Natural product discovery and biosynthetic pathway elucidation from the human pathogen Legionella

项目总结： 天然产品在生产生物体中发挥着重要的生物作用，在历史上一直是一种重要的 药品的来源。微生物天然产物的研究主要集中在环境生物上； 天然产物，特别是由致病微生物产生的潜在小分子毒力因子， 更不用说理解了。军团菌感染可引起军团菌病，可表现为非肺炎 形式为庞蒂亚克热或急性肺炎形式，如军团病。退伍军人病病死率 约10%是由于并发症，而在医疗机构感染的人约为25%。虽然怎么了？ 军团菌影响寄主的蛋白效应因子已被深入研究，鉴定出次生菌 军团菌产生的代谢物以及这些天然产物在军团菌感染中所起的作用尚未得到充分研究。 这项拟议的工作将识别和表征人类潜在的生物活性天然产品。 病原体军团菌。用基因打靶的方法鉴定一种潜在的新型金属酶催化 不寻常的氧化重排产生N-亚硝基产物，其中一个同系物在 Drozanskii中保存的170个嗜肺军团菌亚种和另一个同源物是 被发现了。活性分析表明，该同系物催化类似的反应，但使用不同的底物。 生物信息学分析确定了生物合成基因簇(BGC)编码的基因邻域 抗性酶和前药物活化酶，可能表明潜在生物活性的生物合成 代谢物。 目的1从军团菌生物合成基因簇中鉴定天然产物和生物活性 发现潜在的致命因素。目标2将启动两种酶的特性和机理研究 在BGC中涉及到潜在药效团的生物合成，便于理解和铺垫 这类新型的药效团产生酶对的抑制剂设计方法。成功 完成这些目标将确定潜在的生物活性天然产物的化学结构 人类病原体军团菌。此外，涉及潜在药效团生物合成的两个关键酶 也将被描述为。对这些天然产物及其生物合成的鉴定可能会揭示新的 可针对军团菌感染的毒力途径。最终，整个工作流程将是 泛指研究来自人类的其他新的天然产物或潜在的毒性因子 病原体军团菌通过扩大新的细胞毒素类别来对抗新出现的传染病 候选药物/毒力因子和2)来自生物合成途径的新的抑制靶点。

Project Summary: Natural products play essential biological roles in producing organisms and have been a historically important source of medicines. Microbial natural products research has focused largely on environmental organisms; natural products, especially the potential small molecule virulent factor produced by pathogenic microbes, are much less understood. Legionella infection causes Legionellosis, which can be present in its non-pneumonic form as Pontiac fever or acute pneumonic form as Legionnaires’ disease. The fatality rate of legionnaires disease is about 10% due to complications and about 25% for those infected in the healthcare facility. While how the protein effectors of Legionella affect the host have been intensively studied, identifying the secondary metabolites Legionella produce and the roles these natural products play in Legionella infection are understudied. The proposed work will identify and characterize the potential bioactive nature products from the human pathogen Legionella. Using a gene-targeting approach to identify a potential novel metalloenzyme that catalyzes unusual oxidative rearrangement to generate the N-nitroso product, one homolog conserved in over 170 Legionella pneumophila subspecies and another homolog conserved in Legionella drozanskii was discovered. Activity assay shows this homolog catalyzes similar reactions but utilizes different substrates. Bioinformatic analysis of the gene neighborhood identifies the biosynthetic gene cluster (BGC) encodes resistance enzyme and a prodrug activating enzyme, possibly indicating the biosynthesis of potential bioactive metabolites. Aim 1 will identify the natural product and bioactivity from the biosynthetic gene cluster from Legionella to uncover the potential virulent factor. Aim 2 will initiate the characterization and mechanistic study of two enzymes in the BGC involving the biosynthesis of the potential pharmacophore to facilitate the understanding and pave the way for the inhibitor design of this new class of pharmacophore-producing enzyme pair. Successful completion of these aims will identify the chemical structure of the potential bioactive natural products from the human pathogen Legionella. In addition, two key enzymes involving the potential pharmacophore biosynthesis will also be characterized. Identification of those natural products and their biosynthesis will possibly reveal new virulence pathways that can be targeted to combat Legionella infection. Ultimately, the overall workflow will be generalized to investigate other novel natural products or potential virulent factors from the human pathogen Legionella to fight the emerging infectious disease by expanding the pool of 1) new classes of cytotoxic drug candidates/virulent factors and 2) new inhibition targets from the biosynthetic pathway.