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甲氧西林耐药性的药物。 金黄色葡萄球菌(MRSA)通过工程化噬菌体内毒素。PlyG，一种分子量为25 kDa的裂解酶 由伽马噬菌体编码。PlyG含有一个T7类溶菌酶催化结构域，能够降解B。 炭疽菌细胞壁肽聚糖，导致细菌细胞裂解，附着在-75个氨基酸的C末端 域。C-末端结构域是以酶为靶点的二聚体碳水化合物识别模块 特别是对营养炭疽杆菌细胞和萌发的孢子。我们假设全长PlyG 以N-末端结构域和C-末端结构域之间的特定接触稳定的单体非活性状态存在， 以及C末端(调节)结构域与炭疽杆菌细胞壁特有的碳水化合物的结合 释放自抑制相互作用，促进完全活性的二聚体PlyG酶的形成。 像PlyG这样的噬菌体内切酶有可能作为新的和强大的抗生素制剂， 被称为‘酶生物制剂’。我们将这种方法扩大到包括MRSA，方法是从其他来源组装酶生素 具有特定金黄色葡萄球菌活性的噬菌体赖氨酸。ClyS是一种含有N末端催化的嵌合蛋白 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(非活性)PlyG的核磁共振结构揭示这类裂解酶自身抑制的分子基础 抗菌酶的结构，并解决ClyS赖氨酸及其组份结构域的结构。在目标2中， 将使用核磁共振来阐明特定炭疽和MRSA靶向的基础，以监测相互作用 细胞壁成分和PlyG和ClyS结合域。目标3将利用这一结构性知识来 体内稳定性增强的PlyG和ClyS的工程异构体。因为细菌必须改变 逃避酶的细胞壁的基本结构，PlyG和ClyS抗性的可能性 出现的压力是低的。这些研究将为一类新的 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.