Structural basis for selctive lysis of anthrax and drug-resistant S. aureus

我们的长期目标是开发有效的预防和治疗炭疽和耐甲氧西林的药物。 S.金黄色葡萄球菌（MRSA）的工程噬菌体内溶素。PlyG，一种分子量为25 kDa的溶素 由伽马噬菌体编码PlyG含有能够水解B的T7溶菌酶样催化结构域。 炭疽菌细胞壁肽聚糖，导致细菌细胞裂解，连接到-75个氨基酸的C-末端 域C-末端结构域是靶向酶的二聚碳水化合物识别模块 特别是植物人B。炭疽菌细胞和萌发孢子。我们假设全长PlyG 存在于通过N-和C-末端结构域之间的特异性接触而稳定的单体非活性状态中， 以及C-末端（调节）结构域与B特有的碳水化合物的结合。炭疽菌细胞壁 释放自身抑制相互作用并促进完全活性的二聚体PlyG酶的形成。 噬菌体内溶酶如PlyG具有用作新型和强大的抗生素剂的潜力， 称为“生物酶”。我们将这种方法扩大到包括MRSA，通过组装来自其他抗生素的酶， 具有特异性S.金黄色葡萄球菌活性ClyS是一种嵌合蛋白，其含有N-末端催化的 结构域和C-末端细胞壁靶向结构域。N-末端催化结构域是一种内肽酶， C端细胞壁靶向结构域为94个残基。在目标1中，我们将确定 全长（无活性）PlyG的NMR结构，以揭示该类溶素自抑制的分子基础 的抗菌酶，并解决结构的ClyS溶素及其组成结构域。在目标2中， 将使用NMR来阐明特定炭疽和MRSA靶向的基础， 细胞壁组分和PlyG和ClyS结合结构域。目标3将利用这些结构知识， 工程化具有增强的体内稳定性的PlyG和ClyS同种型。因为细菌必须改变 细胞壁的基本结构，以逃避酶，PlyG-和ClyS-抗性的概率 将出现的菌株是低的。这些研究将提供重要的机制见解一类新的 抗菌化合物走向临床应用。

Our long-term goal is to develop potent agents for prevention and treatment of anthrax and methicillinresistant S. aureus (MRSA) by engineering phage endolysins. PlyG, a lysin of 25 kDa molecular weight encoded by gamma phage. PlyG contains a T7 lysozyme-like catalytic domain capable of hydrolyzing B. anthracis cell wall peptidoglycan, resulting in bacterial cell lysis, attached to a -75 amino acid C- terminal domain. The C-terminal domain is a dimeric carbohydrate recognition module that targets the enzyme specifically to vegetative B. anthracis cells and germinating spores. We hypothesize that full- length PlyG exists in a monomeric inactive state stabilized by specific contacts between the N- and C-terminal domains, and that binding of the C-terminal (regulatory) domain to carbohydrates unique to the B. anthracis cell wall releases the autoinhibitory interaction and promotes formation of the fully active, dimeric PlyG enzyme. Phage endolytic enzymes like PlyG have the potential to serve as novel and powerful antibiotic agents, termed 'enzybiotics'. We broadened this approach to include MRSA by assembling an enzybiotic from other phage lysins with specific S. aureus activity. ClyS is a chimeric protein containing an N-terminal catalytic domain and a C-terminal cell wall targeting domain. The N-terminal catalytic domain is an endopeptidase of 184 amino acids and the C-terminal cell wall targeting domain is 94 residues. In aim 1, we will determine the NMR structure of full-length (inactive) PlyG to reveal the molecular basis of lysin autoinhibition for this class of antibacterial enzymes, and solve structures of the ClyS lysin and its component domains. In Aim 2, the basis for specific anthrax and MRSA targeting will be elucidated using NMR to monitor interactions between cell wall components and the PlyG and ClyS binding domains. Aim 3 will exploit this structural knowledge to engineer isoforms of PlyG and ClyS with enhanced stability in vivo. Because the bacterium must alter the basic construction of the cell wall to evade an enzybiotic, the probability that PlyG- and ClyS- resistant strains will emerge is low. These studies will provide important mechanistic insights into a novel class of antibacterial compounds moving toward clinical application.