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建一种局部使用的抗菌剂配方，该配方对葡萄球菌的耐药性发展难以产生。肽聚糖水解酶降解细菌细胞壁的主要结构成分。当外用时，这些酶可以近乎物种特异性地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，每一种都以葡萄球菌细胞壁的独特结合为靶点。假说是，当三个独特的裂解结构域融合时，嵌合蛋白将对目标病原体的抗性发展具有高度的抵抗力。这一假说基于以下观察：a)尚未发现任何细菌宿主能够抵抗噬菌体肽聚糖水解酶的裂解作用；b)肽聚糖水解酶活性结构域的大小约为200个氨基酸，并且在融合时可以保持其亲本的特异性；以及c)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。其具体目的是：1)构建多个稳定的三重水解酶融合蛋白，每个融合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)研究各种金黄色葡萄球菌和表皮葡萄球菌对嵌合肽聚糖水解酶杀菌活性的敏感性，以确定潜在的耐药机制。将对浮游葡萄球菌和生物膜相关葡萄球菌进行敏感性测试，并将在体外选择对嵌合肽聚糖水解酶具有抗性的菌株。3)评价嵌合水解酶在鼻腔携带的啮齿动物模型中根除金黄色葡萄球菌鼻腔定植的作用。研究对这些蛋白质的免疫反应，并鉴定体内出现的抗药性金黄色葡萄球菌突变体。如果在体外或体内出现对嵌合PG水解酶的耐药性，将分析耐药金黄色葡萄球菌的PG结构、表面聚合物含量和蛋白酶产生的变化。这项研究可能导致一种局部药物来根除金黄色葡萄球菌鼻腔定植，金黄色葡萄球菌是葡萄球菌感染的一个有记录的危险因素。 葡萄球菌是一种重要的细菌病原体，可引起人类严重的局部和全身感染。耐药金黄色葡萄球菌在医院和社区的发病率明显增加。这项研究将探索一种新型的抗菌剂(一种三效嵌合病毒酶)来根除金黄色葡萄球菌的鼻部定植，因为鼻腔携带是已知的感染危险因素。

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.