Molecular Basis for Group A Streptococcus Encapsulation

A 组链球菌封装的分子基础

基本信息

批准号：
10176394
负责人：
Jochen Zimmer
金额：
$ 19.71万
依托单位：
UNIVERSITY OF VIRGINIA
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：
2020-06-01 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10176394
关键词：
3-Dimensional Affinity Anabolism Antibodies Autoradiography Bacteria Binding Biochemical Biological Carbohydrates Cartilage Cell Adhesion Cell Wall Cell membrane Cell surface Cells Complement Complex Connective and Soft Tissue Couples Cryoelectron Microscopy Crystallization Data Detection Diffusion Encapsulated Enzymes Epitopes Excision Exposure to Extracellular Matrix Extracellular Structure Eye Fab Immunoglobulins Family Generations Glucosamine Glucuronic Acids Hand Human Hyaluronan Immobilization Immune Immunoglobulin Fragments In Vitro Individual Infection Infectious Skin Diseases Innate Immune Response Integral Membrane Protein Label Laboratories Length Mediating Membrane Membrane Proteins Microbial Biofilms Molecular Molecular Conformation Molecular Weight Monitor Multienzyme Complexes Necrotizing fasciitis Organism Phase Polymers Polysaccharides Prevention Proteins Protomer Reaction Research Rheumatic Fever Rheumatic Heart Disease Slide Solid Streptococcal Infections Streptococcus Streptococcus pyogenes Structure Surface Techniques Therapeutic Thick Time Vertebrates Virulence base biochemical tools building materials capsule cell motility combat dimer fascinate glycosyltransferase human pathogen hydrophilicity insight microbial mucoid nanodisk pathogen pathogenic microbe protein 50 kDa protein purification reconstitution structural biology success sugar tool translocase

项目摘要

Essentially all living systems produce cell surface structures to rigidify cells, form protective coats, or facilitate cell adhesion and migration. Microbial ‘cell walls’ usually perform protective functions for survival under detrimental conditions, to reduce the efficacy of their host’s innate immune response, or to form 3-dimensional meshworks, called biofilms. Common building materials for these extracellular structures are polysaccharides that either function on their own or are integrated with other polymers into elaborate composite materials. Mucoid Group A Streptococci produce a thick polysaccharide capsule that consists of hyaluronan (HA). HA is an acidic hetero-polysaccharide primarily produced by vertebrates as an abundant component of the extracellular matrix in soft connective tissues, cartilage, and the vitreous of the eye. Because HA is non- immunogenic, microbial HA capsules are an efficient mechanism to escape complement mediated killing, thereby contributing significantly to streptococcal virulence. Group A streptococcal infections can cause severe illnesses, including rheumatic fever and necrotizing fasciitis. We seek to determine the mechanism by which streptococcal HA capsules are formed. HA is synthesized by a membrane-embedded enzyme (HAS) that performs two tasks. It functions as a (1) glycosyltransferase to synthesize HA from UDP-activated substrates and (2) translocase that secretes HA across the membrane through a channel formed by its own membrane-spanning region. How HAS couples these reactions to secrete an acidic polymer up to ~100,000 sugar units long is currently unknown. The proposed research takes advantage of our detailed biochemical analyses of streptococcal HAS. We demonstrated that the enzyme functions as an obligate dimer in which two protomers form a single HA polymer and likely also a HA channel at their interface. This enzyme complex can be purified and reconstituted into planar membrane bilayers, called nanodiscs, which are excellent membrane surrogates for biochemical and structural analyses. We propose to develop a toolset that will allow us to determine the HAS structure at different states during HA biosynthesis. To this end, under Aim 1 we will generate conformation sensitive Fab antibody fragments that specifically recognize 3-dimensional epitopes of HAS. A primary focus will be on identifying Fab fragments that interact with a single HAS copy in the context of a dimeric assembly, which is expected to facilitate structural analyses by cryo electron microscopy. Further, Fab binders will be selected that recognize and stabilize the HAS dimer interface, which are expected to aid in protein crystallization. In Aim 2, we will generate HAS hyaluronan translocation intermediates to (1) identify the polysaccharide length spanning the enzyme’s transmembrane channel, (2) monitor polymer release from the synthase, and (3) allow structure determination by cryo electron microscopy. Combined, our research will provide a complete toolset necessary to obtain structural snapshots of bacterial hyaluronan biosynthesis along its catalytic cycle.

基本上所有的生命系统都会产生细胞表面结构来硬化细胞、形成保护层或促进微生物“细胞壁”通常在环境下发挥生存保护功能。有害条件，降低宿主先天免疫反应的功效，或形成 3 维这些细胞外结构的常见建筑材料是多糖。它们要么单独发挥作用，要么与其他聚合物集成成复杂的复合材料。 A 类粘液链球菌产生厚厚的多糖胶囊，其中含有透明质酸 (HA)。主要由脊椎动物产生的酸性杂多糖，作为软结缔组织、软骨和眼睛玻璃体中的细胞外基质，因为 HA 是非-的。免疫原性微生物 HA 胶囊是逃避补体介导杀伤的有效机制， A 组链球菌感染可导致严重的疾病，包括风湿热和坏死性筋膜炎。我们试图确定链球菌 HA 胶囊的形成机制。膜嵌入酶 (HAS) 执行两项任务：(1) 糖基转移酶。从 UDP 激活的底物和 (2) 跨膜分泌 HA 的转位酶合成 HA 通过其自身的跨膜区域形成的通道，HAS如何耦合这些反应来分泌。目前尚不清楚长达约 100,000 个糖单位的酸性聚合物。拟议的研究利用了我们对链球菌 HAS 的详细生化分析。该酶作为专性二聚体发挥作用，证明两个原聚体形成单个 HA 聚合物并且在它们的界面上也可能有一个 HA 通道，这种酶复合物可以被纯化并重构成。平面膜双层，称为纳米圆盘，是生物化学和生物化学领域的优秀膜替代品我们建议开发一个工具集，使我们能够确定 HAS 结构。 HA 生物合成过程中的不同状态为此，在目标 1 下，我们将生成构象敏感的 Fab。主要关注点是特异性识别 HAS 3 维表位的抗体片段。识别与二聚体组装中的单个 HAS 副本相互作用的 Fab 片段，即预计将有助于通过冷冻电子显微镜进行结构分析。此外，将选择 Fab 粘合剂。识别并稳定 HAS 二聚体界面，这有望有助于蛋白质结晶。在目标 2 中，我们将生成 HAS 透明质酸易位中间体以 (1) 识别多糖长度跨越酶的跨膜通道，(2) 监测合酶的聚合物释放，(3) 允许结合冷冻电子显微镜的结构测定，我们的研究将提供完整的工具集。获得细菌透明质酸生物合成沿其催化循环的结构快照是必要的。