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

描述（由申请方提供）：金黄色葡萄球菌是耐药性医院感染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性发展是难治的。肽聚糖水解酶降解细菌细胞壁的主要结构组分。当外部应用时，这些酶可以溶解革兰氏阳性病原体，具有近乎种属特异性。该项目的目标是创造独特的嵌合肽聚糖水解酶，由三个裂解活性，每一个针对葡萄球菌细胞壁的独特键。假设是，当三个独特的溶解结构域融合时，嵌合蛋白将非常难在目标病原体中产生耐药性。该假设基于以下观察结果：a）尚未鉴定出可抵抗噬菌体肽聚糖水解酶的裂解作用的细菌宿主; B）肽聚糖水解酶活性结构域的大小为约200个氨基酸，并且在融合时可保持其亲本特异性;以及c）S.金黄色葡萄球菌不太可能产生三个补偿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被优化。2)作为一种手段，以确定潜在的耐药机制，调查敏感性的各种S。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杀菌活性的影响。浮游生物和生物膜相关的葡萄球菌将进行敏感性测试，并在体外筛选耐嵌合肽聚糖水解酶的菌株。3)评价嵌合水解酶根除沙门氏菌。金黄色葡萄球菌鼻腔定植在鼻携带的啮齿动物模型中。研究对蛋白质的免疫应答并鉴定耐药的S.体内产生的金黄色葡萄球菌突变体。如果在体外或体内产生对嵌合PG水解酶的抗性，则抗性S.将分析金黄色葡萄球菌分离株的PG结构、表面聚合物含量和蛋白酶产生的改变。这项研究可能会导致局部药物根除S。金黄色葡萄球菌鼻腔定植，是葡萄球菌感染的危险因素。 葡萄球菌是引起人类严重局部和全身感染的重要细菌病原体。耐药的S.金黄色葡萄球菌在医院和社区明显增加。本研究将探索一种新的抗菌剂（一种三重作用的嵌合病毒酶）来根除沙门氏菌。金黄色葡萄球菌鼻腔定植，因为鼻腔携带是已知的感染风险因素。

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.