Novel whole-animal screens for anti-microbials

本课题的目标是发展秀丽线虫全动物高产养殖。 在96孔板上的筛选试验导致鉴定新类别的抗菌剂或免疫制剂- 增强化合物。以前的研究表明，许多人类病原体感染并杀死线虫。 线虫。拟议的总体战略是确定治愈线虫的天然或合成化合物。 一种持续的细菌感染。我们预计，这项工作将建立解决一些问题的方法 目前抗菌药物发现的主要障碍--发现新的化合物类别，解决 毒性/疗效测试的瓶颈，并将药物输送到目标病原体。 初步结果表明，线虫致病模型可以用于检测 具有抗菌活性的化合物。一种用于抗感染或免疫筛选的原型试验 增强化合物的开发利用了革兰氏阳性人类 条件致病菌粪肠球菌在线虫的肠道中形成持续感染。这个 对6000个小分子和1224个天然产物提取物进行了筛选，得到了 对16个化合物和9个提取物进行了鉴定。此外，MDR泵抑制剂 在线虫治愈试验中显示出与抗菌剂的协同作用。 初步数据表明，线虫模型将产生被忽略的命中 传统的体外筛选，线虫是体外筛选和体外筛选之间有用的中间产物 在哺乳动物身上进行试验。拟议的屏幕开发项目的一个主要目标是增加吞吐量 线虫-E。粪便全动物筛查试验，允许筛查数十万或 数以百万计的化合物和提取物。线虫筛查方法也将扩大到包括 革兰氏阴性肠炎沙门氏菌，适用于多种药物的鉴定 耐药(MDR)泵抑制剂。具体目标有三个：1)发展高产粪肠球菌 治愈化验部分是通过自动计分蠕虫的生存能力。2)开发高通量线虫-- 肠杆菌治愈试验。3)建立一种高通量筛查方法，用于鉴定C。 优雅的治愈化验。

The goal of the propoposed project is to develop Caenorhabditis elegans whole-animal high-throughput screening assays in 96-well plates that lead to the identification of novel classes of antimicrobial or immune- enhancing compounds. Previous work has shown that many human pathogens infect and kill the nematode C. elegans. The proposed overall strategy is to identify natural or synthetic compounds that cure C. elegans of a persistent bacterial infection. We anticipate that this work will establish approaches that solve some of the main obstacles in current antimicrobial discovery - finding newclasses of compounds, solving the bottleneck of toxicity/efficacy testing, and delivering the drug to the target pathogen. Preliminary results demonstrate that the C. elegans pathogenicity model can be used to assay compounds with antimicrobial activity. A prototype assay for screening for anti-infective or immune enhancing compounds was developed that takes advantage of the fact that the Gram positive human opportunistic pathogen, Enterococcus faecalis, forms a persistent infection in the C. elegans intestine. The assay was used to screen 6000 small molecules and 1224 natural product extracts, leading to the identification of 16 compounds and 9 extracts that were active in the assay. In addition MDR pump inhibitors were shown to act in synergy with antimicrobials in the C. elegans curing assay. The preliminary data indicate that a nematode model will produce hits that are overlooked in a conventional in vitro screen and that the nematode is a useful intermediate between an in vitro screen and testing in mammals. A major goal of the proposed screen development project is to increase the throughput of the C. elegans-E. faecalis whole-animal screening assay to allow screening of hundreds of thousands or millions of compounds and extracts. The C. elegans screening methodology will also be extended to include the Gram negative pathogen Salmonella enterica and adapted to enable the identification of multi-drug resistant (MDR) pump inhibitors. There are three specific aims: 1) Develop a high throughput E. faecalis curing assay in part by automating the scoring of worm viability. 2) Develop a high throughput C. elegans - S. enterica curing assay. 3) Develop a high throughput screen for identifying MDR prump inhibitors in the C. elegans curing assays.