High-Throughput assays for imaging human intracellular pathogen infections

人类细胞内病原体感染成像的高通量检测

• 批准号: 7628558
• 负责人: HERVE F AGAISSE
• 金额: $ 36.53万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7628558
• 关键词: Affect; Antibiotics; Bacterial Infections; Biological Assay; Biological Models; Biological Process; Candidate Disease Gene; Cell Line; Cell physiology; Cells; Cellular biology; Chemicals; Computer-Assisted Image Analysis; Cytoskeleton; Defect; Development; Drosophila genus; Epithelial Cells; Fluorescence Microscopy; Gene Targeting; Genetic Screening; Genomics; Growth; Health; Hela Cells; Human; Image; Imaging Techniques; In Vitro; Infection; Integration Host Factors; Investigation; Left; Libraries; Listeria; Listeria monocytogenes; Listeriosis; Mammalian Cell; Mediating; Modeling; Pharmaceutical Preparations; Phenotype; Process; Proteomics; RNA Interference; Reproducibility; Research Personnel; Resistance; Screening procedure; Signal Transduction; Specificity; Staging; Testing; Toxic effect; Vacuole; base; chemical genetics; comparative; design; deviant; drug discovery; effective therapy; genome-wide; high throughput screening; killings; multicatalytic endopeptidase complex; novel strategies; pathogen; prevent; programs; protein degradation; success; trafficking

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.

描述（由申请人提供）：细胞内细菌病原体对人类健康和抗生素药物构成严重威胁，主要用于预防和/或治愈细菌感染。这些化合物主要是通过靶向细菌机械来起作用，不幸的是，在此过程中选择了病原体中的耐药性病例，使我们无法对这些偏差的病原菌株进行有效治疗。药物治疗的这些严肃方面表明，不仅需要开发新药物来抵抗耐药性，还需要采用新的概念方法来识别新的治疗方法。在此应用中，我们建议通过开发高吞吐量筛选（HTS）测定来开发一种新的药物发现方法，以成像哺乳动物细胞中细胞内病原体感染。为此，我们使用单核细胞增生李斯特菌，胞质细菌病原体和HeLa细胞（人类上皮细胞系）建模感染过程。我们首先建议通过自动荧光显微镜和计算机辅助图像分析来开发和优化用于成像和量化哺乳动物细胞中李斯特菌感染的HTS分析。我们将通过筛选耶鲁大学基因组学和蛋白质组学中心可用的20,000种化合物的库来测试测定的可重复性和鲁棒性。接下来，为了确定已确定的候选者的特异性程度，我们建议开发次要测定，以测试细胞毒性，细菌杀伤和感染后其他病原体的潜在活性。我们还将开发二级HTS分析，以进一步表征李斯特菌特异性候选者，并精确确定李斯特菌感染的哪个步骤受相应候选者的活性影响。最后，我们建议利用优化的原始和次要测定，并进行平行的化学和遗传筛选，以鉴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，以鉴定生物活性分子及其相应的靶基因。 我们认为，在各种方法（例如化学和遗传筛查）中使用拟议的测定方法将对所研究的生物学过程，细胞内病原体及其宿主细胞的相互作用提供全面的理解。除了它们的生物医学含义之外，这些研究还可以进一步了解我们对靶向宿主因素的天然细胞功能的理解。