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- Kudrin, Pavel, et al.
(författare)
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Subinhibitory Concentrations of Bacteriostatic Antibiotics Induce relA-Dependent and relA-Independent Tolerance to beta-Lactams
- 2017
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Ingår i: Antimicrobial Agents and Chemotherapy. - : AMER SOC MICROBIOLOGY. - 0066-4804 .- 1098-6596. ; 61:4
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Tidskriftsartikel (refereegranskat)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 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.
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| 2. |
- Varik, Vallo, et al.
(författare)
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HPLC-based quantification of bacterial housekeeping nucleotides and alarmone messengers ppGpp and pppGpp
- 2017
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Here we describe an HPLC-based method to quantify bacterial housekeeping nucleotides and the signaling messengers ppGpp and pppGpp. We have replicated and tested several previously reported HPLC-based approaches and assembled a method that can process 50 samples in three days, thus making kinetically resolved experiments feasible. The method combines cell harvesting by rapid filtration, followed by acid extraction, freeze-drying with chromatographic separation. We use a combination of C18 IPRP-HPLC (GMP unresolved and co-migrating with IMP; GDP and GTP; AMP, ADP and ATP; CTP; UTP) and SAX-HPLC in isocratic mode (ppGpp and pppGpp) with UV detection. The approach is applicable to bacteria without the requirement of metabolic labelling with 32P-labelled radioactive precursors. We applied our method to quantify nucleotide pools in Escherichia coli BW25113 K12-strain both throughout the growth curve and during acute stringent response induced by mupirocin. While ppGpp and pppGpp levels vary drastically (40-and >= 8-fold, respectively) these changes are decoupled from the quotients of the housekeeping pool and guanosine and adenosine housekeeping nucleotides: NTP/NDP/NMP ratio remains stable at 6/1/0.3 during both normal batch culture growth and upon acute amino acid starvation.
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