(259g) Unraveling the Mechanism of Nutrient Transition Driven Ampicillin Persister Formation in Escherichia coli

Bacterial persisters comprise a phenotypic subpopulation of cells that survive supralethal antibiotic stress and upon reinoculation, their progeny possess the same antibiotic sensitivity as the original population^1,2. Persisters present a serious health risk since they are thought to be responsible for the propensity of biofilm infections to relapse after the conclusion of antibiotic therapy¹. Despite this clinical importance, the signaling pathways responsible for persister formation, even during normal growth conditions, are not well characterized. Gaining a more complete mechanistic understanding of persister formation can provide therapeutic targets to combat recalcitrant biofilm infections. In previous studies,  we elucidated a complete persister formation pathway due to a carbon source transition, a natural and common nutrient fluctuation experienced by a bacterial population during growth^3,4. This mechanism was specific to the fluoroquinolone antibiotic, ofloxacin, and implicated the metabolite guanosine tetraphosphate (ppGpp), its biochemical network, and the modulation of (-) DNA supercoiling to confer antibiotic tolerance in response to the carbon source transition. Interestingly, we also demonstrated that carbon source transitions stimulate persister formation when exposed to the β-lactam antibiotic ampicillin through a mechanism distinct from ofloxacin persister formation.  Here, we sought to identify the complete mechanistic pathway responsible for ampicillin persister formation from source of stress to inhibition of the antibiotic’s primary target, peptidoglycan biosynthesis. To accomplish this, we analyzed batch growth of Escherichia coliin defined minimal media containing one or more carbon sources. We measured persister levels using ampicillin and used genetic perturbations, biochemical assays, and quantitative PCR to define a mechanistic persister formation pathway. The glucose exhaustion associated with the transition elicits a RelA, the ribosome associated ppGpp synthase, dependent stringent response, required for persister formation. In addition, the trans-translation apparatus, a system that functions to relieve stalled ribosomes and tag mistranslated proteins for degradation, as well as ClpA, a ATPase chaperone for the ClpP protease, are needed for persister formation. These mediators of persister formation work to ultimately inhibit peptidoglycan biosynthesis resulting in ampicillin tolerance. The results presented here highlight a novel aspect of persister formation, where a single native stress initiates multiple cascades yielding a diversity of persisters within a bacterial population. Identifying mediators of persister formation due to nutrient shifts, such as carbon source transitions, may provide therapeutic targets for prevention of relapsing biofilm infections.

1              Lewis, K. Persister cells. Annual review of microbiology 64, 357-372, doi:10.1146/annurev.micro.112408.134306 (2010).

2              Balaban, N. Q., Merrin, J., Chait, R., Kowalik, L. & Leibler, S. Bacterial persistence as a phenotypic switch. Science 305, 1622-1625, doi:10.1126/science.1099390 (2004).

3              Amato, S. M., Brynildsen, M.P. Nutrient transitions are a source of persisters in Escherichia coli biofilms. . PloS one(2014).

4              Amato, S.M., Orman, M.A. & Brynildsen, M.P. Metabolic Control of Persister Formation in Escherichia coli. Molecular cell 50, 475-487 (2013).