Genetic and Proteomic Approaches to Reveal Bacterial Vulnerabilities to Phage Predation

揭示细菌对噬菌体捕食的脆弱性的遗传和蛋白质组学方法

• 批准号: 10625434
• 负责人: Joseph Bondy-Denomy
• 金额: $ 72.33万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-20 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10625434
• 关键词: Address; Affinity; Affinity Chromatography; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Automobile Driving; Bacteria; Bacterial Infections; Bacterial Physiology; Bacteriophages; Binding; Biochemical; Bioinformatics; Biological Assay; Biology; Cataloging; Cell Death; Cell Fractionation; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Complex; Coupled; Cytolysis; Data; Data Analyses; Detection; Engineering; Ensure; Essential Genes; Family; Future; Genes; Genetic; Genetic Transcription; Goals; Growth; Human; Immune system; Infection; Integration Host Factors; Libraries; Lytic; Maps; Mass Spectrum Analysis; Methods; Modeling; Outcome; Pathogenesis; Pathway interactions; Predatory Behavior; Predisposition; Process; Proteins; Proteome; Proteomics; Pseudomonas aeruginosa; Reagent; Repression; Research; Research Personnel; Resistance; Role; Series; Surveys; System; Therapeutic; Time; Validation; Virus Diseases; bacterial fitness; bacterial genetics; bacterial resistance; biophysical properties; combat; design; effective therapy; fighting; fortification; gene discovery; gene interaction; human pathogen; in vivo; inhibitor; knock-down; multidrug-resistant Pseudomonas aeruginosa; next generation sequencing; novel; pathogen; protein complex; protein protein interaction; resistance gene; resistance mechanism; response; screening; skills; small molecule; success; tool

PROJECT SUMMARY We are entering a post antibiotic world where understanding the mechanism of an antibacterial strategy is not sufficient to ensure an effective therapy. We must also consider the mechanisms of resistance and address them during the design process. We will use genetics and proteomics to discover how bacteria combat bacteriophage (phage) lysis. Driving this goal is the desire to combat phage resistance mechanisms so as to make bacteria more susceptible to phage predation. We first tackle the problem by employing state of the art genetic methods to interrogate the role of essential genes in limiting phage replication and bacterial lysis. Using CRISPR transcriptional interference (CRISPRi), we will conduct the first studies in the human pathogen Pseudomonas aeruginosa to determine whether partial knockdown of essential genes can positively impact phage replication. We hypothesize that inhibition of certain essential genes will not only limit bacterial fitness, but also has the potential to enhance the success of any phage-host pairing, regardless of whether the a priori state is one of phage resistance or sensitivity. Put another way, even phages that already replicate in a given host can do better. We will additionally harness the ease of CRISPRi screening to identify non-essential genes that limit phage replication in strain-dependent manners, more akin to canonical hypervariable immune systems (e.g. CRISPR). To further our understanding of the physical underpinnings of phage resistance, we will create a physical map of phage-host protein-protein interactions using whole cell fractionation proteomics. This is particularly critical as many phage proteins are of unknown function and is in line with our goal of identifying essential protein complexes interacting with phage factors. We will validate the importance of factors identified from genetic and proteomic assays with phage replication assays during single gene knockdown to assess generalities of phenomena observed. Lastly, we suspect that phage “accessory genes” (i.e. hypervariable loci not strictly essential for phage replication in all hosts) represent a treasure trove of inhibitors and modulators of host processes, which could be useful genetic fodder for enhancing future phage therapeutics. Accessory genes have been bioinformatically identified and host binding partners will be identified with conventional affinity purification- mass spectrometry, and validated with phage replication assays. Our research strategy combines the complementary expertise of three investigators: Joseph Bondy-Denomy, an expert in phage biology and bacterial immune systems; and Danielle Swaney, an expert in bioanalytical mass spectrometry who has used her skills to define the host-pathogen protein-protein network for many human pathogens; and Jason Peters, a microbiologist with expertise in bacterial genetics and pathogenesis.

项目摘要 我们正在进入一个后抗生素时代，在这个时代，了解抗菌策略的机制并不是 足以确保有效的治疗。我们还必须考虑抵抗机制并加以解决 在设计过程中。我们将使用遗传学和蛋白质组学来发现细菌如何对抗噬菌体 （噬菌体）裂解。推动这一目标的是对抗噬菌体抗性机制的愿望，以便使细菌 更容易被噬菌体捕食我们首先采用最先进的遗传学方法来解决这个问题 询问必需基因在限制噬菌体复制和细菌裂解中的作用。使用CRISPR 转录干扰（CRISPRi），我们将在人类病原体假单胞菌进行第一次研究 以确定必需基因的部分敲低是否可以积极地影响噬菌体复制。 我们假设，抑制某些必需基因不仅会限制细菌的适应性， 增强任何噬菌体-宿主配对成功的潜力，无论先验状态是否是以下状态之一： 噬菌体抗性或敏感性。换句话说，即使是已经在给定主机中复制的数据库也可以做得更好。 我们还将利用CRISPRi筛选的便利性来识别限制噬菌体表达的非必需基因。 以应变依赖的方式复制，更类似于典型的高变免疫系统（例如CRISPR）。 为了进一步了解噬菌体抗性的物理基础，我们将创建一个物理图谱 噬菌体-宿主蛋白质-蛋白质相互作用的研究。这一点尤其重要，因为 许多噬菌体蛋白的功能是未知的，这与我们鉴定必需蛋白的目标一致 与噬菌体因子相互作用的复合物。我们将验证从遗传学和基因组学中确定的因素的重要性， 在单基因敲低期间用噬菌体复制测定进行蛋白质组学测定，以评估 观察到的现象。最后，我们怀疑噬菌体的“辅助基因”（即高变基因座不严格地 对于所有宿主中的噬菌体复制是必需的）代表了宿主的抑制剂和调节剂的宝库 过程，这可能是有用的遗传饲料，以提高未来的噬菌体治疗。辅助基因具有 已被生物信息学鉴定，宿主结合配偶体将用常规亲和纯化鉴定- 质谱分析，并用噬菌体复制测定法验证。我们的研究策略结合了 三位研究人员的互补专业知识：约瑟夫邦迪-Denomy，噬菌体生物学和细菌学专家 免疫系统;和Danielle Swaney，生物分析质谱专家，她利用自己的技能， 为许多人类病原体定义宿主-病原体蛋白质-蛋白质网络;微生物学家Jason Peters 在细菌遗传学和致病机理方面有专长。