Characterizing a Self-Digesting-Mediated Reversible Drug Tolerance Mechanism in Bacteria

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SUMMARY By integrating our expertise in persister cell biology with advanced current technologies, our overall goals in this project are to characterize a self-digestion-mediated persistence mechanism in bacteria and to explore the therapeutic potential of this process. Bacterial persisters are rare phenotypic variants that are temporarily tolerant to high concentrations of antibiotics. These variants are generally nongrowing cells that are genetically identical to their antibiotic-susceptible kin. Persister cells facilitate the recurrence of chronic infections and serve as a reservoir for the emergence of drug resistance mutants. As such, elimination of these cells improves clinical outcomes for the majority of hospital-treated infections, but effective methods for persister elimination remain limited. The central hypothesis of this proposal is that self-digestion is a mechanism for persister cell formation in bacterial species. Therefore, deciphering the essential components of this mechanism can potentially provide a global treatment approach, as self-digestion is a hallmark of many bacterial species. In our previous studies, we discovered that persisters are mostly derived from stationary-phase cells with a high redox activity that is maintained by endogenous protein and RNA degradation (i.e., self-digestion). We further determined that loss of stationary-phase metabolic activity reduces persister levels by preventing the digestion of endogenous proteins and RNA, yielding cells with enhanced antibiotic sensitivity. Inspired by these promising results, we propose the following specific aims to explore our central hypothesis. (Aim 1) We will map the self-digestion- related mechanisms in our model organism, Escherichia coli, using fluorescence-activated cell sorting, reporter plasmids, gene deletions, chemical inhibitors, metabolomics technology, and novel assays that we have developed to quantify persisters, viable but non-culturable cells, and intracellular degradation. We will further test our hypothesis using a clinically relevant microorganism, Pseudomonas aeruginosa, which is the predominant cause of morbidity and mortality in cystic fibrosis patients with compromised immune systems. (Aim 2) We will utilize a degradable fluorescent protein to develop a novel screening approach for rapidly identifying chemical compounds that can eradicate persister cells by perturbing the self-digestion mechanisms in E. coli and P. aeruginosa. The effects of candidate inhibitors on persister levels will be further tested under in vivo conditions in a mouse model of high cell density infections. Our study is novel and significant on many levels. Our approach to address our central hypothesis is conceptually innovative. In addition, mapping of this comprehensive bacterial pathway from its initial exogenous trigger, through its signal transduction, to the source of antibiotic tolerance, will enable us to develop affective antipersister therapeutics. Finally, this research program will have a clinical impact by providing a platform to study persistence in different bacterial species and by serving as a bridge from laboratory investigations to clinical trials.