Chemical biology of type IV secretion systems

项目总结 细菌已经进化出专门的纳米机器，可以在细胞膜上运送微生物货物。一 多功能易位装置，IV型分泌系统(T4SS)，可以战略性地部署以注射 将大分子底物转化为目标细菌或真核细胞。尽管它们在细菌中很重要 抗生素耐药决定因素的发病和传播，T4SS的机制 组装和运输有效载荷在很大程度上仍未确定。为了解决这一知识差距，长期目标是 这项建议的目的是开发和应用强大的分子工具来加速T4SS的基础研究 纳米机器。幽门螺杆菌的CAG T4SS已成为一种重要的系统 以了解单个分子机器如何将不同的货物运送到目标细胞。而有些人 T4SS有能力将数百种蛋白质或DNA-蛋白质复合体分泌到宿主细胞中，这种能力 转移不同的脂类、核酸、蛋白质和多糖底物，区别于 来自其他系统的CAG T4SS。值得注意的是，细菌癌蛋白CagA被迅速输送到宿主胃细胞 通过CAG T4SS机制。幽门螺杆菌效应分子易位激活先天防御和调节失调 影响胃病进展的信号通路；因此，CAG T4SS- 幽门螺杆菌阳性会显著增加患胃癌的风险。由于它在细菌中的中心作用 在致病机制方面，T4SS是抗菌药物的理想靶点。在本应用程序中，我们建议标识 并对新型的基于小分子的T4SS调制器进行了力学表征。迭代结构--活动 关系研究将被用来开发具有最佳抗毒力的化学支架和药效团。 潜力。探针开发将利用Shaffer实验室的专业知识和分析平台，并将 利用提议的CPRI计算核心中的协同资源(配体结合模型开发， 合理设计，虚拟筛选)，CPRI翻译核心(高通量分析支持，新化合物 药物库和ADMET分析)，以及有机合成核心(药物化学和放大)。 优先和有效的化学探针将与生化和遗传方法结合使用 探讨CAG T4SS在底物转运过程中的调控和动态步骤。使用类似的多学科 在这种方法中，我们将确定H.Pylori CAG T4SS设备如何在细菌-宿主细胞界面组装。 总体而言，这些研究将激发新的基础研究方向，并将为如何 T4SS纳米机器将特定的分子货物运送到目标细胞，以驱动微生物 发病机制。此外，这项工作将产生功能强大的化学工具，广泛适用于 传染病研究，并将确定具有解除T4SS功能潜力的有效先导化合物 在各种与医学相关的病原体中。

PROJECT SUMMARY Bacteria have evolved specialized nanomachines to deliver microbial cargo across the cell envelope. One versatile translocation apparatus, the type IV secretion system (T4SS), can be strategically deployed to inject macromolecular substrates into target bacterial or eukaryotic recipient cells. Despite their importance in bacterial pathogenesis and dissemination of antibiotic resistance determinants, the mechanisms by which the T4SS assembles and transports payload remain largely undefined. To address this knowledge gap, the long-term goal of this proposal is to develop and apply robust molecular tools to accelerate fundamental studies of T4SS nanomachines. The cag T4SS of the gastric bacterium Helicobacter pylori has emerged as an important system for understanding how a single molecular machine can transport diverse cargo into target cells. Whereas some T4SS have the capacity to secrete hundreds of proteins or DNA-protein complexes into the host cell, the ability to translocate a diverse repertoire of lipid, nucleic acid, protein, and polysaccharide substrates distinguishes the cag T4SS from other systems. Notably, the bacterial oncoprotein CagA is rapidly delivered to host gastric cells via cag T4SS mechanisms. Translocated H. pylori effector molecules activate innate defenses and dysregulate signaling pathways that influence progression of gastric disease; consequently, colonization by cag T4SS- positive H. pylori significantly augments the risk for gastric cancer. As a result of its central role in bacterial pathogenesis, the T4SS represents an ideal target for antimicrobials. In this application, we propose to identify and mechanistically characterize novel small molecule-based T4SS modulators. Iterative structure-activity relationship studies will be used to develop chemical scaffolds and pharmacophores with optimized anti-virulence potential. Probe development will take advantage of expertise and assay platforms in the Shaffer lab and will leverage synergistic resources in the proposed CPRI Computational Core (ligand-binding model development, rational design, virtual screening), CPRI Translational Core (high throughput assay support, novel compound repositories, and ADMET profiling), and the Organic Synthesis Core (medicinal chemistry and scale-up). Prioritized and validated chemical probes will be used in conjunction with biochemical and genetic approaches to interrogate cag T4SS regulation and dynamic steps in substrate translocation. Using a similar multidisciplinary approach, we will determine how the H. pylori cag T4SS apparatus assembles at the bacteria-host cell interface. Collectively, these studies will stimulate new basic research directions and will provide important insight into how the T4SS nanomachine orchestrates the delivery of specific molecular cargo to target cells to drive microbial pathogenesis. Furthermore, this work will generate powerful chemical tools that are broadly applicable to infectious disease research, and will identify potent lead compounds with the potential to disarm T4SS function in a variety of medically-relevant pathogens.