Analysis of Protein Synthesis in Bacterial Persisters

 描述（由申请人提供）：我们建议开发方法，以实现表型异质性细菌群体中罕见持久细胞的蛋白质组学分析。同基因细菌群体的特征在于表型异质性，包括代谢率和对抗生素治疗的反应的变化。一旦接触抗生素，大多数细菌细胞就会死亡。但在许多情况下，一个小的亚群（通常< 0.1%）持续存在，并在抗生素挑战缓解后恢复生长。这些“持久细胞”已经在用许多类型的抗生素处理的各种微生物中观察到。病原菌在抗微生物治疗后持续存在和恢复的能力导致慢性感染， 出现耐药菌株。了解使持久性的机制将构成治疗和预防慢性感染的重要一步，但由于持久性细胞的研究需要分析非生长（或生长非常缓慢）细胞的小亚群，持久性的表征一直很困难。我们建议开发和评估在蛋白质组水平上选择性研究这些细胞的一般策略。具体来说，我们将使用生物正交非规范氨基酸标记（BONCAT）和定量质谱法建立持久细胞的时间依赖性蛋白质组学谱之前，期间，和从抗生素挑战中恢复。我们的目标是解决以下问题：1。持留细胞如何应对抗生素的挑战？2.持留细胞如何在抗生素激发后启动生长？和3.是什么使持留细胞亚群与大多数易感细胞不同？更一般地说，我们将建立在细菌持久性，慢性感染和罕见的亚群的研究中具有广泛实用性的生物分析方法， 异质细菌群落

 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.