Biofilms are estimated to cause over 80% of bacterial infections, and persisters are thought to be responsible for their propensity to relapse. Persister are subpopulations of cells, which are largely non- replicating, that are able to tolerate high concentrations of antibiotics and resume growth upon removal of the antibiotic. Persisters are enriched in biofilms, and in this environment they not only tolerate antibiotic treatment but are also physically protected from the immune system. Anti-persister therapies are desperately needed to save lives and reduce the burden of infectious disease on the healthcare system. In order to identify potential targets for the development of novel therapeutic agents against these dangerous phenotypic variants, a greater understanding of persister physiology is needed. However, modern high-throughput technology, such as a transcriptome, cannot be used to characterize persisters because they cannot be isolated to any reasonable degree of purity. Therefore, the current state-of-the-art method to study persister gene expression is limited to investigating one promoter at a time using promoter-GFP transcriptional fusions, fluorescent- activated cell sorting (FACS), and antibiotic tolerance assays. This method is laborious and time-consuming, but has led to important insights into persister physiology. Here we propose to parallelize this technique to enable genome-scale investigations into persister gene expression. To accomplish this, we will utilize a promoter reporter library, FACS, antibiotic tolerance assays, DNA barcoding, sequencing, and statistical analysis. This technique will first be used in Escherichia coli, a common model organism for persistence research, then translated to study Pseudomonas aeruginosa, an important clinical pathogen where persistence has been demonstrated to occur in vivo. The proposed study will result in several important scientific advancements. First, the first high-throughput method for the investigation of persister physiology at the genome scale will be developed and its translatability to clinical pathogens will be demonstrated. Second, the first persister transcription networks will be reconstructed, leading to a greater understanding of the mechanisms behind the establishment and maintenance of the persister phenotype. Third, a P. aeruginosa promoter reporter library will be created and available for use by the greater research community, facilitating the study of this important clinical pathogen. Collectively, successful completion of this research will develop tools for a deeper understanding of persistence, opening the door for the development of novel anti- persistence therapies, bringing about breakthroughs for the treatment of biofilm infections.
Persisters are bacterial cells thought to cause the relapse of biofilm infections, since in this environment they are enriched, can tolerate antibiotic treatment, and can reinstate a full infection after removal of the antibiotic. The proposed study will develop the first genome-scale tools to investigate the persister phenotype. Further, we will use this technology to define transcriptional differences between normal cells and persisters in Escherichia coli and Pseudomonas aeruginosa, which could form the basis of anti-persister therapies for the treatment of biofilm infections.
| Barrett, Theresa C; Mok, Wendy W K; Brynildsen, Mark P (2017) Biased inheritance protects older bacteria from harm. Science 356:247-248 |
| Henry, Theresa C; Brynildsen, Mark P (2016) Development of Persister-FACSeq: a method to massively parallelize quantification of persister physiology and its heterogeneity. Sci Rep 6:25100 |
| Henry, Theresa C; Brynildsen, Mark P (2016) Quantifying Current Events Identifies a Novel Endurance Regulator. Trends Microbiol 24:324-326 |
| Sandvik, Elizabeth L; Fazen, Christopher H; Henry, Theresa C et al. (2015) Non-Monotonic Survival of Staphylococcus aureus with Respect to Ciprofloxacin Concentration Arises from Prophage-Dependent Killing of Persisters. Pharmaceuticals (Basel) 8:778-92 |
