Biological and structural diversity of bacterial type IV secretion systems

项目总结 大分子通过生物膜的传输是所有细胞的基本过程。在 对细菌遗传交换的最早研究可以追溯到1940年的S，F质粒(当时被称为性别 因子‘)被证明是自我转移的，并通过重组介导整个大肠杆菌的转移 将染色体传给受体细菌。在接下来的75年里，研究确立了广泛的医学重要性 F和其他可移动遗传元件(MGE)在细菌基因组形成中的作用，以及作为 抗生素耐药性和其他健康特征在细菌种群中的传播。MGES还编码 结合菌毛或其他细胞表面粘附素，促进DNA转移所需的细胞间接触 并建立强大的、耐抗生素的生物膜社区。MGE通过细胞间传递 纳米机器被称为IV型分泌系统(T4SS)。T4SS存在于大多数细菌中，如果不是全部的话 物种，它们在功能上多样化为两个大的亚科，DNA转移或接合 将效应器蛋白转运到真核宿主细胞的系统和“效应器转运子”是关键的 感染过程的特征。在过去的27年里，我的团队使用了分子、遗传和生化 确定T4SS的许多机械和体系结构特征的方法，包括 DNA底物通过T4SS的易位途径。我们一直在实施新兴市场 技术，就在过去的半年里，我们开始以前所未有的速度解决T4SS结构 通过原位冷冻电子断层扫描(CryoET)进行分辨。这些新结构显著推进了 但也提出了与管理i)的基本机制和信号有关的新的重要问题 包膜跨T4SS通道和结合菌毛的组装，II)早期底物募集和 加工反应，以及三)与细菌和真核生物建立直接接触(交配连接) 细胞。展望未来，我们将通过(1)继续解决新的结构来解决这些基本问题 利用原位CryoET、生化分级、超分辨荧光技术对E.ColiF T4SS编码 显微镜和单粒子低温电子显微镜，(2)定义新可视化的ATPase能量的贡献 位于通道入口处的中心，用于绑定和展开衬底和分离附件 利用体内和体外生物化学和超微结构的方法，(3)探讨 结合菌毛和细胞表面粘附素在交配连接的形成和分解中的应用细胞学， 生化和生物物理方法，以及(4)利用我们开发的不同的模型系统来 确定机械主题和专门机制。我们将继续利用我们的专业知识 为“团队科学”和多学科重点开展密切合作。我们的研究将产生重要的新的 深入了解T4SS超家族的结构、生物发生和作用机制。这些发现 将导致这一领域的重大范式转变，并为介入疗法的设计奠定基础。

PROJECT SUMMARY The transmission of macromolecules across biological membranes is a fundamental process in all cells. In the earliest studies of genetic exchange in bacteria dating back to the 1940's, the F plasmid (then termed `sex factor') was shown to self-transfer and, through recombination, mediate the transfer of the entire E. coli chromosome to recipient bacteria. In the ensuing ~75 years, studies established the broad medical importance of F and other mobile genetic elements (MGEs) in the shaping of bacterial genomes and as vectors for dissemination of antibiotic resistance and other fitness traits among bacterial populations. MGEs also encode conjugative pili or other cell surface adhesins, which promote intercellular contacts necessary for DNA transfer and establishment robust, antibiotic-resistant biofilm communities. MGEs are transmitted intercellularly through nanomachines termed type IV secretion systems (T4SSs). The T4SSs are present in most if not all bacterial species, where they have functionally diversified into two large subfamilies, the DNA transfer or conjugation systems and the `effector translocators' that translocate effector proteins into eukaryotic host cells as a critical feature of infection processes. Over the past 27 years, my group has used molecular, genetic and biochemical approaches to identify many mechanistic and architectural features of T4SSs, including the first view of the translocation route for a DNA substrate through a T4SS. We have consistently implemented emerging technologies, and just within the past 1½ years we began to solve T4SS structures at unprecedented resolution by in situ cryoelectron tomography (CryoET). These new structures are significantly advancing the field, but also are raising important new questions relating to underlying mechanisms and signals governing i) assembly of envelope-spanning T4SS channels and conjugative pili, ii) early-stage substrate recruitment and processing reactions, and iii) establishment of direct contacts (mating junctions) with bacterial and eukaryotic cells. Moving forward, we will address these fundamental questions by (1) continuing to solve novel structures encoded by the E. coli F T4SS using in situ CryoET, biochemical fractionation, super-resolution fluorescence microscopy, and single-particle CryoEM, (2) defining contributions of the newly visualized ATPase energy center positioned at the channel entrance in binding and unfolding substrates and dissociating accessory factors using in vivo and in vitro biochemistry and ultrastructural approaches, (3) exploring the roles of conjugative pili and cell surface adhesins in formation and disassembly of mating junctions using cytological, biochemical and biophysical approaches, and (4) exploiting our development of distinct model systems to identify mechanistic themes and specialized mechanisms. We will continue to draw on the expertise of our close collaborations for a `team-science' and multidisciplinary focus. Our studies will generate important new insights into the architecture, biogenesis, and mechanism of action of the T4SS superfamily. These findings will lead to major paradigm shifts in this field, and set the stage for design of intervention therapies.