Subinhibitory Concentrations of Bacteriostatic Antibiotics Induce relA -Dependent and relA -Independent Tolerance to β-Lactams

Pavel Kudrin,Vallo Varik,Dominik Rejman,Jelena Beljantseva,Ievgen Dzhygyr,Tanel Tenson,Sofia Raquel Alves Oliveira,Vasili Hauryliuk,Teresa Del Peso Santos,Felipe Cava

doi:10.1128/aac.02173-16

Abstract

ABSTRACTThe nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, the Escherichia coli (p)ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p)ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p)ppGpp synthesis. We show that in a biochemical system from purified E. coli components, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p)ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to β-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p)ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.

Highlights

The nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence
GTP hydrolysis by EF-G was efficiently inhibited by thiostrepton both when the experiment was performed in the presence of vacant 70S ribosomes (Fig. 1A) and when 70S ribosomes were programmed with poly(U) model mRNA and deacylated tRNAPhe (Fig. 1B)
We confirmed the strict tRNA dependence of RelA inhibition by thiostrepton observed in the poly(U)-driven system using a more physiologically relevant model system, i.e., RelA activated by ribosomal initiation complexes programmed with model mRNA with an open reading frame coding for the MF dipeptide (Fig. 1E and F)

Summary

Introduction

The nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. The ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p)ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin This abolishment, does not reduce the persister level. An acute increase in (p)ppGpp levels upon stress—the so-called stringent response— drives the reallocation of available metabolic resources, gearing up bacterial physiology for stress resistance and survival This regulatory system is of significant medicinal importance; (p)ppGpp plays a key role in the regulation of bacterial virulence [2] and contributes to bacterial survival during antibiotic treatment by both increasing the antibiotic tolerance of the bacterial population as a whole [3, 4] and driving the formation of a small subpopulation of highly tolerant cells, the so-called persister cells [5,6,7], in a generally sensitive culture. Our follow-up studies have shown that neither Relacin nor peptide 1018 inhibits the stringent response in live cells [24, 25]

Methods

Results

Conclusion