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Phenotypic heterogeneity in populations of isogenic bacterial cells includes variations in metabolic rates and responses to antibiotic treatment. In particular, sub-populations of “persister” cells exhibit increased antibiotic tolerance. Understanding the mechanisms that underlie bacterial persistence would constitute an important step toward preventing and treating chronic infections. On the other hand, bacteria often have multiple molecular mechanisms to adapt to fluctuating environments. Understanding these mechanisms, and their redundancy, requires examinations in depth at the molecular level. This thesis describes a time- and cell state-selective proteome-labeling approach that enables researchers to investigate heterogeneous systems and molecular redundancy. In Chapter 1, we review the concept of bacterial persistence. The definition of bacterial persistence is introduced. Both the differences and connections between bacterial persistence and resistance are covered. In particular, we discuss research related to Pseudomonas aeruginosa (P. aeruginosa), an important opportunistic pathogen found in many cystic fibrosis patients. State-of-the-art technologies to investigate bacterial persistence are discussed, and we conclude that advanced tools are needed to advance research on bacterial persistence further. In Chapter 2, we highlight the concept of bioorthogonal noncanonical amino acid tagging (BONCAT). BONCAT is a powerful tool developed in the Tirrell and Schuman laboratories allowing the incorporation of noncanonical amino acids (ncAA) into newly-synthesized proteins. We review established strategies for proteomics, especially cell-selective proteomics. We introduce the concept and mechanism of BONCAT and address the advantages of BONCAT in the investigation of phenotypic heterogeneity and bacterial persistence. In Chapter 3, we describe our work using BONCAT for understanding bacterial persistence. In particular, we investigated the process of persister resuscitation, as it is closely related to the reoccurrence of P. aeruginosa infections. The characteristics of the heterogeneity of persister cells during persister awakening were examined by survival assays and by ScanLag, an automated colony-based system allowing high-throughput acquisition of time-lapse images, quantification, and analysis of growth of bacterial colonies. Two BONCAT methods were developed in the P. aeruginosa strain PA14 by treating cells either with L-azidohomoalanine (Aha), which avoids extensive usage of antibiotic markers and allows direct integration with PA14 transposon insertion library, or with L-azidonorleucine (Anl), which has the advantage of specificity, as well as direct application in nutrition-rich medium. Through BONCAT enrichment experiments, we found proteins involved in the biosynthesis of pyochelin, a secondary siderophore involved in bacterial iron acquisition, were up-regulated in the regrowth phase. We further explored whether the up-regulation was a result of the modulation of HigB-HigA toxin-antitoxin system. In Chapter 4, we describe our work for understanding molecular redundancy. The chapter follows up on our observation of up-regulation of pyochelin-related proteins during persister regrowth. We discuss the hypothesis that pyochelin confers a growth advantage in persister cells subject to carbon-limited conditions. In addition, we discuss the potential role of Fur, a ferric uptake regulator, in bacterial persistence. |
