Ecological triggers and transcriptional profiling to guide antibiotic discovery

概述：本提案将基于微阵列的转录谱分析与生态学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。近年来，我们发现昆虫循环液中的L-Pro可诱导光habduus属和xenorhabduus属昆虫病原菌产生具有生物活性的小分子。这些革兰氏阴性γ变形菌与链霉菌竞争，链霉菌是研究最多的抗生素生产属，就其次级代谢潜力而言。与许多我们对其生态位知之甚少的链霉菌物种不同，光habdus和Xenorhabdus物种是与线虫和昆虫三方共生的中心，为实验室研究提供了生态框架。这种细菌在环境中捕食昆虫幼虫的感染性幼线虫（IJ）的肠道中和平地生存。当蠕虫成功进入猎物的循环系统时，它会反刍细菌，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，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来统计表达的抗生素和小分子毒力因子基因簇，我们将采用遗传学驱动的方法来识别编码的生物活性产物，用于基于核磁共振的结构解析。该方法将立即将新的代谢物与其相应的基因簇连接起来，并且也可能导致新的生物合成转化，因为许多簇含有不寻常的酶。为了验证遗传驱动的方法，我们在早期的代谢组学分析研究中选择了一种由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）酶学的一个不同寻常的方面-腺苷酸化结构域混杂作为支架多样性的管道。生物化学和位点导向基因突变研究将阐明这种与下游串联冷凝结构域有关的现象，这可能为生物合成途径提供新的分叉。这些生物合成研究将为研究结构多样化的其他医学相关途径中的现象提供基础。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目标，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普遍性，我们将从大约200种细菌和真菌昆虫病原体中产生一个粗略的天然产物库，这些细菌和真菌在我们的代谢物诱导条件下生长。由于昆虫病原体必须克服昆虫的先天免疫系统，这与目前的抗癌靶点有共同的特点，我们将筛选一系列细胞系，包括白血病、乳腺癌和肺癌细胞，以及抗癌靶点吲哚胺2,3-双加氧酶。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再生来源，并为在独立的学术研究项目中研究它们的调控、生物合成和结构提供一个跳板。

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.