Co-opting Endogenous Pathogen Autolysins as Next Generation Antibiotics

总结： 抗生素耐药性是对人类健康的最大威胁之一。特别是，六个所谓的 ESKAPE病原体（屎肠球菌、金黄色葡萄球菌、肺炎克雷伯菌、不动杆菌 鲍曼不动杆菌、铜绿假单胞菌和肠杆菌科）代表高度耐药细菌， 造成了巨大的全球疾病负担。这场危机的潜在范围在最近的一次会议上得到了强调。 这份报告是由威康信托基金会和英国政府委托编写的;作者预测，到2050年， 抗药性细菌感染可能使全球经济累计损失100万亿美元，并导致1000万人死亡 人每年。为了解决这个问题，迫切需要创新的抗菌治疗。一 令人信服的治疗策略利用降解细胞壁肽聚糖的重组酶， 导致细菌溶解和死亡。目前，所有这些溶解酶疗法本质上都是反式作用的，即，他们 来自噬菌体或真核生物的免疫系统。这项建议旨在 为开发溶菌酶药物建立了一个全新的范例。我们假设 病原体自身的内源性细胞壁水解酶（即，“自溶素”）可以被共选择以产生有效的 对新的耐药表型难治的抗微生物剂。为了验证这一假设，我们将继续 对高影响病原体甲氧西林耐药S.金黄色葡萄球菌（MRSA），虽然战略 应该广泛适用于任何细菌病原体。在这里，互补的计算和实验 方法将用于鉴定、分离和工程化来自葡萄球菌的有效自溶素， 蛋白质组目的1：对S.将搜索金黄色葡萄球菌和相关细菌的自溶素 使用生物信息学。候选酶将被克隆、评估，并且它们的活性将通过 计算引导的融合到高性能细胞壁靶向结构域。在目标2中， 将采用通量筛选策略从病原菌基因组文库中鉴定自溶素， 葡萄球菌候选酶的活性将通过组合嵌合基因工程来提高， 高性能细胞壁靶向结构域，然后对所得产物进行高通量功能筛选 嵌合文库。在aim 3中，先导性自溶素候选物将被进一步工程化用于有效的抗葡萄球菌 使用定向进化策略。这些研究中最有希望的主要候选人将是 使用一组临床相关的体外和体内测定进行严格评估。最终，这个项目可以 产生新的抗葡萄球菌药物和开发抗菌药物的全新范例 生物疗法

Summary: Antibiotic resistance represents one of the greatest threats to human health. In particular, the six so-called ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and enterobacteriaceae) represent highly drug-resistant bacteria that exert a tremendous global burden of disease. The potential scope of this crisis was highlighted in a recent report commissioned by the Wellcome Trust and British Government; the authors projected that, by 2050, drug-resistant bacterial infections could cost the global economy a cumulative $100 trillion and kill 10 million people annually. To address this issue, there is a critical need for innovative antibacterial treatments. One compelling therapeutic strategy leverages recombinant enzymes that degrade cell wall peptidoglycan, thereby causing bacterial lysis and death. Currently, all such lytic enzyme therapies are trans-acting in nature, i.e., they are derived from bacteriophage or the immune systems of eukaryotic organisms. This proposal seeks to establish an entirely new paradigm for developing bacteriolytic enzyme drugs. We hypothesize that a pathogen's own endogenous cell wall hydrolases (i.e., “autolysins”) can be co-opted to yield potent antimicrobial agents that are refractory to new resistance phenotypes. To test this hypothesis, we will pursue initial studies with the high impact pathogen methicillin resistant S. aureus (MRSA), although the strategy should be broadly applicable to any bacterial pathogen. Here, complementary computational and experimental approaches will be utilized to identify, isolate, and engineer potent autolysins derived from staphylococcal proteomes. In aim 1, the sequenced genome of S. aureus and related bacteria will be searched for autolysins using bioinformatics. Candidate enzymes will be cloned, evaluated, and their activities will be improved via computationally guided fusion to high performance cell wall targeting domains. In aim 2, a complementary high throughput screening strategy will be taken to identify autolysins from genomic libraries of pathogenic staphylococci. The activities of candidate enzymes will be improved via combinatorial chimeragenesis with high performance cell wall targeting domains, followed by high throughput functional screening of the resultant chimeric libraries. In aim 3, lead autolysin candidates will be further engineered for potent anti-staphylococcal activity using a directed evolution strategy. The most promising lead candidates from these studies will be rigorously evaluated using a panel of clinically relevant in vitro and in vivo assays. Ultimately, this project could yield both novel anti-staphylococcal agents and an entirely new paradigm for development of antibacterial biotherapies.