Co-opting Endogenous Pathogen Autolysins as Next Generation Antibiotics

摘要： 抗生素耐药性是对人类健康的最大威胁之一。尤其是六个所谓的 致病菌(粪肠球菌、金黄色葡萄球菌、肺炎克雷伯菌、不动杆菌 鲍曼氏杆菌、铜绿假单胞菌和肠杆菌科)代表高度耐药的细菌 给全球造成巨大的疾病负担。这场危机的潜在范围在最近的一次 受惠康信托和英国政府委托的一份报告；作者预测，到2050年， 抗药性细菌感染可能给全球经济造成累计100万亿美元的损失，并导致1000万人死亡 每年都有人。为了解决这个问题，迫切需要创新的抗菌治疗方法。一 引人注目的治疗策略利用了能降解细胞壁肽聚糖的重组酶，从而 导致细菌溶解和死亡。目前，所有这种裂解酶疗法本质上都是反式作用的，即它们 来源于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一个全新的范式。我们假设一个 病原体自身的内源性细胞壁水解酶(即“自溶素”)可以被增选以产生有效的 对新的耐药表型耐药的抗菌剂。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金黄色葡萄球菌(MRSA)的初步研究，尽管策略 应广泛适用于任何细菌病原体。在这里，互补的计算和实验 将利用各种方法来鉴定、分离和设计来自葡萄球菌的有效自溶素。 蛋白质组。在目标1中，将搜索金黄色葡萄球菌和相关细菌的已测序基因组中的自溶素。 利用生物信息学。候选酶将被克隆、评估，它们的活性将通过 计算引导融合到高性能细胞壁靶向结构域。在《目标2》中，一个互补的高潮 将采取通过量筛选策略从致病病毒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来提高 高性能细胞壁靶向结构域，随后高通量功能筛选结果 嵌合库。在目标3中，将进一步设计候选自溶素来有效地抗葡萄球菌。 使用定向进化策略的活动。这些研究中最有希望的候选人将是 使用一组临床相关的体外和体内测试进行严格评估。最终，这个项目可能会 既产生了新的抗葡萄球菌药物，又产生了抗菌药物开发的全新范式 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.