutation; Predisposition; Process; Production; Proteins; Proxy; RNA; RNA Binding; RNA Stability; Repression; Research; Ribosomes; Role; Second Messenger Systems; Signal Transduction; Starvation; Structure; Techniques; Testing; Transcript; Translations; Ultrafiltration; Validation; Work; alpha ketoglutarate; aptamer; candidate identification; computational pipelines; design; environmental stressor; genetic approach; genetic resistance; in vitro activity; in vivo; in vivo evaluation; innovation; insight; interest; metabolomics; novel; nuclease; programs; protein metabolite; response; small molecule; time use; transcriptome; transcriptome sequencing; translation factor

Project Summary Bacterial cells are extremely efficient in adapting to environmental stresses. As a prime example, they synthesize a second messenger called ppGpp in response to starvation. Accumulation of ppGpp in bacteria arrests growth and reprograms cellular physiology to promote survival. These effects of ppGpp is required for antibiotic persistence that allows bacteria to survive the treatment of antibiotics to which they do not encode genetic resistance. At molecular levels, ppGpp reprograms gene expression, and targets many other proteins involved in translation and small-molecule metabolism. As a preliminary study, I synthesized a photo-crosslinking probe of ppGpp and used this probe to capture and identify about 30 new ppGpp-binding proteins in E. coli. Recently, a ppGpp riboswitch has been discovered in Gram-positive organisms. Intriguingly, E. coli transcriptome has a rich secondary-structure landscape, but the capability of these secondary structures in binding to small-molecule metabolite has not been explored. Therefore, in Aim 1, I will use a photo-crosslinking approach to capture binding partners of ppGpp from E. coli transcriptome, and identify these transcripts using RNA-seq. Hit interactions will be validated biophysically, and their putative effects on translation efficiency or the transcript stability will be examined in vivo. Additionally, in my preliminary study, I also compared metabolite profiles in E. coli before and shortly after ppGpp induction, and found strong perturbation of dozens of essential metabolites. This perturbation of cellular metabolism goes much beyond known direct effects of ppGpp. It is unclear how ppGpp induction drives the inhibition of various metabolic pathways to effectively arrest bacterial growth and promote persistence. In Aim 2, I hypothesize that “ppGpp-sensitive” metabolites, i. e., those whose levels perturbed by ppGpp induction, serve as a proxy of ppGpp to regulate cellular metabolism. I will seek discovering protein-metabolite interactions responsible for this indirect effect of ppGpp. To this end, I will use time-resolved metabolomics to identify candidate metabolites and enzymes whose levels/activities perturbed contemporaneously with ppGpp accumulation. I will then screen for protein-metabolite interactions among these candidates using an ultrafiltration-based assay. Briefly, I will purify each protein of interest (POI) to high homogeneity. Then, I will subject a mixture containing a library of all candidate metabolites and a single POI to ultrafiltration. Analyzing the filtrate using MS should reveal a decrease of the POI’s cognate ligands. I will validate any novel interactions identified using biochemical, structural, and genetic approaches. Together, the proposed research will explore two new aspects of ppGpp signaling, namely RNA targeting and indirect effects on cellular metabolism via ppGpp-sensitive metabolites. This study may lead to the discovery of key regulatory interactions required for bacterial persistence, and these interactions may serve as targets for anti-persistence drug design.

项目摘要 细菌细胞在适应环境压力方面非常有效。作为一个最好的例子，他们合成了 另一种称为ppGpp的信使响应饥饿。PpGpp在细菌中的积累抑制生长 并对细胞生理学进行重新编程以促进生存。这些ppGpp的作用是抗生素所必需的 让细菌在抗生素的治疗中存活下来的持久性，而这些抗生素不是由它们的基因编码的 抵抗。在分子水平上，ppGpp重新编程基因表达，并以许多其他相关蛋白质为靶点 在翻译和小分子代谢方面。作为初步研究，我合成了一种光交联型探针 并利用该探针捕获和鉴定了约30个新的ppGpp结合蛋白。最近， 在革兰氏阳性菌中发现了ppGpp核糖开关。有趣的是，大肠杆菌转录组有一个 丰富的二级结构景观，但这些二级结构与小分子结合的能力 代谢物还没有被发现。因此，在目标1中，我将使用照片交联法来捕获绑定 从大肠杆菌转录组中获得ppGpp的配对，并用RNA-seq鉴定这些转录本。点击互动将 通过生物物理验证，它们对翻译效率或转录本稳定性的假定影响将是 活体检查。此外，在我的初步研究中，我还比较了之前和之前在大肠杆菌中的代谢物图谱 在ppGpp诱导后不久，发现了数十种必需代谢物的强烈扰动。这种微扰 细胞新陈代谢的影响远远超出已知的ppGpp的直接影响。目前尚不清楚ppGpp如何诱导 抑制各种代谢途径，有效阻止细菌生长，促进持久性。 在目标2中，我假设对ppGpp敏感的代谢物，即那些其水平受到ppGpp干扰的代谢物。 诱导，作为ppGpp的替代物来调节细胞代谢。我将寻求发现蛋白质代谢物 造成ppGpp这种间接影响的相互作用。为此，我将使用时间分辨代谢组学来 确定与ppGpp同时干扰其水平/活性的候选代谢物和酶 积累。然后，我将使用一个 基于超滤的化验。简而言之，我将提纯每个感兴趣的蛋白质(POI)，使其高度均一。那么，我会 将含有所有候选代谢物库和单一POI的混合物进行超滤。分析 使用MS的滤液应该显示POI的同源配体的减少。我将验证任何新的交互 使用生化、结构和遗传方法进行鉴定。总之，拟议的研究将探索 PpGpp信号的两个新方面，即RNA靶向和通过 PpGpp敏感代谢物。这项研究可能导致发现关键的监管相互作用 细菌持久性，这些相互作用可能成为抗持久性药物设计的靶点。