Ecological triggers to exploit antibiotic and virulence factor regulation

指导抗生素发现的生态触发因子和转录谱分析 ？摘要 该建议将基于微阵列的转录谱分析与生态相关的小分子结合起来， 诱导剂，以确定表达的抗生素和毒力因子基因簇的小分子发现的努力。 我们最近发现，昆虫循环液中的L-Pro诱导昆虫产生生物活性小分子， 光杆状菌属和黄杆状菌属的昆虫病原细菌。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菌属， 次级代谢潜力与许多链霉菌不同，我们对它们的 生态小生境，Photorhabdus和Xodorhabdus物种处于三边共生的中心， 线虫和昆虫，为实验室研究提供生态框架。细菌会持续存在 在环境中捕食昆虫幼虫的感染性幼（IJ）线虫的肠道中和平地存在。当一个 当蠕虫成功进入猎物的循环系统时，它会杀死细菌，然后细菌会产生一种 杀死幼虫的各种毒素，以及发出IJ蠕虫开始繁殖信号的小分子 成虫，对抗昆虫防御机制的小分子，以及保护猎物免受 竞争的细菌和真菌。通过对表达的抗生素和小分子毒力因子基因的计数， 集群使用昆虫调节代谢物刺激信号，我们将采用遗传学驱动的方法， 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，也可能导致新的生物合成 由于许多簇中含有不寻常的酶，因此这些簇的转化是不寻常的。为了验证基因驱动的方法， 在我们早期的代谢组学分析研究中，我们选择了一种被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。生化和定点遗传 突变研究将阐明这一现象与下游串联缩合结构域 这些生物合成研究将为生物合成途径提供基础， 调查其他与医学相关的结构多样化途径中的现象。的 微阵列研究代表了关键的培训机会，它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组学分析策略跟踪的聚类靶标。最后， 为了开始探索昆虫病原体中代谢物诱导的普遍性，我们将产生一个天然的粗品。 从大约200个细菌和真菌昆虫病原体与我们的代谢产物生长的产品库 诱导条件。因为昆虫病原体必须克服昆虫的先天免疫系统， 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， 和肺癌细胞的作用。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， 提供了一个发射台，调查他们的监管，生物合成和结构在一个独立的学术 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.