Ecological triggers to exploit antibiotic and virulence factor regulation

利用抗生素和毒力因子调节的生态触发因素

基本信息

批准号：
8643795
负责人：
Jason Michael Crawford
金额：
$ 23.7万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-01 至 2015-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8643795
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

项目摘要

Ecological triggers and transcriptional profiling to guide antibiotic discovery ¿ 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.

生态触发和转录分析以引导抗生素发现概括该提案结合了基于微阵列的转录分析与生态相关的小分子诱导者以鉴定表达的抗生素和病毒因子基因簇进行小分子发现工作。我们最近发现，昆虫电流液中的L-Pro诱导生物活性小分子的产生昆虫的致病细菌和Xenorhabdus属的昆虫病菌。这些革兰氏阴性 γ-杆菌链霉菌链霉菌是最研究的抗生素生产属，就其而言继发代谢潜力。与许多链霉菌物种不同，我们对它们的了解很少生态壁ches，Photorhabdus和Xenorhabdus物种是三边形共生的中心线虫和昆虫为实验室研究提供了生态框架。细菌持续存在在环境中狩猎昆虫幼虫的幼年（IJ）线虫的肠道中和平地。当a 蠕虫在进入其猎物电路系统方面取得了成功，它反射了细菌，然后产生杀死幼虫的各种毒素，这些毒素的小分子，这些分子信号为ij蠕虫繁殖成人，反昆虫防御机制的小分子和保护猎物免受的抗生素竞争细菌和真菌。通过TallyS表达的抗生素和小分子病毒因子基因使用绝缘调节代谢物刺激信号的集群，我们将采用一种遗传学驱动的方法确定用于基于NMR的结构阐明的编码生物活性产品。该方法将立即将新代谢产物连接到其相应的基因簇，也可能导致新型生物合成转换，许多簇具有异常的酶。为了验证遗传学驱动的方法，我们在早期的代谢组学研究中选择了由L-Pro上调的代谢产物。这导致了系列新的生物活性化合物和非核糖体肽合成酶（NRPS）酶学的异常方面 - 腺苷酸化域的滥交是脚手架多样性的渠道。生化和定向通用突变研究将阐明与下游串联凝结域有关的这种现象这可能会在生物合成路径中提供新颖的叉子。这些生物合成研究将为研究其他与医学相关的结构多样化途径中的现象。代表关键培训机会的微阵列研究肯定会导致新的抗生素基因将使用类似的遗传和差异代谢分析策略跟踪的聚类目标。最后，为了开始探测昆虫病原体中代谢物诱导的普遍性，我们将产生一种天然的原油我们的代谢产物生长的大约200种细菌和真菌昆虫病的产品库诱导条件。因为昆虫病原体必须克服昆虫的先天免疫系统，这与当前的抗癌目标共享特征，我们将筛选一系列细胞系，包括白血病，乳房，除抗癌靶标2,3-二氧酶外，肺癌细胞和肺癌细胞。总而言之会阐明昆虫病原体是否可以成为生物医学小分子和提供一个发射台，以调查其监管，生物合成和在独立学术中的结构研究计划。