Molecular Basis for Group A Streptococcus Encapsulation

基本上，所有的生命系统都会产生细胞表面结构，以使细胞变得坚硬，形成保护层，或促进 细胞黏附和迁移。微生物的“细胞壁”通常起到保护生命的作用 有害的条件，以降低其宿主的先天免疫反应的效力，或形成三维 网状结构，称为生物膜。这些胞外结构的常见建筑材料是多糖 这些聚合物要么单独发挥作用，要么与其他聚合物集成在一起，形成精致的复合材料。 粘液型A组链球菌产生一种由透明质酸(HA)组成的厚厚的多糖壳。HA是 一种酸性杂多糖，主要由脊椎动物产生，是 眼部软结缔组织、软骨和玻璃体中的细胞外基质。因为HA不是 免疫原性微生物HA胶囊是逃避补体介导的杀伤的有效机制， 从而极大地促进了链球菌的毒力。A组链球菌感染可导致严重 疾病，包括风湿热和坏死性筋膜炎。 我们试图确定链球菌HA胶囊形成的机制。HA是由一种 膜包埋酶(HAS)，执行两个任务。它的作用是作为(1)糖基转移酶 从UDP激活的底物合成HA和(2)跨膜分泌HA的转位酶 通过由其自身的跨膜区域形成的通道。情侣们是如何分泌这些反应的 一种长达约10万个糖单位的酸性聚合物目前尚不清楚。 这项拟议的研究利用了我们对链球菌HAS的详细生化分析。我们 证明了该酶作为专有二聚体的功能，在该二聚体中两个原基形成一个单一的HA聚合物 并且可能还在它们的接口处具有HA信道。这种酶复合体可以被纯化并重组为 平面膜双层，称为纳米盘，是生物化学和生物化学的优秀膜替代品。 结构分析。我们建议开发一个工具集，使我们能够在 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 特异性识别HAS三维表位的抗体片段。主要的重点将是 在二聚体组件的上下文中识别与单个HAS拷贝相互作用的Fab片段，这是 预计将有助于通过低温电子显微镜进行结构分析。此外，将选择符合以下条件的FAB粘合剂 识别和稳定HAS二聚体界面，预计这将有助于蛋白质结晶。 在目标2中，我们将产生HAS透明质酸转位中间体来(1)鉴定多糖的长度 跨越酶的跨膜通道，(2)监测合成酶的聚合物释放，以及(3)允许 低温电子显微镜结构测定。结合起来，我们的研究将提供一套完整的工具 这是获得细菌透明质酸生物合成催化循环结构快照所必需的。

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.