Unraveling Mechanisms of Persister Formation during Normal Growth

Abstract

Persisters are phenotypic variants present within bacterial populations that exhibit extreme tolerance toward antibiotic stress and are believed to be responsible for chronic and recurrent infections. Despite this clinical importance, the pathways responsible for persister formation during normal growth remain elusive. Therefore, we studied the role of a native stress, carbon source transitions, in persister formation for both planktonic and biofilm lifestyles upon exposure to two different classes of antibiotics, ofloxacin, a fluoroquinolone, and ampicillin, a β-lactam. Interestingly, we found that this single stress stimulates persister formation through numerous pathways leading to highly heterogeneous persister subpopulations.

We discovered that carbon source transitions stimulated both ofloxacin and ampicillin persister formation, and data suggested that these persisters formed through largely distinct mechanisms. We first analyzed ofloxacin persister formation and reconstructed a molecular-level persister formation pathway from glucose exhaustion to a novel type of toxin-antitoxin (TA) module, the ppGpp biochemical network. We found that increased levels of ppGpp in conjunction with nucleoid-associated proteins are required mediators of the observed fluoroquinolone tolerance. Next, we examined how ampicillin persisters formed from the same metabolic stress and discovered that formation of ampicillin persisters required RelA and that loss of clpA, ssrA, or smpB eliminated persister formation through relaxation of the stringent response. Further, we found that tolerance to ampicillin was achieved through broad inhibition of peptidoglycan biosynthesis. ppGpp and trans-translation were found to be common mediators of both pathways whereas ClpA was unique for ampicillin persisters and nucleoid-associated proteins were unique for ofloxacin persisters. In addition, we found that carbon source transitions stimulated persister formation in biofilms, and ofloxacin persisters required ppGpp and nucleoid-associated proteins similarly to planktonic cultures.

This work highlights the need to consider an antibiotic’s mode of action when analyzing persister formation pathways and demonstrates that individual stresses can produce persister heterogeneity. Knowledge of the common mediators responsible for persister formation from natural metabolic fluctuations, such as carbon source shifts, can be utilized as therapeutic targets to prevent persister formation. Combating persisters will expand and enhance the efficacy of our antibiotic arsenal in treating recalcitrant infections.