Elucidating the mechanisms of protein secretion across the outer membrane by bacterial autotransporters

项目摘要/摘要 致病性革兰氏阴性细菌每年造成500多万人死亡。一批 物种也表现出高度的抗生素耐药性，占世界上12个成员中的9个 卫生组织的优先病原体名单。革兰氏阴性细菌感染通常由所谓的 自体转运蛋白，一类穿过外膜进入细胞外空间的蛋白质，它们在那里起作用 作为毒力因子，如粘附素、蛋白酶等有害物质。自动传输器由一个 转位器域，它保留在膜上，以及乘客域，它分泌在整个 膜的另一侧，即使不使用化学能，例如，三磷酸腺苷。目标是这些自动转运体 因为抑制是一种很有希望的方法，可以控制感染，同时限制耐药性的发展。 然而，首先，研究自动转运蛋白折叠、分泌和表达的分子机制 在细胞表面以外的地方是迫切需要的。该项目将通过三个具体目标满足这一需求。在……里面 第一个目标是，将如何表征乘客区域的折叠，回答为什么在体内折叠是命令 比体外培养快数量级。第二个目标集中在乘客域的分泌上 膜通过与BAMA转位区域的杂交β桶，该蛋白负责其 膜插入。将确定通过组合桶的路径，以及 过程中的外膜。在第三个目标中，另一类自我转运蛋白，两个伙伴的分泌 将对系统进行建模，目标是确定它们与其他系统之间的共性和差异 上课。这个项目使用的主要方法工具是原子尺度分子动力学(MD)。 模拟。这些模拟都将在现实环境中进行，包括准确的 非对称性革兰氏阴性外膜。还将使用多种创新方法，包括深度 用于对折叠过程建模的学习，用于提取动力学信息的马尔可夫状态建模，以及粗略的 观察自发分泌的粒状布朗动力学。与多个实验机构密切合作 实验室将为MD模拟结果提供关键输入和验证。

Project Summary/Abstract Pathogenic Gram-negative bacteria are collectively responsible for over 5 million deaths annually. A number of species also exhibit high levels of antibiotic resistance, comprising nine out of twelve members on the World Health Organization’s list of priority pathogens. Gram-negative bacterial infection is often mediated by so-called autotransporters, a class of proteins that cross the outer membrane to the extracellular space where they act as virulence factors, such as adhesins, proteases, and other harmful agents. Autotransporters consist of a translocator domain, which remains in the membrane, and a passenger domain, which secretes across the membrane to the other side, even without the use chemical energy, e.g., ATP. Targeting these autotransporters for inhibition represents a promising means of controlling infection while limiting the development of resistance. However, first, research into the molecular mechanisms of autotransporter folding, secretion, and expression beyond the cell surface is critically needed. This project will meet that need through three specific aims. In the first aim, how the passenger domain folds will be characterized, answering why folding in vivo is orders of magnitude faster than in vitro. The second aim focuses on the secretion of the passenger domain across the membrane through a hybrid-β-barrel of the translocator domain with BamA, the protein responsible for its membrane insertion. The pathway through the combined barrels will be determined, as well as the influence of the outer membrane on the process. In the third aim, another class of autotransporters, two-partner secretion systems, will be modeled, with a goal of identifying both commonalities and differences between them and other classes. The primary methodological tool to be used for this project is atomic-scale molecular dynamics (MD) simulations. These simulations will all be carried out in realistic environments, including an accurate model of the asymmetric Gram-negative outer membrane. Multiple innovative approaches will also be used, including deep learning for modeling the folding process, Markov state modeling for extracting kinetics information, and coarse- grained Brownian dynamics for observing spontaneous secretion. Close collaboration with multiple experimental labs will provide key inputs to and validation of the MD simulation results.