Ecological determinants of natural product synthesis in cave myxobacteria

洞穴粘细菌天然产物合成的生态决定因素

• 批准号: 9811794
• 负责人: Caleb Nathaniel Fischer
• 金额: $ 6.16万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9811794
• 关键词: Actinobacteria class; Antibiotics; Apoptosis; Benchmarking; Biological; Biology; Burkholderia; Cell Line; Cell physiology; Cells; Cellular Assay; Chemicals; Column Chromatography; Cues; DNA Damage; Data; Dictyostelium; Ecosystem; Escherichia coli; Evaluation; Family; Fluorescent Antibody Technique; Foundations; Future; Gene Cluster; Genome; Genomics; Goals; Human; Incubated; Laboratories; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Metabolism; Methods; Molecular; Myxococcales; Myxococcus xanthus; Natural Products; Neoplasm Metastasis; Organism; Pathway interactions; Predatory Behavior; Production; Protein-Serine-Threonine Kinases; Proteobacteria; Regulation; Research; Research Proposals; Role; Source; Stimulus; Streptomyces; Structure; Techniques; Testing; Therapeutic; Variant; base; cancer cell; cancer therapy; cellular targeting; comparative; competitive environment; drug discovery; emerging antibiotic resistance; genome sequencing; insight; ion mobility; metabolome; metabolomics; microbial; microorganism; novel; pressure; response; social; tandem mass spectrometry

SUMMARY/ABSTRACT Natural products (NPs) isolated from diverse sources stock the majority of our nation's biomedical arsenal. Due to emerging antibiotic resistance and limited cancer treatments, additional NP discoveries are of fundamental importance. Microbial genomics has unveiled `gifted' microorganisms—those capable of synthesizing many structurally diverse NPs. However, most gifted microorganisms do not synthesize NPs under laboratory conditions. Understanding elicitors that activate NP synthesis would enable the discovery of new and potentially therapeutic NPs. In select gifted microorganisms (e.g. actinobacteria), chemical and biological elicitors modulate NP production; in others (e.g. myxobacteria), the effects of chemical and biological stimuli are unknown. Three lines of evidence support a role for chemical and biological stimuli in myxobacterial NP regulation. First, myxobacteria rely on NPs for predation. Second, myxobacterial genomes are enriched for regulatory networks, such as serine-threonine protein kinases, which detect and respond to external stimuli. Third, preliminary data described herein is consistent with our hypothesis. Chemical (the sub-lethal antibiotic norfloxacin) and biological (the prey E. coli and the predator Dictyostelium descoideum AX-2) stimuli modulate known and putative novel natural products in Myxococcus xanthus DK1622. For these reasons, we hypothesize that biological and chemical stimuli modulate myxobacterial NP synthesis. The proposed project assesses the consequence of competitive stimuli on myxobacterial secondary metabolism, evaluates their bioactivity against a cancer line, and elucidates the structure of myxobacterial NPs. In Aim 1, the consequences of chemical and biological stimuli on myxobacterial secondary metabolism will be further investigated. Ecological stimuli (bi-partite competitor cultures, tripartite predatory/prey cultures, sub-lethal antibiotics) will be presented to myxobacteria (domesticated and cave-isolated) and the resultant metabolome will be evaluated by mass spectrometry. Select features will be targeted for isolation based on divergence from known myxobacterial NPs (via molecular networking and ion mobility analyses). Structural elucidation will be accomplished via column chromatography, tandem mass spectrometry, and NMR. In Aim 2, myxobacterial metabolomes and metabolites will be assessed for their role in mammalian chemical biology using multiplexed activity metabolomics (MAM), a high-throughput technique that enables the simultaneous evaluation of a large number of metabolites against an array of cell subtypes and cellular pathways. MAM incubates fractionated metabolites against a cancer-derived cell line and assays them against 8-15 fluorescent antibodies targeting cellular function (e.g. apoptosis, DNA damage, and viability). Cytometric analysis and correlation of the resulting spectral and bioactivity chromatograms prioritizes leads to test against primary human cancer cells and to structurally decipher (Aim 1). !

总结/摘要 从不同来源分离的天然产物（NPs）储存了我国大部分的生物医学产品。 阿森纳官方由于新出现的抗生素耐药性和有限的癌症治疗，更多的NP发现是 至关重要。微生物基因组学揭示了“天才”微生物-那些能够 合成许多结构多样的NP。然而，大多数天才微生物不合成NP 在实验室条件下。了解激活NP合成的激发子将有助于发现 新的和潜在的治疗NP。 在选择有天赋的微生物（例如放线菌）中，化学和生物诱导子调节NP 在另一些（例如粘细菌）中，化学和生物刺激的影响是未知的。三 一系列证据支持化学和生物刺激在粘细菌NP调节中的作用。第一、 粘细菌依靠NP进行捕食。其次，粘细菌基因组丰富了调控网络， 例如丝氨酸-苏氨酸蛋白激酶，其检测并响应外部刺激。三、初步数据 这里所描述的与我们的假设一致。化学药物（亚致死抗生素诺氟沙星）和 生物（猎物E.大肠杆菌和捕食者Dictyosteeletum descoideum AX-2）刺激调节已知的和 黄色粘球菌DK 1622中推定的新天然产物。基于这些原因，我们假设， 生物和化学刺激调节粘细菌NP合成。拟议项目评估了 结果的竞争性刺激对粘细菌的次级代谢，评估其生物活性， 癌细胞系，并阐明了粘细菌纳米颗粒的结构。 在目标1中，化学和生物刺激对粘细菌次级代谢的影响 将进一步调查。生态刺激（二分竞争者文化，三分掠夺/猎物文化， 亚致死性抗生素）将呈现给粘细菌（驯化和洞穴隔离）， 将通过质谱法评价代谢物组。将根据以下条件选择要隔离的功能 与已知粘细菌NP的差异（通过分子网络和离子迁移率分析）。结构 通过柱色谱法、串联质谱法和NMR来完成解析。 在目标2中，将评估粘细菌代谢物组和代谢物在哺乳动物中的作用， 化学生物学使用多重活性代谢组学（MAM），一种高通量技术，使 同时评价大量代谢物对一系列细胞亚型和细胞毒性的影响， 途径。MAM将分级代谢物与癌源性细胞系孵育，并将其与 8-15靶向细胞功能（例如凋亡、DNA损伤和活力）的荧光抗体。细胞术 对所得光谱和生物活性色谱图进行分析和关联， 原代人类癌细胞和结构破译（目标1）。 !