title: Subinhibitory Concentrations of Bacteriostatic Antibiotics InducerelA-Dependent andrelA-Independent Tolerance to β-Lactams.
abstract: The nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, theEscherichia 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 purifiedE. colicomponents, 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.
authors.affiliation: University of Tartu, Institute of Technology, Tartu, Estonia.
authors.forename: Pavel
authors.initials: P
authors.lastname: Kudrin
authors.affiliation: University of Tartu, Institute of Technology, Tartu, Estonia.|Department of Molecular Biology, Umeå University, Umeå, Sweden.|Laboratory for Molecular Infection Medicine Sweden, Umeå University, Umeå, Sweden.
authors.forename: Vallo
authors.initials: V
authors.lastname: Varik
authors.affiliation: University of Tartu, Institute of Technology, Tartu, Estonia.
authors.forename: Sofia Raquel Alves
authors.initials: SR
authors.lastname: Oliveira
authors.affiliation: University of Tartu, Institute of Technology, Tartu, Estonia.
authors.forename: Jelena
authors.initials: J
authors.lastname: Beljantseva
authors.affiliation: Department of Molecular Biology, Umeå University, Umeå, Sweden.|Laboratory for Molecular Infection Medicine Sweden, Umeå University, Umeå, Sweden.
authors.forename: Teresa
authors.initials: T
authors.lastname: Del Peso Santos
authors.affiliation: Department of Molecular Biology, Umeå University, Umeå, Sweden.|Laboratory for Molecular Infection Medicine Sweden, Umeå University, Umeå, Sweden.
authors.forename: Ievgen
authors.initials: I
authors.lastname: Dzhygyr
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, v.v.i., Prague, Czech Republic.
authors.forename: Dominik
authors.initials: D
authors.lastname: Rejman
authors.affiliation: Department of Molecular Biology, Umeå University, Umeå, Sweden.|Laboratory for Molecular Infection Medicine Sweden, Umeå University, Umeå, Sweden.
authors.forename: Felipe
authors.initials: F
authors.lastname: Cava
authors.affiliation: University of Tartu, Institute of Technology, Tartu, Estonia.
authors.forename: Tanel
authors.initials: T
authors.lastname: Tenson
authors.affiliation: University of Tartu, Institute of Technology, Tartu, Estonia vasili.hauryliuk@umu.se.|Department of Molecular Biology, Umeå University, Umeå, Sweden.|Laboratory for Molecular Infection Medicine Sweden, Umeå University, Umeå, Sweden.
authors.forename: Vasili
authors.identifier: 0000-0003-2389-5057
authors.initials: V
authors.lastname: Hauryliuk
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002701:Chloramphenicol / Q000494:pharmacology; D004347:Drug Interactions; D004361:Drug Tolerance; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006159:Guanosine Tetraphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D007533:Isoleucine-tRNA Ligase / Q000235:genetics; D008025:Ligases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D016712:Mupirocin / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D013883:Thiostrepton / Q000494:pharmacology*; D014295:Trimethoprim / Q000494:pharmacology; D047090:beta-Lactams / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D047090:beta-Lactams; D006159:Guanosine Tetraphosphate; C055772:adenosine 5',5'''-triphosphoguanosine-3'''-diphosphate; D002701:Chloramphenicol; D012343:RNA, Transfer; D014295:Trimethoprim; D016712:Mupirocin; D013762:Tetrahydrofolate Dehydrogenase; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases; D007533:Isoleucine-tRNA Ligase; D013752:Tetracycline; D013883:Thiostrepton
keywords: RelA; antibiotics; mupirocin; persistence; ppGpp; ribosomes; thiostrepton; tolerance; trimethoprim; β-lactam