Molecular Basis for Group A Streptococcus Encapsulation

基本上所有的生命系统都产生细胞表面结构，以使细胞硬化，形成保护性涂层，或促进细胞生长。 细胞粘附和迁移。微生物的“细胞壁”通常在环境中起保护作用， 有害的条件下，以降低其宿主的先天免疫反应的功效，或形成三维 称为生物膜的网状物。这些细胞外结构的常见建筑材料是多糖 它们要么自己发挥作用，要么与其他聚合物结合成复杂的复合材料。 粘液A族链球菌产生由透明质酸（HA）组成的厚多糖囊。HA是 一种酸性杂多糖，主要由脊椎动物产生，是 在软结缔组织、软骨和眼睛的玻璃体中的细胞外基质。因为HA是非- 免疫原性微生物HA胶囊是逃避补体介导的杀伤的有效机制， 从而显著促进链球菌毒力。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胶囊形成的机制。HA是由一种 膜包埋酶（HAS）执行两项任务。它的功能是（1）糖基转移酶， 从UDP激活的底物合成HA和（2）跨膜分泌HA的移位酶 通过由其自身跨膜区域形成的通道。HAS是如何将这些反应结合起来分泌的 目前还不知道一种长达约100，000个糖单元的酸性聚合物。 这项研究利用了我们对链球菌HAS的详细生化分析。我们 证明该酶作为专性二聚体发挥作用，其中两个原聚体形成单个HA聚合物 并且可能还在它们的接口处具有HA通道。这种酶复合物可以被纯化并重构成 平面膜双层，称为纳米盘，其是生物化学和生物医学的优良膜替代物。 结构分析。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，我们将产生构象敏感性Fab 特异性识别HAS的三维表位的抗体片段。一个主要的重点将是 鉴定在二聚体组装的情况下与单个HAS拷贝相互作用的Fab片段， 预期有助于通过低温电子显微镜进行结构分析。此外，将选择Fab结合物， 识别并稳定HAS二聚体界面，预期其有助于蛋白质结晶。 在目标2中，我们将产生HAS透明质酸易位中间体以（1）鉴定多糖长度 跨越酶的跨膜通道，（2）监测聚合物从合酶释放，和（3）允许 通过低温电子显微镜测定结构。结合起来，我们的研究将提供一个完整的工具集， 获得细菌透明质酸生物合成沿着其催化循环的结构快照是必要的。

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.