In Situ Architecture of Specialized Bacterial Secretion Systems

专业细菌分泌系统的原位架构

基本信息

批准号：
10687209
负责人：
Bo Hu
金额：
$ 39万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-04 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10687209
关键词：
Address Antibiotic Resistance Architecture Bacteria Bacterial Adhesins Bacterial DNA Bacteroidetes Biogenesis Biological Cells Collaborations Communities Complex Cryo-electron tomography Cytoplasm DNA Data Collection Diameter Disease Progression Escherichia coli F Factor Foundations Helicobacter pylori In Situ In Vitro Infection Intervention Legionella pneumophila Medical Membrane Methods Microbial Biofilms Mobile Genetic Elements Peptide Hydrolases Periodontal Diseases Pilum Play Population Porphyromonas gingivalis Protein Secretion Proteins Resolution Resources Role Sex Pili Structure Surface System Therapeutic Intervention Type IV Secretion System Pathway Virulence Virulence Factors Visualization cell envelope computerized data processing gingipain improved insight member nanomachine novel pathogen resistance gene system architecture therapy design therapy development

项目摘要

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.

细菌已经进化出专门的纳米机器，作为分泌系统来输送蛋白质或 DNA 从细菌细胞质到周围环境或进入其他真核或细菌靶细胞。迄今为止，已鉴定出九种不同类型的细菌分泌系统，其中分布最广泛且最广泛。 IV 型分泌系统 (T4SS) 具有多种功能，可穿过许多革兰氏阴性菌的细胞膜 DNA转移或接合系统这一大亚科的成员是-阳性物种。医学上存在问题，因为它们传递移动遗传元件（MGE）及其抗生素货物这些系统还阐述了细菌群体中的抗性基因和毒力决定因素；接合菌毛或其他表面粘附素，促进建立坚固的耐抗生素生物膜第二个 T4SS 亚家族，“效应器易位器”被许多医学界使用。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 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 系统位于其天然细胞包膜内。正在改变 T4SS 架构配置方式的现有范式，它们提出了第一个清晰的中央底物易位通道的视图，并且他们识别出新颖的 F 编码结构，配置为 F 菌毛分泌系统 (T9SS) 的基础平台，主要存在于门中。例如，拟杆菌在感染中也发挥着关键作用，牙龈卟啉单胞菌将其 T9SS 部署到感染中。分泌牙龈蛋白酶和毒力因子来诱发牙周病最近，我解决了这个问题。通过原位 Cryo-ET 分析了这种 T9SS 在其自然细胞环境中的结构。这个大的（直径约 50 nm），跨越包膜的纳米机器与任何其他可视化的细菌分泌系统明显不同在这个 MIRA 提案中，我寻求全面定义该 MIRA 的结构和子单元组成。 F 质粒 Tra 和幽门螺杆菌 Cag T4SS 以及牙龈卟啉单胞菌 T9SS 通过解决关键的未解决问题使用原位冷冻电子断层扫描 (Cryo-ET) 可以理想地或独特地实现我们将 i) 求解原位结构。重点关注这些纳米机器的区域，例如内膜复合物、易位通道、和机器-菌毛连接点不适合使用体外方法进行结构分析，ii) 通过与 T4SS 和 T9SS 领域的专家合作，利用我们的资源，将我们的更广泛的机械和生物学背景下的结构发现，以及 iii) 完善数据收集方法我们的研究将产生重要的新成果。对细菌分泌纳米机器的结构、生物发生和作用机制的见解，并为干预疗法的设计奠定基础。