Subinhibitory concentrations of bacteriostatic antibiotics induce relA-dependent and relA-independent tolerance to β-lactams
Kudrin, Pavel ; Varik, Vallo ; Oliveira, Sofia Raquel Alves ; Beljantseva, Jelena ; Del Peso Santos, Teresa ; Dzhygyr, Ievgen ; Rejman, Dominik ; Cava, Felipe ; Tenson, Tanel and Hauryliuk, Vasili LU
(2017)
In Antimicrobial Agents and Chemotherapy
61(4).
- Abstract
The 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... (More)
The 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 ô-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.
(Less)
- author
- Kudrin, Pavel
; Varik, Vallo
; Oliveira, Sofia Raquel Alves
; Beljantseva, Jelena
; Del Peso Santos, Teresa
; Dzhygyr, Ievgen
; Rejman, Dominik
; Cava, Felipe
; Tenson, Tanel
and Hauryliuk, Vasili
LU
- publishing date
- 2017-04
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Antibiotics, Mupirocin, Persistence, ppGpp, RelA, Ribosomes, Thiostrepton, Tolerance, Trimethoprim, β-lactam
- in
- Antimicrobial Agents and Chemotherapy
- volume
- 61
- issue
- 4
- article number
- e02173-16
- publisher
- American Society for Microbiology
- external identifiers
-
- pmid:28115345
- scopus:85016798861
- ISSN
- 0066-4804
- DOI
- 10.1128/AAC.02173-16
- language
- English
- LU publication?
- no
- additional info
- Funding Information: We thank Gemma Atkinson and Niilo Kaldalu for useful comments on the manuscript, Libor Kr?sn? for sharing B. subtilis BSB1 strain, Jaanus Remme and Aivar Liiv for sharing the E. coli strain for purification of A1067U 70S ribosomes as well as the 6His EF-G overexpression construct, Martin Pavelka for sharing E. coli strain PM2482 lacking ynhG and ycbB genes, Viktoriya Shyp for her effort during the early stages of the project, Sergo Kasvandik for LC-MS analysis of thiostrepton samples, Sergei Kopanchuk for introducing us to Pluronic F-127, and Mike Cashel for sharing the overexpression construct for the RelSeq enzyme and detailed protocols for ppGpp preparation. This study was supported by funds from the European Regional Development Fund through the Centre of Excellence for Molecular Cell Technology (V.H. and T.T.), Estonian Science Foundation grants (PUT37 to V.H. and IUT2-22 to T.T.), the Molecular Infection Medicine Sweden (V.H. and F.C.), the Swedish Research Council (grants 2013-4680 to V.H. and 2013-2440 to F.C.), the Ragnar S?derberg Foundation (V.H.), the Knut and Alice Wallenberg Foundation (F.C.), and the Czech Science Foundation (grant 15-11711S to D.R.). V.H. conceived the study, coordinated the study, and drafted the manuscript with input from P.K., V.V., T.D.P.S., F.C., and T.T. V.H., T.T., F.C., P.K., V.V., and T.D.P.S. designed experiments and analyzed the data. P.K., J.B., and I.D. performed biochemical experiments. V.V. and S.R.A.O. performed nucleotide measurements and bactericidal antibiotic killings. V.V., S.R.A.O., and T.D.P.S. performed muropeptide analysis. D.R. provided materials. All authors have read and approved the manuscript as submitted. Publisher Copyright: © 2017 Kudrin et al. Copyright: Copyright 2017 Elsevier B.V., All rights reserved.
- id
- ce21db0d-81d8-4420-a3e2-bd3009e02514
- date added to LUP
- 2021-09-24 20:39:22
- date last changed
- 2024-03-23 10:17:12
@article{ce21db0d-81d8-4420-a3e2-bd3009e02514,
abstract = {{<p>The 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 ô-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.</p>}},
author = {{Kudrin, Pavel and Varik, Vallo and Oliveira, Sofia Raquel Alves and Beljantseva, Jelena and Del Peso Santos, Teresa and Dzhygyr, Ievgen and Rejman, Dominik and Cava, Felipe and Tenson, Tanel and Hauryliuk, Vasili}},
issn = {{0066-4804}},
keywords = {{Antibiotics; Mupirocin; Persistence; ppGpp; RelA; Ribosomes; Thiostrepton; Tolerance; Trimethoprim; β-lactam}},
language = {{eng}},
number = {{4}},
publisher = {{American Society for Microbiology}},
series = {{Antimicrobial Agents and Chemotherapy}},
title = {{Subinhibitory concentrations of bacteriostatic antibiotics induce relA-dependent and relA-independent tolerance to β-lactams}},
url = {{http://dx.doi.org/10.1128/AAC.02173-16}},
doi = {{10.1128/AAC.02173-16}},
volume = {{61}},
year = {{2017}},
}