Ecological triggers to exploit antibiotic and virulence factor regulation

指导抗生素发现的生态触发因素和转录图谱 ？摘要 该方案将基于微阵列的转录图谱与生态相关的小分子相结合 用于小分子发现的识别表达的抗生素和毒力因子基因簇的诱导剂。 我们最近发现，昆虫循环液中的L-Pro可以诱导小分子生物活性物质的产生。 光杆菌属和纹杆菌属的昆虫病原菌。这些革兰氏阴性菌 伽玛变形杆菌与链霉菌竞争，链霉菌是研究最多的抗生素生产属，在它们的 次生代谢势。与许多链霉菌不同，我们对链霉菌的了解很少 生态位、光杆菌属和拟杆菌属物种处于三边共生的中心， 为实验室研究提供生态框架的线虫和昆虫。这种细菌持续存在 在感染幼虫(IJ)的肠道中和平地捕食环境中的昆虫幼虫。当一个 蠕虫成功地进入猎物的循环系统，它反胃细菌，然后产生一种 杀死幼虫的各种毒素，小分子是IJ蠕虫繁殖的信号 成虫，对抗昆虫防御机制的小分子，以及保护猎物免受 相互竞争的细菌和真菌。通过统计表达的抗生素和小分子毒力因子基因 使用昆虫调节代谢物刺激信号的集群，我们将使用遗传学驱动的方法来 鉴定编码的生物活性产物，用于基于核磁共振的结构鉴定。这一方法将立即 将新的代谢物连接到相应的基因簇，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。为了验证基因驱动的方法， 在我们早期的代谢组学研究中，我们选择了一种受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多肽合成酶(NRPS)酶学的一个不同寻常的方面- 腺化结构域杂乱作为支架多样性的管道。生化和定点遗传 突变研究将阐明这种现象与下游串联缩合结构域有关 这可能会在生物合成的道路上提供一个新的分叉。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。这个 微阵列研究代表着关键的培训机会，肯定会带来新的抗生素基因 将使用类似的遗传和差异代谢组谱策略跟踪的集群目标。最后， 为了开始探索在昆虫病原体中诱导代谢物的共性，我们将产生一个粗略的天然 与我们的代谢物一起生长的大约200种细菌和真菌昆虫病原的产品库 诱导条件。因为昆虫病原体必须克服昆虫的先天免疫系统，这是 与目前的抗癌靶点相同的特征，我们将筛选一系列细胞系，包括白血病，乳腺， 和肺癌细胞，除了抗癌靶标吲哚胺2，3-双加氧酶。总而言之，这些研究 将阐明昆虫病原体是否可以成为生物医学小分子和 为研究它们的调控、生物合成和结构提供了一个独立的学术平台 研究计划。

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.