Biological and structural diversity of bacterial type IV secretion systems

项目摘要 大分子的跨膜传递是所有细胞的基本过程。在 细菌遗传交换的最早研究可以追溯到20世纪40年代，F质粒（当时被称为“性 因子“）显示出自我转移，并通过重组介导整个E.杆菌 染色体到受体细菌。在随后的75年里，研究确立了广泛的医学重要性， F和其他移动的遗传元件（MGE）在细菌基因组的形成中的作用以及作为 抗生素耐药性和其他适应性特征在细菌种群中的传播。MGE还编码 接合性皮利或其它细胞表面粘附素，其促进DNA转移所必需的细胞间接触 并建立强大的、抗真菌的生物膜群落。MGE通过细胞间传递 纳米机器称为IV型分泌系统（T4SS）。T4SS存在于大多数（如果不是所有）细菌中。 物种，在那里他们有功能多样化成两个大的亚科，DNA转移或共轭 系统和将效应蛋白转运到真核宿主细胞中的“效应转运子”作为一种关键的 感染过程的特征。在过去的27年里，我的团队已经使用了分子，遗传和生物化学 识别T4SS的许多机械和体系结构特征的方法，包括 通过T4SS的DNA底物的易位途径。我们一直在实施新兴的 在过去的一年半里，我们开始以前所未有的速度解决T4SS结构问题， 通过原位冷冻电子断层扫描（CryoET）进行分辨率。这些新的结构正在大大推进 但也提出了与以下方面有关的重要新问题： 跨膜T4 SS通道和接合皮利的组装，ii）早期底物募集，和 处理反应，和iii）与细菌和真核生物建立直接接触（交配连接） 细胞展望未来，我们将通过以下方式解决这些基本问题：（1）继续解决新结构 由E. coli F T4SS，生化分级，超分辨荧光 显微镜和单粒子CryoEM，（2）定义新可视化的ATP酶能量的贡献 在结合和展开基质和解离附件中位于通道入口处的中心 利用体内和体外生物化学和超微结构的方法，（3）探索的作用， 接合的皮利和细胞表面粘附素在使用细胞学的交配连接的形成和分解中， 生物化学和生物物理方法，以及（4）利用我们开发的不同模型系统， 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 密切合作，以“团队科学”和多学科为重点。我们的研究将产生重要的新的 深入了解T4SS超家族的结构、生物起源和作用机制。这些发现 将导致这一领域的重大范式转变，并为干预治疗的设计奠定基础。

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.