Novel whole-animal screens for anti-microbials

本项目的目标是建立秀丽隐杆线虫全动物高通量 在96孔板中进行筛选试验，以鉴定新型抗微生物或免疫- 增强化合物。先前的工作表明，许多人类病原体感染并杀死线虫 C.优雅提出的总体策略是确定治疗C的天然或合成化合物。elegans 持续性细菌感染我们预计，这项工作将建立解决一些问题的方法， 目前抗菌剂发现的主要障碍-发现新的化合物类别，解决 毒性/功效测试的瓶颈，以及将药物递送至目标病原体。 初步结果表明，C.线虫致病性模型可用于测定 具有抗菌活性的化合物。用于筛选抗感染或免疫的原型测定 开发了增强化合物，其利用了革兰氏阳性人 机会致病菌粪肠球菌（Enterococcus faecalis）在C.线虫肠的 试验用于筛选6000个小分子和1224个天然产物提取物，导致 鉴定了16种化合物和9种提取物在试验中具有活性。此外，MDR泵抑制剂 在C. elegans固化试验。 初步数据表明，线虫模型将产生在一个 并且线虫是体外筛选和 在哺乳动物中进行测试。提出的屏幕开发项目的一个主要目标是增加吞吐量 氏梭elegans-E.粪肠球菌全动物筛选试验，以允许筛选数十万或 数以百万计的化合物和提取物。梭elegans筛选方法也将扩展到包括 革兰氏阴性病原体肠道沙门氏菌，并适于能够鉴定多种药物 耐药（MDR）泵抑制剂。具体目标有三：1）开发高通量的大肠杆菌;粪 通过自动化蠕虫活力的评分来部分地治愈测定。2)开发高通量C.优雅的， S.肠固化试验3)建立一种高通量筛选方法，用于鉴定C. elegans固化测定。

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.