Analysis of Protein Synthesis in Bacterial Persisters

细菌存留物中蛋白质合成的分析

• 批准号: 9198757
• 负责人: DAVID A TIRRELL
• 金额: $ 18.35万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9198757
• 关键词: Address; Amino Acids; Antibiotic Therapy; Antibiotics; Automobile Driving; Cell Survival; Cells; Chronic; Clinical; Communities; Exhibits; Exposure to; Genes; Growth; Heterogeneity; Infection; Knock-out; Label; Lead; Ligase; Mass Spectrum Analysis; Methionine-tRNA Ligase; Methods; Microbe; Phase; Phenotype; Population; Protein Analysis; Protein Biosynthesis; Proteins; Proteome; Proteomics; Pseudomonas aeruginosa; Recovery; Resuscitation; Running; Time; Variant; antimicrobial; design; interest; killings; metabolic rate; mutant; overexpression; pathogen; pathogenic bacteria; prevent; programs; promoter; public health relevance; resistant strain; response; tool

 DESCRIPTION (provided by applicant): We propose to develop methods to enable proteomic analysis of rare persister cells in phenotypically heterogeneous bacterial populations. Isogenic bacterial populations are characterized by phenotypic heterogeneity that includes variations in metabolic rates and responses to antibiotic treatment. Upon exposure to antibiotics, most bacterial cells die. But in many cases, a small subpopulation (usually < 0.1%) persists and, upon relief of antibiotic challenge, resumes growth. These "persister cells" have been observed for a wide variety of microbes treated with many types of antibiotics. The ability of pathogenic bacteria to persist and recover following antimicrobial therapy leads to chronic infections and the emergence of resistant strains. Understanding the mechanisms that enable persistence would constitute an important step toward treating and preventing chronic infections, but because studies of persister cells require analysis of small subpopulations of non-growing (or very slowly growing) cells, characterization of persisters has been difficult. We propose to develop and evaluate a general strategy for selective study of these cells at the proteomic level. Specifically we will use bio-orthogonal non-canonical amino acid tagging (BONCAT) and quantitative mass spectrometry to establish the time-dependent proteomic profiles of persister cells before, during, and upon recovery from antibiotic challenge. We aim to address the following questions: 1. How do persister cells respond to antibiotic challenge? 2. How do persister cells initiate growth following antibiotic challenge? and 3. What makes the persister cell subpopulation different from the antibiotic-susceptible majority? More generally, we will establish bioanalytical methods of broad utility in the study of bacterial persistence, chronic infection, and rare sub-populations in heterogeneous bacterial communities.

 描述（由适用提供）：我们建议开发方法，以实现表型异质细菌种群中稀有持久细胞的蛋白质组学分析。等生细菌种群的特征是表型异质性，包括代谢率的变化和对抗生素治疗的反应。暴露于抗生素后，大多数细菌细胞会死亡。但是在许多情况下，少量亚群（通常<0.1％）持续存在，并且在缓解抗生素挑战后，恢复了生长。对于用多种类型的抗生素处理的多种微生物，已经观察到了这些“持久细胞”。致病细菌在抗菌治疗后持续和恢复的能力导致慢性感染和 抗性菌株的出现。了解能够持久性的机制将构成治疗和预防慢性感染的重要一步，但是由于对持久细胞的研究需要分析非生长（或非常缓慢生长）细胞的小亚群，因此很难表征persisters的表征。我们建议在蛋白质组学水平上制定和评估对这些细胞选择性研究的一般策略。具体而言，我们将使用生物正交的非传统氨基酸标记（Boncat）和定量质谱法来建立持久细胞的时间依赖性蛋白质组学特征，然后再恢复抗生素挑战。我们的目标是解决以下问题：1。持久细胞如何应对抗生素挑战？ 2。在抗生素挑战之后，持久细胞如何启动生长？ 3。是什么使持久细胞亚群与可抗生素的多数不同？更一般地，我们将在细菌持久性，慢性感染和罕见的亚群中建立广泛效用的生物分析方法 异质细菌群落。