Understanding molecular rules governing bacteriophage specificity and virulence by high-throughput mutational and metagenomic scanning

项目摘要/摘要 噬菌体疗法可能是解决抗生素耐药性危机的一个有希望的解决方案，许多人都证明了这一点。 最近的成功故事。然而，天然噬菌体的使用在有效性、可靠性、 可扩展性和速度。由于进化限制，天然噬菌体的效力较低，结果不一致 在笨重的鸡尾酒中，当细菌产生抗药性时发现新的噬菌体是缓慢而费力的。我们 提出了一种以天然噬菌体为底盘的高通量精密基因组工程的新框架 创造出适合治疗应用的强效噬菌体变体。通过结合混合选择实验 通过深度测序，我们的方法通过系统化的方式对目标噬菌体基因的序列空间进行采样 突变概况和挖掘丰富的多样性的元基因组序列，以确定新的功能变体。 这些实验中序列函数知识库增强了我们对以下方面的基本理解 突变影响噬菌体功能，并使设计-构建-测试-学习平台能够快速设计新的噬菌体 对抗新的和耐药的细菌菌株。为了实现这一想法，我们开发了我们称之为Oracle的产品 利用目标基因座上的预定义序列生成大型噬菌体变异体文库的技术 利用高通量重组酶介导的基因组编辑和Cas9引导的浓缩噬菌体基因组。 甲骨文可以应用于任何噬菌体基因的多样化。在此R21应用程序中，我们将描述和设计 T7噬菌体受体结合蛋白(RBP)序列与功能关系的研究 已知可引起尿路感染的致病性大肠杆菌。RBP是寄主范围的主要决定因素，因为它 介导噬菌体和宿主受体之间的相互作用。在目标1中，我们将使用Oracle系统地剖析 T7RBP(10,507个变种)的单个氨基酸在理解哪些残基是关键残基方面的功能作用 以获得特异性、毒性和稳定性。在RBP末端发现的类免疫球蛋白结构域在噬菌体中起着关键作用 吸附和特异性，并在尾状病毒噬菌体之间猖獗地交换。在目标2中，我们将 通过替换天然的T7类Ig结构域从病毒元基因组中筛选出约25,000个类Ig结构域 以调查针对新宿主的功能增益。我们将分析这两个文库(点突变和元基因组 与尿路感染患者中发现的82株临床分离的大肠杆菌进行比对，发现T7 潜在治疗用途的变种。我们最初的屏幕显示T7功能增益变异体能够感染 以及杀死一名尿路感染患者的自发耐药临床大肠杆菌分离物，该细菌不能被 野性的。我们将Oracle技术平台视为开发和优化的标准工具 底盘噬菌体针对不同的细菌分支，菌株变种，并迅速制定对策 抵抗耐药菌株。

PROJECT SUMMARY/ABSTRACT Bacteriophage therapy could be a promising solution to the antibiotic resistance crisis as evidenced by many recent success stories. However, the use of natural phages has fundamental limitations in efficacy, reliability, scalability and speed. Natural phages have lower efficacy due evolutionary constraints, give inconsistent results in unwieldy cocktails, and discovery new phages when bacterial resistance arises is slow and laborious. We propose a new framework by high-throughput precision genome engineering of natural phages (as chassis) to create potent phage variants suitable for therapeutic applications. By combining pooled selection experiments with deep sequencing, our approach samples the sequence space of targeted phage genes via systematic mutational profiling and mines the rich diversity of metagenomic sequences to identify new functional variants. The sequence-function knowledgebase from these experiments enhance our basic understanding of how mutations affect phage function, and enable a design-build-test-learn platform for rapid design of new phages against new and resistant bacterial strains. To implement this idea, we developed what we term as ORACLE technology for generating large libraries of phage variants with pre-defined sequences at a target locus on the phage genome using high-throughput recombinase-mediated genome editing and Cas9-guided enrichment. ORACLE can be applied to diversify any phage gene. In this R21 application, we will characterize and engineer receptor binding proteins (RBP) of T7 phage to elucidate sequence-function relationship and to eliminate pathogenic E. coli known to cause urinary tract infection. RBP is the primary determinant of host range as it mediates interaction between phage and host receptors. In Aim 1, we will use ORACLE to systematically dissect the functional role of individual amino acids of T7 RBP (10,507 variants) to understand which residues are critical for specificity, virulence and stability. Ig-like domains found at the distal tip of RBP play a key role in phage adsorption and specificity, and are rampantly exchanged among Caudovirales phages. In Aim 2, we will functionally screen ~25,000 Ig-like domains mined from viral metagenomes by replacing native T7 Ig-like domain to investigate gain-of-function against new hosts. We will assay both libraries (point mutants and metagenomic variants) against a panel of 82 clinical E. coli isolates found in patients with urinary tract infection to find T7 variants for potential therapeutic use. Our initial screens show T7 gain-of-function variants capable of infecting and killing a spontaneously resistant clinical E. coli isolate from a patient with UTI that could not be killed by wildtype. We envision the ORACLE technology platform as a standard tool for development and optimization of chassis phages to target different bacterial clades, strain variants, and to rapidly develop countermeasures against resistant strains.