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Subinhibitory Concentrations of Bacteriostatic Antibiotics Induce relA-Dependent and relA-Independent Tolerance to beta-Lactams
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). University of Tartu, Institute of Technology, Tartu, Estonia.
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2017 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 61, no 4, article id e02173-16Article in journal (Refereed) Published
Abstract [en]

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 beta-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.

Place, publisher, year, edition, pages
AMER SOC MICROBIOLOGY , 2017. Vol. 61, no 4, article id e02173-16
Keywords [en]
beta-lactam, RelA, antibiotics, mupirocin, persistence, ppGpp, ribosomes, thiostrepton, tolerance, trimethoprim
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:umu:diva-134211DOI: 10.1128/AAC.02173-16ISI: 000397598800032PubMedID: 28115345OAI: oai:DiVA.org:umu-134211DiVA, id: diva2:1110883
Available from: 2017-06-16 Created: 2017-06-16 Last updated: 2018-09-25Bibliographically approved
In thesis
1. Functional studies of Escherichia coli stringent response factor RelA
Open this publication in new window or tab >>Functional studies of Escherichia coli stringent response factor RelA
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

RelA is a ribosome associated multi-domain enzyme, which plays a crucial role in adaptation ofEscherichia coli to nutritional stress as such as amino acid deficiency. It detects the deficiency of aminoacids in the cell by monitoring whether a tRNA at the acceptor site (A-site) of the ribosome is chargedwith amino acid or not. When RelA detects uncharged, i.e. deacylated tRNA, it starts to producealarmone guanosine penta- or tetraphosphate, collectively referred to as (p)ppGpp. (p)ppGpp is aglobal metabolism regulator in bacteria. Increase in (p)ppGpp concentration alters crucial metabolicprocesses, such as DNA replication, gene expression, cell wall synthesis and translation. Thesechanges also include activation of different virulence factors and are proposed to drive formation of abacterial sub-population that is highly resilient to antibiotic treatment, the so-called persisters.

For a long time the molecular mechanism of RelA’s activation by and interaction with the ribosomedeacylatedtRNA complex was unknown. Only recently several cryo-EM structures of RelA-ribosomecomplex have shed light on how C-terminal domains of RelA interact with ribosome-deacylated tRNAcomplex. Guided by these structures we investigated the role of RelA’s domains in this interaction byconstructing a set of RelA C-terminal truncates and subjecting these to biochemical and microbiologicalexperimentation. These experiments were complemented with mutations in ribosomal RNA atpositions that interact with RelA, namely A-site finger and sarcin-ricin loop.

We have shown that only the full-length wild type RelA can be activated by ribosome-tRNA complex,whereas, the set of truncated proteins missing either one, two or three C-terminal domains do notrespond to the presence of uncharged tRNA in the A-site of the ribosome. However, these truncatedversions can still be activated by vacant 70S ribosome as well as pppGpp, suggesting that N-terminaldomain of RelA has an allosteric regulation site for (p)ppGpp and is able to interact with the ribosome.The mechanism of this interaction is yet to be elucidated.

We have shown that A-site finger of the ribosome is required for RelA activation and recruitment tothe ribosome. Using EMSA assays, we have shown that RelA and deacylated tRNA do not form a stablecomplex off the ribosome. His432 located in TGS domain of RelA is crucial for recognition of deacylatedtRNA and a mutation of this histidine to glycine abolishes RelA activation by deacylated tRNA.Since (p)ppGpp plays an important role in bacterial survival and pathogenicity we have also testedseveral strategies for RelA inhibition by antibiotics, which target ribosomes and the interactionbetween RelA and ribosome-deacylated tRNA complex. We have shown that antibiotic thiostreptoninhibits (p)ppGpp synthesis by preventing RelA-tRNA interaction on the ribosome. (p)ppGppproduction is also inhibited by chloramphenicol and tetracycline.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2018. p. 52
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1979
Keywords
Stringent response, RelA, (p)ppGpp, ribosome, tRNA, metabolism regulation, allosteric regulation, translation, nutritional stress
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-152071 (URN)978-91-7601-934-4 (ISBN)
Public defence
2018-10-19, Major groove, Byggnad 6L, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
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Available from: 2018-09-28 Created: 2018-09-25 Last updated: 2018-10-22Bibliographically approved

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Varik, ValloSantos, Teresa Del PesoDzhygyr, IevgenCava, FelipeHauryliuk, Vasili

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Varik, ValloSantos, Teresa Del PesoDzhygyr, IevgenCava, FelipeHauryliuk, Vasili
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