In Situ Architecture of Specialized Bacterial Secretion Systems

细菌已经进化出专门的纳米机器，作为分泌系统来运送蛋白质或DNA 从细菌细胞质进入周围环境或进入其他真核细胞或细菌靶细胞。至 到目前为止，已经确定了九种不同类型的细菌分泌系统。分布最广、分布最广的 其中多功能的是IV型分泌系统(T4SS)，它遍历许多革兰氏阴性菌的细胞膜 和-阳性物种。DNA转移或接合系统是一个大亚家族的成员 医学上有问题，因为它们提供可移动的遗传元件(MGE)和它们携带的抗生素 细菌群体中的抗性基因和毒力决定因素；这些系统还阐述了 结合菌毛或其他表面粘附素，促进建立坚固的、耐抗生素的生物膜 社区。作为第二个T4SS亚家族，效应器移位器被许多医学上使用- 重要的病原体将蛋白质效应物穿过细胞膜传递到周围环境或 进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)，我已经 最近解决了三种不同的T4SS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。这些新结构 正在改变T4SS的架构配置方式的现有范例，它们提供了第一个清晰的 中央底物转运通道的视图，它们识别被配置为 F菌毛的基础平台。IX型分泌系统(T9SS)，主要存在于门中 类杆菌在感染中也起着关键作用。例如，牙龈卟啉单胞菌将其T9SS部署到 分泌牙痛蛋白水解酶和毒力因子，刺激牙周疾病。最近，我解决了 通过原位冷冻技术在其自然细胞环境中对该T9SS的结构进行了研究。这个大的(~50 nm直径)， 包膜跨越的纳米机器与任何其他可见的细菌分泌系统显著不同 约会。在这项米拉提案中，我试图全面定义 F质粒TrA和H.Pylori CAG T4SS和牙龈假单胞菌T9SS 使用原位冷冻技术是理想的或唯一可接近的。我们将i)通过以下方式解决现场结构问题 重点放在这些纳米机器的区域，如内膜复合体，转位通道， 以及不能用体外方法进行结构分析的机器-菌毛连接，二) 通过与T4SS和T9SS领域的专家协作，充分利用我们的资源 更广泛的机械和生物学背景下的结构性发现，以及三)完善数据收集方法 以及提高原位Cryo-ET分辨率极限的工艺。我们的研究将产生重要的新的 对细菌分泌物纳米机器的结构、生物发生和作用机制的见解， 并为介入疗法的设计奠定了基础。

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.