Triple action chimera: eradication of nasal S. aureus by cell wall hydrolases

描述（由申请人提供）：金黄色葡萄球菌是耐药医院和社区获得性感染的主要原因。该项目的长期目标是创造一种局部使用的抗微生物制剂，该制剂对葡萄球菌的耐药发展具有难治性。肽聚糖水解酶降解细菌细胞壁的主要结构成分。当外用时，这些酶能以接近物种特异性的方式裂解革兰氏阳性病原体。该项目的目标是创造独特的嵌合肽聚糖水解酶，由三种裂解活性组成，每种酶都针对葡萄球菌细胞壁的独特键。假设是，当三个独特的裂解结构域融合时，嵌合蛋白将高度难以抵抗目标病原体的抗性发展。这一假设是基于以下观察：a)尚未发现能抵抗噬菌体肽聚糖水解酶裂解作用的细菌宿主；B)肽聚糖水解酶活性域约200个氨基酸大小，融合后能保持亲本特异性；c)金黄色葡萄球菌不太可能产生三个代偿性突变来抵抗三重水解酶融合蛋白的作用。具体目标是：1)构建多个稳定的三重水解酶融合蛋白，每个蛋白具有三种独特的葡萄球菌细胞壁降解活性。嵌合蛋白的体外活性将得到优化。2)研究不同金黄色葡萄球菌和表皮葡萄球菌菌株对嵌合肽聚糖水解酶的敏感性，作为鉴定潜在耐药机制的手段。将对浮游和生物膜相关的葡萄球菌进行敏感性测试，并进行对嵌合肽聚糖水解酶耐药的体外筛选。3)评价嵌合水解酶在消除金黄色葡萄球菌鼻腔定植的作用。研究对蛋白质的免疫反应，并鉴定体内出现的耐药金黄色葡萄球菌突变体。如果在体外或体内出现对嵌合PG水解酶的抗性，将分析耐药金黄色葡萄球菌分离物在PG结构、表面聚合物含量和蛋白酶产生方面的变化。这项调查可能会导致局部药物根除金黄色葡萄球菌鼻腔定植，一个记录的危险因素葡萄球菌感染。

DESCRIPTION (provided by applicant): Staphylococcus aureus is a leading cause of drug resistant nosocomial and community acquired infections. The long-term goal of this project is to create an antimicrobial formulation for topical use that is refractory to resistance development by staphylococci. Peptidoglycan hydrolases degrade the major structural component of bacterial cell walls. When applied externally, these enzymes can lyse Gram-positive pathogens with near species-specificity. The goal of this project is to create unique chimeric peptidoglycan hydrolases consisting of three lytic activities, each targeting a unique bond of the staphylococcal cell wall. The hypothesis is that when three unique lytic domains are fused, the chimeric protein would be highly refractory to resistance development in the target pathogen. This hypothesis is based on the observation that: a) no bacterial host has been identified that can resist the lytic action of phage peptidoglycan hydrolase enzymes; b) peptidoglycan hydrolase activity domains are ~200 amino acids in size and can maintain their parental specificities when fused; and c) S. aureus is unlikely to develop three compensatory mutations to resist the action of a triple hydrolase fusion protein. The specific aims are to: 1) Construct multiple stable triple hydrolase fusion proteins, each with three unique staphylococcal cell wall degrading activities. In vitro activity of the chimeric proteins will be optimized. 2) As a means to identify potential resistance mechanisms, investigate the susceptibility of various S. aureus and S. epidermidis strains to the bactericidal activity of the chimeric peptidoglycan hydrolases. Planktonic and biofilm-associated staphylococci will be tested for susceptibility, and in vitro selection for strains resistant to the chimeric peptidoglycan hydrolases will occur. 3) Evaluate chimeric hydrolases for eradication of S. aureus nasal colonization in a rodent model of nasal carriage. Investigate the immune response to the proteins and identify resistant S. aureus mutants that arise in vivo. If resistance to the chimeric PG hydrolases arises in vitro or in vivo, the resistant S. aureus isolates will be analyzed for alterations in PG structure, surface polymer content, and protease production. This investigation may lead to a topical agent to eradicate S. aureus nasal colonization, a documented risk factor for staphylococcal infection. Staphylococci are important bacterial pathogens that cause severe local and systemic infections in humans. The incidence of antibiotic-resistant S. aureus has increased markedly in the hospital and community. This study will explore a novel antimicrobial agent (a triple action chimeric viral enzyme) to eradicate S. aureus nasal colonization, since nasal carriage is a known risk factor for infection.