Screening phage-host receptor interactions using thermal proteome profiling

这项工作的长期目标是将致病性NTM的环境储库、矿储库 从相关环境中寻找NTM特异性分枝杆菌噬菌体，并开发一种高通量工具， 询问分枝杆菌噬菌体-NTM相互作用。本提案的总体目标是增加 分枝杆菌噬菌体新的NTM分子靶点的发现率，以推进新的治疗方法， 包括噬菌体治疗。该提案的重点是结核分枝杆菌（MABS）。而 MABS是美国肺部感染的第二大常见原因，MABS感染可能有 与更常见的NTM病原体相比，患者不良结局的发生率更高。的假设 这项研究是MABS特异性的分枝杆菌噬菌体来源于MABS丰富的样品，可用于鉴定 用于治疗或去除MABS的新的基于噬菌体的分子靶标。该项目的基本原理是， MABS特异性噬菌体工具的开发将有助于定量MABS风险 评估和促进耐药性MABS的替代治疗策略。 为了实现这一提议的目标并检验我们的假设，我们将把MABS丰富的 基于与致病性MABS菌株的丰度和遗传相似性的环境， 环境、人痰和临床分离样品的测序数据。新颖、高通量 需要测序工具来捕获MABS亚种分辨率的空间和时间变化， 存在和丰富。我们将通过采用多位点，16 S rRNA，rpoB， hsp 65和hsp 65（41）。一旦将环境MABS储层关联起来， 环境将被挖掘MABS特异性噬菌体。这些之间的相互作用 分枝杆菌噬菌体和NTM生物体将被筛选，以确定新的NTM分子靶点， 热蛋白质组分析相应的分枝杆菌噬菌体蛋白和先前发表的报告 对于具有NTM亲和力的分枝杆菌噬菌体蛋白，将用于定量结合效率并确定 从没有潜在的溶解。 这项研究的结果可以告知免疫功能低下的患者行为，以避免高风险的NTM水库 并确定高通量测序技术是否可以应用于MABS的物种水平分辨率 在人类痰液中。高通量噬菌体/宿主蛋白筛选平台的开发将 加速噬菌体分子靶点的鉴定，这是开发新型NTM的潜在来源 抗菌剂

The long-term goal of this work is to correlate environmental reservoirs of pathogenic NTM, mine reservoirs for NTM-specific mycobacteriophages from correlated environments, and develop a high-throughput tool to interrogate mycobacteriophage-NTM interactions. The overall objective of this proposal is to increase the discovery rate of new NTM molecular targets of mycobacteriophages to advance novel therapeutics, including phage-based treatments. The focus in this proposal is on Mycobacterium abscessus (MABS). While MABS is the second most common cause of pulmonary infections in the U.S., MABS infections may have higher rates of adverse patient outcomes compared to more common NTM pathogens. The hypothesis of this study is MABS-specific mycobacteriophages sourced from MABS-rich samples can be used to identify novel, phage-based molecular targets for treatment or removal of MABS. The rationale for this project is that the development of MABS-specific mycobacteriophage tools will contribute to quantitative MABS risk assessments and promote alternative treatment strategies for antibiotic-resistant MABS. To achieve the objectives of this proposal and test our hypothesis, we will correlate MABS-rich environments based on abundance and genetic similarity to pathogenic MABS strains using high-throughput sequencing data of environmental, human sputum, and clinically isolated samples. Novel, high throughput sequencing tools are required to capture spatial and temporal variation in MABS subspecies resolution, presence, and abundance. We will achieve this by employing multilocus, metabarcoding of 16S rRNA, rpoB, hsp65, and erm(41). Once environmental MABS reservoirs have been correlated, highly ranked environments will be mined for MABS-specific bacteriophages. Interactions between these mycobacteriophages and NTM organisms will be screened to identify novel NTM-molecular targets using thermal proteome profiling. The corresponding mycobacteriophage proteins and previously published reports for mycobacteriophage proteins with NTM affinity will be used to quantify binding efficiency and determine lysis from without potential. Results from this study can inform immunocompromised patient behavior to avoid high-risk NTM reservoirs and determine if high-throughput sequencing techniques can be applied for species-level resolution of MABS in human sputum. The development of a high-throughput phage/host-protein screening platform would accelerate the identification of phage molecular targets, a potential source for the development of novel NTM antimicrobials.