The long-term goal of our research is to understand the mechanism of formation of dormant persister cells exhibiting multidrug tolerance, and to develop a therapy for their eradication. Persisters play an important role in drug tolerance of biofilms. The goal of this project is to obtain an essentially complete set of persister genes and to identify essential targets for future development of anti-persister drugs. We have made significant progress in understanding the nature of persisters and reported the first genes involved in persistence, but a comprehensive set of persister genes remains to be discovered. We have also developed two methods to isolate persisters, which lead to the first persister transcriptome, but a robust, rapid method for obtaining large quantities of persister cells is lacking. In this project, we will aim to resolve these obstacles which are impeding progress in this important field. We will use two organisms - E. coli;and Y. pestis. This will allow us to identify conserved persister genes. Y. pestis survives in the environment well enough to be able to cause an airborne infection. It is possible that the analog of spores - persister cells - aid the survival and spread of the pathogen. This study will lay the background for examining the role of persisters in Y. pestis survival. Based on what we have learned about persisters, it does not appear that any single method will provide a definitive means of identifying persister genes. We therefore propose to use several approaches, and will then synthesize the results of these independent methods to arrive with confidence at a comprehensive set of persister genes. The Specific Aims of this project are: 1. Genomics of high persistence mutants. We will obtain high persistence (hip) mutants with increased production of persisters. Mutated genes will be identified by whole genome sequencing of up to 100 strains in collaboration with a team from The Broad Institute. 2. Isolating persisters and transcription profiling. A. Cell sorting. Advancements in message amplification will allow us to obtain a high-quality transcriptome from small numbers of persisters. We will isolate persisters as described previously, by sorting dim cells from a growing population expressing degradable GFP. A time-dependent transcriptome will point to candidate persister genes. B. Persister capture. Expressing a surface epitope under the control of a persister-specific promoter will allow us to isolate large amounts of persisters. 3. Identification of essential persister maintenance genes. Persister maintenance genes will be identified from screening a large ts mutant library of E. coli. This study will show which functions are critical for persister survival, and will produce attractive targets for developing a dual therapy to eradicate infections. PUBLIC HEALTH RELEVANCE This project is aimed at understanding the nature of bacterial tolerance to antibiotics. Existing antibiotics are unable to eradicate persisters, which are specialized dormant cells present in all bacterial populations. Persisters are largely responsible for many serious relapsing infections. Identifying genes responsible for persister formation will lead to targets for developing an anti-persister therapy.