In Situ Architecture of Specialized Bacterial Secretion Systems

专业细菌分泌系统的原位架构

• 批准号: 10472718
• 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
• 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/10472718
• 关键词: Address; Antibiotic Resistance; Architecture; Bacteria; Bacterial Adhesins; Bacterial DNA; Bacteroidetes; Biogenesis; Biological; Caliber; Cells; Collaborations; Communities; Complex; Cryo-electron tomography; Cytoplasm; DNA; Data Collection; 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; Proteins; Resolution; Resources; Role; Sex Pili; Structure; Surface; System; Therapeutic Intervention; Type IV Secretion System Pathway; Virulence; Virulence Factors; cell envelope; computerized data processing; gingipain; improved; insight; member; nanomachine; novel; pathogen; resistance gene; system architecture; therapy design; therapy development

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.

细菌已经进化了专业的纳米机，可作为分泌系统发挥蛋白质或DNA 从细菌细胞质到周围的环境或其他真核或细菌靶细胞。到 日期，已经确定了九种不同类型的细菌分泌系统。最广泛分布和 这些多功能是IV型分泌系统（T4SS），遍历许多革兰氏阴性的细胞信封 和 - 阳性物种。一个大型亚科的成员，DNA转移或共轭系统是 在医学上有问题，因为它们提供移动遗传因素（MGE）及其抗生素的货物 抗性基因和病毒在细菌种群中决定。这些系统也详细说明 共轭菌毛或其他表面粘合剂，以促进建立强大的抗生素抗性生物膜 社区。第二个T4SS子家族，“效应器转换器”由许多医学上部署 重要的病原体，可以在细胞包膜上传递蛋白质作用到周围的环境或 进入真核宿主细胞促进感染。通过使用原位冷冻电子层析成像（Cryo-ET），我有 最近解决了三种不同T4SS的结构，肺炎军团菌dot/icm，大肠杆菌 F质粒Tra和幽门螺杆菌CAG系统的天然细胞信封中。这些新结构 正在更改T4SS如何配置T4SS的现有范例，它们提出了第一个清晰的 中央基材易位通道的视图，并确定了新颖的F模式结构，该结构配置为 f Pili的低音平台。 IX类型分泌系统（T9SSS），主要在门中发现 细菌植物，在感染中也起着关键作用。例如 秘密的金刚蛋白酶蛋白酶和病毒因素促进牙周疾病。最近，我解决了 该T9S在其自然细胞环境中通过原位冷冻-ET的结构。这个大（直径约50 nm）， 与任何其他细菌分泌系统显着跨膜纳米机械差异 日期。在此Mira提议中，我试图全面定义的结构和亚基组成 F质粒Tra和幽门螺杆菌CAG T4SS和牙龈疟原虫T9SS通过解决关键未解决的问题 理想情况下使用原位冷冻-ET使用。我们将与 强调这些纳米机器的区域，例如内膜复合物，易位通道， 和机器 - 使用体外方法不适合结构分析的钢琴连接，ii） 通过与T4SS和T9SS领域的专家合作来利用我们的资源 在更广泛的机械和生物环境中的结构发现，以及iii）提炼数据收集的方法 并处理以提高原位冷冻-ET的分辨率限制。我们的研究将产生重要的新 对细菌分泌纳米机器的结构，生物发生和作用机理的见解， 并为设计干预疗法的设计奠定了基础。