(683a) Metabolic Control of Persister Formation

Bacterial persisters comprise of a subpopulation of cells from a genetically identical population that phenotypically exhibit multidrug tolerance. Persisters survive supralethal antibiotic stress and upon reinoculation, their progeny possess the same antibiotic sensitivity as the original population [1, 2]. Persisters are particularly significant for biofilm infections, where they are thought to be responsible for the propensity of such infections to relapse after the conclusion of antibiotic therapy [1]. Therefore, understanding the mechanisms that give rise to persisters can provide therapeutic targets to combat relapse infections. Current research suggests that the terminal effectors of persistence are toxin-antitoxin (TA) modules, due to their ability to inhibit growth, increase antibiotic tolerance, and exhibit bistability. However, the molecular mechanisms that mediate this tolerance upstream of TA modules remain elusive with only a few signaling cascades defined [3-5]. Previous work has demonstrated that the majority of persister formation during normal growth in rich media occurs during mid- to late-exponential phase [6], which is characterized by numerous metabolic perturbations, the onset of oxygen limitation, and the appearance of higher density phenotypes, such as quorum sensing. Inspired by this work, we sought to identify molecular mechanisms by which persisters form during normal growth. Since the extent of stresses experienced during growth suggests that diverse mechanisms may be present, we focused on a single native stress in isolation. In particular, we sought to answer the fundamental question of whether carbon source transitions stimulate persister formation, and if so, determine the intracellular pathway associated with formation. To accomplish this, we analyzed batch growth of Escherichia coli in defined minimal media containing one or more carbon sources. We measured persister levels using the fluoroquinolone antibiotic, ofloxacin, quantified metabolite concentrations, and used genetic perturbations, flow cytometry, and a mathematical model to define a mechanistic persister formation pathway from initial stress (glucose exhaustion) to a metabolic TA module (guanosine tetraphosphate and its biochemical network) and its inhibition of the antibiotic’s primary target (DNA gyrase). The results presented here identify a comprehensive signaling pathway responsible for persister formation from a native stress and suggest that TA-like metabolic networks may play a significant role in persister formation.

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3.         Dorr, T., M. Vulic, and K. Lewis, Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli. PLoS Biol, 2010. 8(2): p. e1000317.

4.         Nguyen, D., et al., Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria. Science, 2011. 334(6058): p. 982-6.

5.         Vega, N.M., et al., Signaling-mediated bacterial persister formation. Nat Chem Biol, 2012. 8(5): p. 431-3.

6.         Keren, I., et al., Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett, 2004. 230(1): p. 13-8.