In Situ Architecture of Specialized Bacterial Secretion Systems

细菌已经进化出专门的纳米机器，作为分泌系统来递送蛋白质或DNA 从细菌细胞质到周围环境或进入其它真核或细菌靶细胞。到 迄今为止，已鉴定出九种不同类型的细菌分泌系统。分布最广， IV型分泌系统（T4 SS）是其中的通用系统，它穿过许多革兰氏阴性菌的细胞包膜， 阳性物种。一个大的亚家族的成员，DNA转移或缀合系统，是 医学上存在问题，因为它们递送移动的遗传元件（MGE）及其抗生素货物 细菌种群中的耐药基因和毒力决定因素;这些系统还阐述了 接合的皮利或其它表面粘附素，其促进牢固的、抗微生物生物膜的建立 社区.第二个T4 SS亚家族，“效应易位"是部署了许多医学- 重要的病原体将蛋白质效应物穿过细胞包膜递送到周围环境， 植入真核宿主细胞以引发感染通过使用原位冷冻电子断层扫描（Cryo-ET），我 最近解决了三种不同的T4 SS的结构，嗜肺军团菌Dot/Icm，大肠杆菌 F质粒Tra和幽门螺杆菌Cag系统。这些新的结构 正在改变T4 SS架构配置的现有范例，它们首次明确了 中央底物易位通道的视图，他们确定了新的F编码结构， F皮利的基部平台。IX型分泌系统（T9 SSs），主要存在于动物门中 拟杆菌在感染中也起关键作用。例如，牙龈卟啉单胞菌部署其T9 SS， 分泌牙龈菌蛋白酶和毒力因子以诱发牙周病。最近，我解决了 通过原位Cryo-ET在其天然细胞环境中观察该T9 SS的结构。这种大（~50 nm直径）， 跨越细菌的纳米机器明显不同于任何其他可视化的细菌分泌系统， 约会在这个MIRA提案中，我试图全面定义的结构和亚基组成的 F质粒Tra和H. pylori Cag T4 SS和P. gingivalis T9 SS的研究进展 理想地或唯一地使用原位Cryo-ET可接近。我们将i）解决原位结构， 强调这些纳米机器的区域，如内膜复合物，易位通道， 和机器-菌毛连接处，这些连接处不适合使用体外方法进行结构分析，ii） 通过与T4 SS和T9 SS领域的专家合作，利用我们的资源， 在更广泛的机制和生物学背景下的结构研究结果，以及iii）改进数据收集方法 和处理以提高原位Cryo-ET的分辨率极限。我们的研究将产生重要的新的 深入了解细菌分泌纳米机器的结构、生物起源和作用机制， 并为干预治疗的设计奠定基础。

Bacteria have evolved specialized nanomachines functioning as secretion systems to deliver proteins or DNA from the bacterial cytoplasm to the surrounding milieu or into other eukaryotic or bacterial target cells. To date, nine different types of bacterial secretion systems have been identified. The most widely-distributed and versatile of these, the type IV secretion systems (T4SSs), traverse the cell envelopes of many Gram-negative and -positive species. Members of one large subfamily, the DNA transfer or conjugation systems, are medically problematic because they deliver mobile genetic elements (MGEs) and their cargoes of antibiotic resistance genes and virulence determinants among bacterial populations; these systems also elaborate conjugative pili or other surface adhesins that promote establishment of robust, antibiotic-resistant biofilm communities. A second T4SS subfamily, the ‘effector translocators’ are deployed by many medically- important pathogens to deliver protein effectors across the cell envelope either to the surrounding milieu or into eukaryotic host cells to incite infection. By use of in situ cryo-electron tomography (Cryo-ET), I have recently solved the structures of three different T4SSs, the Legionella pneumophila Dot/Icm, Escherichia coli F plasmid Tra, and Helicobacter pylori Cag systems within their natural cell envelopes. These new structures are changing existing paradigms for how T4SSs are architecturally configured, they present the first clear views of central substrate translocation channels, and they identify novel F-encoded structures configured as basal platforms for F pili. Type IX secretion systems (T9SSs), which are found mainly in the phylum Bacteroidetes, also play critical roles in infection. Porphyromonas gingivalis, for example, deploys its T9SS to secrete gingipain proteinases and virulence factors to incite periodontal disease. Very recently, I solved the structure of this T9SS in its natural cellular context by in situ Cryo-ET. This large (~50 nm diameter), envelope-spanning nanomachine differs markedly from any other bacterial secretion systems visualized to date. In this MIRA proposal, I seek to comprehensively define the structures and subunit compositions of the F plasmid Tra and H. pylori Cag T4SSs and the P. gingivalis T9SS by addressing key unresolved questions that are ideally or uniquely approachable using in situ Cryo-ET. We will i) solve in situ structures with emphasis on regions of these nanomachines such as the inner membrane complexes, translocation channels, and machine - pilus junctions that have not been amenable to structural analyses using in vitro approaches, ii) leverage our resources through collaborations with experts in the T4SS and T9SS fields to place our structural findings in broader mechanistic and biological contexts, and iii) refine methods for data collection and processing to improve the resolution limits of in situ Cryo-ET. Our studies will generate important new insights into the architectures, biogenesis, and mechanisms of action of bacterial secretion nanomachines, and set the stage for design of intervention therapies.