Innate immunity-based screen for bacterial type III secretion system inhibitors

描述（由申请人提供）：III型分泌系统（T3SS）是数十种病原体（包括沙门氏菌、EPEC、志贺氏菌和耶尔森氏菌）致病所需的细菌附属物。这些病原体每年总共造成2亿多人患病，50多万人死亡。然而，这些和其他细菌病原体正在以惊人的速度对目前可用的抗生素产生耐药性。由于自1962年以来只有两类新的抗生素进入市场，因此迫切需要新的治疗方法来支持后代的现代医学。T3SS在许多细菌物种中是保守的，但由病原体特异性表达。这使得T3SS成为抗微生物剂的最佳靶标，所述抗微生物剂可以阻碍表达T3SS的病原体的发病机制，而不会对微生物群造成大的破坏。虽然T3SS抑制剂已被确定，但很少有已知的分子靶点，并且没有一个已经进入临床。T3SS的功能是将细菌效应蛋白注射到靶宿主细胞内。一旦进入 在宿主细胞中，这些效应蛋白操纵正常的宿主细胞过程，以利于病原体。我们已经设计了一种新的策略，用于识别可以破坏T3SS与宿主细胞功能性相互作用的能力的小分子抑制剂。该试验依赖于哺乳动物细胞响应功能性T3 SS激活宿主转录因子NF κ B的能力，使宿主细胞作为小分子是否可以抑制T3 SS功能的指示剂。在目标1中，我们将利用这种NF κ B激活试验开发高通量筛选（HTS）来鉴定T3 SS抑制剂，使用假结核耶尔森菌作为模型T3 SS表达病原体。为了实现这一点，我们将产生一个稳定转染的NF κ B荧光素酶报告细胞系，可以支持一个强大的和可重复的HTS。此外，我们将开发一种快速的二次筛选，以消除抑制NF κ B通路的化合物，使我们能够集中精力研究特异性靶向T3 SS的化合物。在目标2中，我们将开发和审查一个实验管道，（i）将用于验证先导化合物的T3SS抑制活性，（ii）将允许快速鉴定先导化合物靶向的III型分泌阶段，即，T3SS组装、宿主膜上的T3SS孔形成、效应蛋白在宿主细胞内的T3SS易位和T3SS效应蛋白在宿主细胞内的活性 宿主细胞完成这项工作后，我们将使用我们内部的天然产物和合成化合物库（超过55，000种化合物）进行本提案中描述的HTS筛选和实验管道。此外，我们将提交我们的屏幕快速通道进入NIH路线图分子图书馆探针生产中心网络。这将能够鉴定一套靶向III型分泌的不同阶段的T3SS抑制剂，这将作为未来开发T3SS抑制剂的生化工具箱的基础，该工具箱具有已知的分子靶标，将用作新疗法的研究探针和支架。

DESCRIPTION (provided by applicant): The type III secretion system (T3SS) is a bacterial appendage required by dozens of pathogens to cause disease, including Salmonella, EPEC, Shigella, and Yersinia. Collectively, these pathogens cause over 200 million cases of human illness and well over half a million deaths per year. Yet these and other bacterial pathogens are developing resistance to currently available antibiotics at an alarming rate. As only two new classes of antibiotics have reached the market since 1962, new therapeutics are critically needed to support modern medicine for future generations. The T3SS is well-conserved across a large number of bacterial species yet is specifically expressed by pathogens. This makes the T3SS an optimal target for antimicrobial agents that can hinder the pathogenesis of T3SS-expressing pathogens without causing large disruptions to the microbiota. While T3SS inhibitors have been identified, few have known molecular targets and none have reached the clinic. The function of the T3SS is to inject bacterial effector proteins inside target host cells. Once inside host cells, these effector proteins manipulate normal host cell processes, to the benefit of the pathogen. We have devised a novel strategy for identifying small molecule inhibitors that can disrupt the ability of the T3SS to functionally interact with host cells. This assay relies on the ability of mammalian cells to activate the host transcription factor NFκB in response to a functional T3SS, letting host cells serve as an indicator of whether a small molecule can inhibit T3SS function. In Aim 1, we will exploit this NFκB activation assay to develop a high throughput screen (HTS) to identify T3SS inhibitors, using Yersinia pseudotuberculosis as a model T3SS-expressing pathogen. To achieve this, we will generate a stably-transfected NFκB luciferase reporter cell line that can support a robust and reproducible HTS. In addition, we will develop a rapid secondary screen to eliminate compounds that inhibit the NFκB pathway, allowing us to focus our efforts on compounds that specifically target the T3SS. In Aim 2, we will develop and vet an experimental pipeline that (i) will serve to validate the T3SS inhibitory activity of lead compounds and (ii) will allow rapid identification of the stage of type III secretion targeted by lead compounds,-i.e., T3SS assembly, T3SS pore formation on host membranes, T3SS translocation of effector proteins inside host cells, and activity of T3SS effector proteins inside host cells. Upon completion of this work, we will carry out the HTS screen and experimental pipeline described in this proposal, using our in-house natural products and synthetic compound libraries (over 55,000 compounds). In addition, we will submit our screen for Fast Track entry into the NIH Roadmap Molecular Libraries Probe Production Centers Network. This will enable identification of a suite of T3SS inhibitors that target different stages of type III secretion, whch will serve as the foundation for future development of a biochemical toolbox of T3SS inhibitors with known molecular targets that will be used as research probes and scaffolds for novel therapeutics.