Ecological triggers and transcriptional profiling to guide antibiotic discovery

指导抗生素发现的生态触发因素和转录谱

• 批准号: 8091077
• 负责人: Jason Michael Crawford
• 金额: $ 9万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8091077
• 关键词: Adolescent; Adult; Anabolism; Antibiotics; Antineoplastic Agents; Bacteria; Biochemical; Bioinformatics; Biological; Biological Assay; Biological Factors; Blood; Breast; Cardiovascular system; Cell Line; Defense Mechanisms; Educational workshop; Environment; Enzymatic Biochemistry; Enzymes; Gammaproteobacteria; Gene Cluster; Gene Mutation; Genes; Genetic; Goals; Hemolymph; Human; Immune system; Inflammatory Response; Insecta; Investigation; Knock-out; Laboratories; Larva; Lead; Libraries; Light; Liquid substance; Logic; Malignant Neoplasms; Malignant neoplasm of lung; Mentors; Metabolic; Methods; Microbe; Modeling; Molecular Target; Nematoda; Outcome; Pathogenesis; Pathway interactions; Phase; Photorhabdus; Physical condensation; Physiology; Point Mutation; Production; Regulation; Research; Series; Signal Transduction; Site; Source; Streptomyces; Structural Chemistry; Structure; Sum; Symbiosis; System; Systems Biology; Techniques; Therapeutic; Toxin; Training; Tryptophan 2,3 Dioxygenase; Validation; Virulence Factors; Xenorhabdus; Xenorhabdus luminescens; Xenorhabdus nematophilus; base; cancer cell; epimerization; fungus; insight; killings; leukemia; meetings; metabolomics; microbial; novel; pathogen; pathogenic bacteria; peptide synthase; programs; scaffold; small molecule; symposium

DESCRIPTION (provided by applicant): Ecological triggers and transcriptional profiling to guide antibiotic discovery 7 Summary This proposal combines microarray-based transcriptional profiling with ecologically relevant small molecule inducers to identify expressed antibiotic and virulence factor gene clusters for small molecule discovery efforts. We recently discovered that L-Pro in insect circulatory fluid induces bioactive small molecule production in insect pathogenic bacteria of the Photorhabdus and Xenorhabdus genera. These Gram-negative Gammaproteobacteria rival Streptomyces, the most studied antibiotic producing genus, in terms of their secondary metabolic potential. Unlike many Streptomyces species, in which we know very little about their ecological niches, Photorhabdus and Xenorhabdus species are at the center of a trilateral symbiosis with nematodes and insects that provides an ecological framework for laboratory investigation. The bacteria persist peacefully in the guts of infective juvenile (IJ) nematodes that hunt insect larvae in the environment. When a worm succeeds in entering its prey's circulatory system, it regurgitates the bacteria, which then produce an assortment of toxins that kill the larva, small molecules that signal for the IJ worms to become reproducing adults, small molecules that counter insect defense mechanisms, and antibiotics to protect their prey from competing bacteria and fungi. By tallying expressed antibiotic and small molecule virulence factor gene clusters using insect regulatory metabolite stimulation signals, we will employ a genetics-driven approach to identify the encoded bioactive products for NMR-based structure elucidation. The approach will immediately connect the new metabolites to their corresponding gene clusters and will also likely lead to novel biosynthetic transformations, as many of the clusters harbor unusual enzymes. To validate the genetics-driven approach, we selected a metabolite up-regulated by L-Pro in our earlier metabolomic profiling studies. This led to a series of new bioactive compounds and an unusual facet of non-ribosomal peptide synthetase (NRPS) enzymology - adenylation domain promiscuity as a conduit for scaffold diversity. Biochemical and site-directed genetic mutation studies will illuminate this phenomenon in connection to downstream tandem condensation domains that may provide a novel fork in the biosynthetic path. These biosynthetic studies will provide the basis for investigating the phenomenon in other medically relevant pathways for structural diversification. The microarray studies, which represent the key training opportunity, will certainly lead to new antibiotic gene cluster targets that will be tracked using similar genetic and differential metabolomic profiling strategies. Finally, to begin probing the generality of metabolite induction in insect pathogens, we will produce a crude natural product library from approximately 200 bacterial and fungal entomopathogens grown with our metabolite inducing conditions. Because insect pathogens must overcome the insect's innate immune system, which shares features with current anticancer targets, we will screen a series of cell lines, including leukemia, breast, and lung cancer cells, in addition to the anticancer target indoleamine 2,3-dioxygenase. In sum, these studies will shed light on whether insect pathogens could be a revitalized source of biomedical small molecules and provide a launch pad for investigating their regulation, biosynthesis, and structure in an independent academic research program. PUBLIC HEALTH RELEVANCE: Insect-pathogenic microbes harbor many as yet unidentified and unusual biosynthetic pathways that encode an assortment of antibiotics to successfully compete against other microbes in their ecological niche and to overcome the insect's innate immune system, which shares many similarities with current anticancer molecular targets. By using bacterial expression analysis on two model insect pathogens and by mimicking the host's physiology, we will not only uncover host recognition signals in bacterial pathogenesis, but also will target the small molecule biosynthetic gene clusters to discover their encoded bioactive products that hold promising biomedical potential. An anticancer screen will be conducted with natural product libraries generated from a panel of bacterial and fungal entomopathogens using metabolite-inducing conditions to identify new anticancer compounds and to begin exploring the generality of insect pathogens as a revitalized source of bioactive small molecules.

描述（由申请人提供）：生态触发和转录谱分析以指导抗生素发现7概述该提议将基于微阵列的转录谱分析与生态相关的小分子诱导剂相结合，以鉴定表达的抗生素和毒力因子基因簇，用于小分子发现工作。我们最近发现，昆虫循环液中的L-脯氨酸诱导发光杆菌属和黄杆菌属的昆虫病原细菌产生生物活性小分子。这些革兰氏阴性γ-变形菌的竞争对手链霉菌，研究最多的抗生素生产属，在他们的次级代谢潜力。与许多链霉菌物种不同，我们对它们的生态位知之甚少，Photorhabdus和Xtophabdus物种处于与线虫和昆虫的三边共生的中心，为实验室研究提供了生态框架。细菌和平地存在于环境中捕食昆虫幼虫的感染性幼（IJ）线虫的肠道中。当蠕虫成功进入其猎物的循环系统时，它会使细菌繁殖，然后产生各种毒素杀死幼虫，小分子信号使IJ蠕虫成为繁殖成虫，小分子对抗昆虫防御机制，以及抗生素保护猎物免受竞争细菌和真菌的侵害。通过使用昆虫调节代谢物刺激信号对表达的抗生素和小分子毒力因子基因簇进行计数，我们将采用遗传学驱动的方法来鉴定编码的生物活性产物，用于基于NMR的结构解析。该方法将立即将新的代谢产物与其相应的基因簇连接起来，并且还可能导致新的生物合成转化，因为许多簇含有不寻常的酶。为了验证遗传学驱动的方法，我们选择了在我们早期的代谢组学分析研究中由L-Pro上调的代谢物。这导致了一系列新的生物活性化合物和非核糖体肽合成酶（NRPS）酶学的一个不寻常的方面-腺苷酸化结构域混杂作为支架多样性的管道。生物化学和定点基因突变研究将阐明这种现象与下游串联缩合结构域的联系，这些结构域可能在生物合成途径中提供一个新的分叉，这些生物合成研究将为研究其他医学相关途径中的结构多样化现象提供基础。微阵列研究代表了关键的培训机会，肯定会导致新的抗生素基因簇靶点，这些靶点将使用类似的遗传和差异代谢组学分析策略进行跟踪。最后，为了开始探索昆虫病原体中代谢物诱导的一般性，我们将从在我们的代谢物诱导条件下生长的大约200种细菌和真菌昆虫病原体产生粗天然产物文库。由于昆虫病原体必须克服昆虫的先天免疫系统，该系统与当前的抗癌靶点具有相同的特征，因此除了抗癌靶点吲哚胺2，3-双加氧酶外，我们还将筛选一系列细胞系，包括白血病，乳腺癌和肺癌细胞。总之，这些研究将揭示昆虫病原体是否可以成为生物医学小分子的复兴来源，并为在独立的学术研究计划中研究其调控，生物合成和结构提供一个发射台。 公共卫生相关性：昆虫病原微生物具有许多尚未鉴定和不寻常的生物合成途径，这些途径编码各种抗生素，以成功地与其生态位中的其他微生物竞争，并克服昆虫的先天免疫系统，该系统与当前的抗癌分子靶点有许多相似之处。通过对两种模式昆虫病原体的细菌表达分析，并通过模拟宿主的生理，我们不仅可以揭示细菌致病过程中的宿主识别信号，而且还可以靶向小分子生物合成基因簇，以发现其编码的具有生物医学潜力的生物活性产物。将使用诱导代谢物的条件，用从一组细菌和真菌昆虫病原体产生的天然产物文库进行抗癌筛选，以鉴定新的抗癌化合物，并开始探索昆虫病原体作为生物活性小分子的再生来源的普遍性。