Proteomic Investigation of Tolerant Escherichia coli Populations from Cyclic Antibiotic Treatment

Abstract

Persisters are a subpopulation of cells that have enhanced abilities to survive antibiotics and other stressful conditions. Recently, it was found that when persisters were repeatedly regrown and retreated with the same antibiotic for several cycles, the new population will become tolerant to the drug. In this study, we applied such cyclic antibiotic treatment on Escherichia coli populations using different classes of antibiotics (ampicillin, ciprofloxacin, and apramycin) during the exponential phase. After a few cycles, we observed that the evolved populations exhibit high tolerance to the specific class of antibiotic used during the evolution experiments, which are achieved by single-point mutations in one or several genes. Interestingly, all evolved populations show multidrug tolerance at the stationary phase, indicating that they have higher triggered persister fraction. Proteomic analysis and crosscomparison of the regulated proteomes of the tolerant populations during the stationary phase identified protein candidates with similar expression profiles that might be important for the tolerance phenotype. Susceptibility tests of mutants lacking gene coding for these protein candidates showed that they have significantly reduced survival toward antibiotics not only during the stationary phase, but also during the exponential phase. We demonstrated how proteomics, combined with cyclic antibiotic treatment as a means to enrich tolerant populations, is a promising avenue to obtain fresh insights into the phenomenon of persistence.

Proteomic Study of the Survival and Resuscitation Mechanisms of Filamentous Persisters in an Evolved Escherichia coli Population from Cyclic Ampicillin Treatment

“…As Evo3A highly resembles the Δ cyaA mutant, where the single point mutation on Evo3A’s cyaA gene led to an amino acid change to a stop codon at the middle of the gene, our observation is consistent with the previously reported result. As shown previously, Evo3A has a high persistence phenotype, where the mutations confer tolerance to about 1% of the population, resulting in a bimodal killing curve ( 11 ).…”

“…Evo3A is an evolved E. coli population from a recent adaptive laboratory evolution (ALE) experiment ( 11 ), possessing nonsynonymous single point mutations on yhgE and cyaA genes that likely render them nonfunctional. To generate Evo3A, E. coli K-12 MG1655 was treated with a high dose of ampicillin (∼10× MIC) during exponential phase, the survivors were regrown on fresh medium, and the cycle was repeated for four times.…”

“…1e and f ), and observed that the content of protein and ATP are increasing over the duration of antibiotic treatment on Evo3A. The decrease in ATP level from 0 to 1 h after treatment is attributable to the sharp decrease in cell number during the first hour of treatment (first phase of killing of the biphasic curve [ 11 ]). Taken together, we showed that during antibiotic treatment, the surviving filaments are loaded with machinery necessary for growth but do not undergo cell division, perhaps to evade the effect of ampicillin, which targets the cell wall synthesis machinery.…”

“…Our group has recently performed similar ALE experiments as a means to generate evolved populations with high fractions of persister cells and then subject them to proteomics study for cross-comparison analysis of the regulated proteomes ( 11 ). Using this approach, we have identified a set of protein candidates with similar expression profiles across multiple tolerant populations that are important for their persistence phenotype.…”

Bacteria under antibiotic attack: Different strategies for evolutionary adaptation

“…When repeatedly diluting an overnight culture of Escherichia coli in fresh medium containing a high ampicillin dose, mutants are selected that exhibit population-wide tolerance conferred by an increased lag time that is protective against the antibiotic [5]. Furthermore, when populations of E. coli [30][31][32], Staphylococcus aureus [33], or ESKAPE pathogens (Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) [34] are treated daily with a high concentration of antibiotics, the fraction of tolerant cells within the population rapidly increases.…”