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.

本质上，所有生物系统都会产生细胞表面结构以固化细胞，形成保护性外套或制备细胞粘合剂和迁移。微生物“细胞壁”通常执行受保护的功能以生存有害条件，以降低宿主先天免疫反应的效率或形成3维网状工作，称为生物膜。这些细胞外结构的常见建筑材料是多糖该要么自行起作用，要么与其他聚合物集成到精美的复合材料中。粘液组A链球菌产生了由透明质酸（HA）组成的厚多糖胶囊。哈是由脊椎动物产生的酸性杂糖糖浆作为绝对成分产生的原发性软性结缔组织，软骨和眼睛的玻璃体中的细胞外基质。因为ha是非 - 免疫原性的微生物HA胶囊是一种有效的机制，可以逃避完成介导的杀戮，从而对链球菌病毒产生了显着贡献。 A组链球菌感染会引起严重疾病，包括风湿热和坏死性筋膜炎。我们试图确定形成链球菌HA胶囊的机制。 HA由执行两个任务的膜包裹的酶（HAS）。它充当（1）糖基转移酶至从UDP激活的底物和（2）易位的HA中合成HA，该易位酶在整个膜上分泌HA 通过自己的膜跨膜区域形成的通道。这些对秘密的反应如何目前尚不清楚高达约100,000糖单元的酸性聚合物。拟议的研究利用了我们对链球菌具有的详细生化分析。我们证明该酶是一种强制性二聚体，其中两种代理形成单个HA聚合物并且可能在其界面上有一个HA频道。该酶复合物可以被纯化并重构为平面膜双层，称为纳米盘，它们是生化和结构分析。我们建议开发一种工具集，该工具集将使我们能够确定 HA生物合成期间的不同状态。为此，在目标1下，我们将产生构象敏感的晶圆厂特异性识别has的三维表位的抗体片段。主要重点将放在识别与单个相互作用的Fab片段在二聚体组件的上下文中具有复制品预计通过冷冻电子显微镜促进结构分析。此外，将选择Fab Binders 识别并稳定其二聚体界面，预计有助于蛋白质结晶。在AIM 2中，我们将生成具有透明质酸易位中间体到（1）识别多糖长度跨越酶的跨膜通道，（2）从合酶中释放聚合物，（3）允许通过冷冻电子显微镜测定结构。合并，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周期的结构快照所必需的。