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

项目总结/摘要 噬菌体治疗可能是一个有前途的解决抗生素耐药性危机，因为许多证据表明， 最近的成功故事然而，天然维生素的使用在功效、可靠性、 可扩展性和速度。由于进化的限制，自然界中的细菌具有较低的功效，产生不一致的结果 在笨重的鸡尾酒中，当细菌耐药性出现时，发现新的抗生素是缓慢而费力的。我们 提出了一个新的框架，通过高通量的精确基因组工程的天然昆虫（作为底盘）， 产生适用于治疗应用的有效噬菌体变体。通过合并选择实验 通过深度测序，我们的方法通过系统的方法对靶向噬菌体基因的序列空间进行采样， 突变分析和挖掘宏基因组序列的丰富多样性，以确定新的功能变体。 这些实验的序列-功能知识库增强了我们对 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。 对抗新的耐药菌株。为了实现这个想法，我们开发了我们称之为Oracle的东西 用于产生噬菌体变体的大文库的技术，所述噬菌体变体在噬菌体上的靶基因座处具有预定义的序列， 使用高通量重组酶介导的基因组编辑和Cas9指导的富集来构建噬菌体基因组。 ORACLE可用于使任何噬菌体基因多样化。在此R21应用程序中，我们将描述和设计 T7噬菌体的受体结合蛋白（RBP），以阐明序列-功能关系并消除 致病性大肠大肠杆菌引起尿路感染。RBP是宿主范围的主要决定因素， 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 T7 RBP（10，507种变体）的单个氨基酸的功能作用，以了解哪些残基是关键的 特异性、毒性和稳定性。在RBP末端发现的Ig样结构域在噬菌体中起关键作用， 吸附性和特异性，并在尾状病毒目之间进行激烈的交换。在目标2中，我们将 通过替换天然T7 Ig样结构域功能性筛选从病毒宏基因组中挖掘的约25，000个Ig样结构域 来研究新宿主的功能获得我们将分析两个文库（点突变体和宏基因组文库）， 变异）对一组82个临床E.大肠杆菌分离株在尿路感染患者中发现T7 用于潜在治疗用途的变体。我们的初步筛选显示T7功能获得性变体能够感染 并杀死一个自发耐药的临床大肠杆菌。从UTI患者分离的大肠杆菌分离株不能被 野生型我们设想将ORACLE技术平台作为开发和优化 针对不同的细菌分支、菌株变体和快速开发对策的底盘分析 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.