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

通过高通量突变和宏基因组扫描了解控制噬菌体特异性和毒力的分子规则

基本信息

批准号：
10317124
负责人：
Srivatsan Raman
金额：
$ 19.44万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-10 至 2023-11-30
项目状态：
已结题

项目摘要

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.

项目概要/摘要正如许多人所证明的那样，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最近的成功案例。然而，天然噬菌体的使用在功效、可靠性、可扩展性和速度。由于进化限制，天然噬菌体的功效较低，结果不一致在笨重的鸡尾酒中，当细菌产生耐药性时发现新的噬菌体是缓慢而费力的。我们通过天然噬菌体（作为底盘）的高通量精密基因组工程提出了一个新的框架创建适合治疗应用的有效噬菌体变体。通过结合汇总选择实验通过深度测序，我们的方法通过系统地对目标噬菌体基因的序列空间进行采样突变分析并挖掘宏基因组序列的丰富多样性，以识别新的功能变异。这些实验中的序列函数知识库增强了我们对如何进行的基本理解突变影响噬菌体功能，并为快速设计新噬菌体提供了设计-构建-测试-学习平台对抗新的和耐药的细菌菌株。为了实现这个想法，我们开发了我们所说的 ORACLE 用于在目标基因座上生成具有预定义序列的大型噬菌体变体文库的技术使用高通量重组酶介导的基因组编辑和 Cas9 引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 应用中，我们将描述和设计 T7 噬菌体的受体结合蛋白 (RBP) 阐明序列功能关系并消除已知可引起尿路感染的致病性大肠杆菌。 RBP 是宿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来系统地剖析 T7 RBP（10,507 个变体）的各个氨基酸的功能作用，以了解哪些残基至关重要特异性、毒力和稳定性。 RBP 远端发现的 Ig 样结构域在噬菌体中发挥关键作用吸附和特异性，并且在有尾病毒目噬菌体之间频繁交换。在目标 2 中，我们将通过替换天然 T7 Ig 样结构域，对从病毒宏基因组中挖掘的约 25,000 个 Ig 样结构域进行功能筛选研究针对新宿主的功能获得。我们将分析两个文库（点突变体和宏基因组变体）针对在尿路感染患者中发现的一组 82 种临床大肠杆菌分离株来寻找 T7 潜在治疗用途的变体。我们的初始屏幕显示 T7 功能获得性变体能够感染杀死来自尿路感染患者的自发耐药临床大肠杆菌，该大肠杆菌无法被杀死野生型。我们设想 ORACLE 技术平台作为开发和优化底盘噬菌体针对不同的细菌进化枝、菌株变体，并快速制定对策对抗耐药菌株。