High-Throughput assays for imaging human intracellular pathogen infections

描述（由申请人提供）：细胞内细菌病原体对人类健康构成严重威胁，抗生素药物主要用于预防和/或治疗细菌感染。这些化合物主要通过靶向细菌机制起作用，不幸的是，在这一过程中，正在选择病原体中的耐药病例，使我们无法有效治疗这些异常致病菌株。药物治疗的这些严重问题表明，不仅需要开发新的药物来对抗耐药性，而且需要开发新的概念方法来确定新的治疗方法。在这个应用中，我们建议通过开发高通量筛选（HTS）方法来成像哺乳动物细胞内病原体感染，从而开发一种新的药物发现方法。为此，我们使用单核增生李斯特菌（一种胞质细菌病原体）和HeLa细胞（一种人类上皮细胞系）来模拟感染过程。我们首先提出开发和优化HTS方法，通过自动荧光显微镜和计算机辅助图像分析来成像和量化哺乳动物细胞中的李斯特菌感染。我们将通过筛选耶鲁基因组学和蛋白质组学中心提供的20,000个化合物库来测试我们分析的可重复性和稳健性。接下来，为了确定确定的候选物的特异性程度，我们建议开发二级分析来测试细胞毒性，细菌杀灭和感染后对其他病原体的潜在活性。我们还将开发二次HTS分析，以进一步表征李斯特菌特异性候选物，并精确确定李斯特菌感染的哪一步受到相应候选物活性的影响。最后，我们建议利用优化后的一级和二级分析，并进行平行的化学和遗传筛选，以确定影响李斯特菌感染过程的细菌和宿主因素。我们的最终目标是对化学和遗传筛选中观察到的表型进行比较分析，以潜在地识别生物活性分子及其相应的靶基因。

DESCRIPTION (provided by applicant): Intracellular bacterial pathogens constitute a serious threat to human health and antibiotic drugs are largely used to prevent and/or cure bacterial infection. These chemical compounds mainly act by targeting the bacterial machinery and, unfortunately, cases of resistance among pathogens are being selected in the process, leaving us with no effective treatment against these deviant pathogenic strains. These serious aspects of drug treatment point to the necessity of developing not only new drugs to counter resistance, but also new conceptual approaches in order to identify new treatments. In this application, we propose to develop a new approach to drug discovery by developing high throughput screening (HTS) assays for imaging intracellular pathogen infection in mammalian cells. To this end, we model the infection process using Listeria monocytogenes, a cytosolic bacterial pathogen and HeLa cells, a human epithelial cell line. We first propose to develop and optimize an HTS assay for imaging and quantifying Listeria infection in mammalian cells by automated fluorescence microscopy and computer assisted image analysis. We will test the reproducibility and robustness of our assay by screening a library of 20,000 compounds available at the Yale Center for Genomics and Proteomics. Next, in order to determine the degree of specificity of the identified candidates, we propose to develop secondary assays to test for cell toxicity, bacterial killing and potential activity on other pathogens upon infection. We will also develop secondary HTS assays to further characterize Listeria-specific candidates and precisely determine which step of Listeria infection is affected by the activity of the corresponding candidates. Finally, we propose to utilize the optimized primary and secondary assays and conduct parallel chemical and genetic screens to identify bacterial and host factors affecting the Listeria infection process. Our ultimate objective is to conduct comparative analyses of the phenotypes observed in chemical and genetic screens to potentially identify bioactive molecules and their corresponding target genes. We believe that the use of the proposed assays in various approaches, such as chemical and genetic screens, will provide a comprehensive understanding of the biological process under investigation, the interaction of an intracellular pathogen and its host cell. Beyond their biomedical implications, these studies may also further our understanding of the native cellular functions of the targeted host factors.