Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA.
Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan, USA.
mBio. 2017 Oct 24;8(5):e01593-17. doi: 10.1128/mBio.01593-17.
Biofilm infections exhibit high tolerance against antibiotic treatment. The study of biofilms is complicated by phenotypic heterogeneity; biofilm subpopulations differ in their metabolic activities and their responses to antibiotics. Here, we describe the use of the bio-orthogonal noncanonical amino acid tagging (BONCAT) method to enable selective proteomic analysis of a biofilm subpopulation. Through controlled expression of a mutant methionyl-tRNA synthetase, we targeted BONCAT labeling to cells in the regions of biofilm microcolonies that showed increased tolerance to antibiotics. We enriched and identified proteins synthesized by cells in these regions. Compared to the entire biofilm proteome, the labeled subpopulation was characterized by a lower abundance of ribosomal proteins and was enriched in proteins of unknown function. We performed a pulse-labeling experiment to determine the dynamic proteomic response of the tolerant subpopulation to supra-MIC treatment with the fluoroquinolone antibiotic ciprofloxacin. The adaptive response included the upregulation of proteins required for sensing and repairing DNA damage and substantial changes in the expression of enzymes involved in central carbon metabolism. We differentiated the immediate proteomic response, characterized by an increase in flagellar motility, from the long-term adaptive strategy, which included the upregulation of purine synthesis. This targeted, selective analysis of a bacterial subpopulation demonstrates how the study of proteome dynamics can enhance our understanding of biofilm heterogeneity and antibiotic tolerance. Bacterial growth is frequently characterized by behavioral heterogeneity at the single-cell level. Heterogeneity is especially evident in the physiology of biofilms, in which distinct cellular subpopulations can respond differently to stresses, including subpopulations of pathogenic biofilms that are more tolerant to antibiotics. Global proteomic analysis affords insights into cellular physiology but cannot identify proteins expressed in a particular subpopulation of interest. Here, we report a chemical biology method to selectively label, enrich, and identify proteins expressed by cells within distinct regions of biofilm microcolonies. We used this approach to study changes in protein synthesis by the subpopulation of antibiotic-tolerant cells throughout a course of treatment. We found substantial differences between the initial response and the long-term adaptive strategy that biofilm cells use to cope with antibiotic stress. The method we describe is readily applicable to investigations of bacterial heterogeneity in diverse contexts.
生物膜感染对抗生素治疗表现出很高的耐受性。生物膜的研究很复杂，因为存在表型异质性；生物膜亚群在代谢活性和对抗生素的反应方面存在差异。在这里，我们描述了使用生物正交非典型氨基酸标记 (BONCAT) 方法来选择性地分析生物膜亚群的蛋白质组学。通过控制表达突变的甲硫氨酰-tRNA 合成酶，我们将 BONCAT 标记靶向生物膜微菌落中表现出对抗生素更高耐受性的细胞区域。我们富集并鉴定了这些区域中细胞合成的蛋白质。与整个生物膜蛋白质组相比，标记的亚群的核糖体蛋白丰度较低，并且富含未知功能的蛋白质。我们进行了脉冲标记实验，以确定对氟喹诺酮类抗生素环丙沙星的超 MIC 处理具有耐受性的亚群的动态蛋白质组学反应。适应性反应包括上调参与 DNA 损伤感应和修复的蛋白质，以及中央碳代谢相关酶的表达发生重大变化。我们区分了对鞭毛运动有直接影响的即刻蛋白质组反应，以及包括嘌呤合成上调的长期适应策略。这种对细菌亚群的靶向、选择性分析表明，研究蛋白质组动力学如何增强我们对生物膜异质性和抗生素耐受性的理解。细菌生长通常以单细胞水平的行为异质性为特征。在生物膜生理学中，异质性尤为明显，其中不同的细胞亚群对压力的反应不同，包括对抗生素更耐受的致病性生物膜的亚群。全蛋白质组分析提供了对细胞生理学的深入了解，但无法识别特定感兴趣的亚群中表达的蛋白质。在这里，我们报告了一种化学生物学方法，用于选择性标记、富集和鉴定生物膜微菌落中特定区域细胞表达的蛋白质。我们使用这种方法研究了抗生素耐受细胞亚群在整个治疗过程中蛋白质合成的变化。我们发现生物膜细胞应对抗生素应激的初始反应和长期适应策略之间存在很大差异。我们描述的方法易于应用于不同背景下的细菌异质性研究。
