Analysis of Protein Synthesis in Bacterial Persisters

 描述(由申请人提供)：我们建议开发方法来实现对表型不同的细菌群体中罕见的宿存细胞的蛋白质组学分析。同基因细菌群体的特征是表型异质性，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，一小部分人(通常为0.1%)持续存在，并在抗生素挑战缓解后恢复增长。在用多种抗生素治疗的各种微生物中都观察到了这些“持久细胞”。病原菌在抗菌治疗后持续存在和恢复的能力会导致慢性感染和 出现耐药菌株。了解持久力的机制将是治疗和预防慢性感染的重要一步，但由于对持久细胞的研究需要分析不生长(或生长非常缓慢)的细胞的小亚群，因此很难确定持续者的特征。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。具体地说，我们将使用生物正交非典范氨基酸标签(BONCAT)和定量质谱仪来建立抗生素攻击之前、期间和恢复后外周细胞的随时间变化的蛋白质组图谱。我们的目标是解决以下问题：1.宿存细胞如何对抗生素挑战做出反应？2.宿存细胞如何在抗生素挑战后启动生长？以及3.宿存细胞亚群与大多数抗生素敏感人群有何不同？更广泛地说，我们将建立广泛实用的生物分析方法，用于研究细菌持久性、慢性感染和稀有亚群。 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.