RNA Futile Cycling in Model Persisters Derived from MazF Accumulation

ABSTRACT Metabolism plays an important role in the persister phenotype, as evidenced by the number of strategies that perturb metabolism to sabotage this troublesome subpopulation. However, the absence of techniques to isolate high-purity populations of native persisters has precluded direct measure...

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Autores principales:	Wendy W. K. Mok, Junyoung O. Park, Joshua D. Rabinowitz, Mark P. Brynildsen
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Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/9ed2f20e632b491d90cb66dbe686cafe
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spelling	oai:doaj.org-article:9ed2f20e632b491d90cb66dbe686cafe2021-11-15T15:41:24ZRNA Futile Cycling in Model Persisters Derived from MazF Accumulation10.1128/mBio.01588-152150-7511https://doaj.org/article/9ed2f20e632b491d90cb66dbe686cafe2015-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01588-15https://doaj.org/toc/2150-7511ABSTRACT Metabolism plays an important role in the persister phenotype, as evidenced by the number of strategies that perturb metabolism to sabotage this troublesome subpopulation. However, the absence of techniques to isolate high-purity populations of native persisters has precluded direct measurement of persister metabolism. To address this technical challenge, we studied Escherichia coli populations whose growth had been inhibited by the accumulation of the MazF toxin, which catalyzes RNA cleavage, as a model system for persistence. Using chromosomally integrated, orthogonally inducible promoters to express MazF and its antitoxin MazE, bacterial populations that were almost entirely tolerant to fluoroquinolone and β-lactam antibiotics were obtained upon MazF accumulation, and these were subjected to direct metabolic measurements. While MazF model persisters were nonreplicative, they maintained substantial oxygen and glucose consumption. Metabolomic analysis revealed accumulation of all four ribonucleotide monophosphates (NMPs). These results are consistent with a MazF-catalyzed RNA futile cycle, where the energy derived from catabolism is dissipated through continuous transcription and MazF-mediated RNA degradation. When transcription was inhibited, oxygen consumption and glucose uptake decreased, and nucleotide triphosphates (NTPs) and NTP/NMP ratios increased. Interestingly, the MazF-inhibited cells were sensitive to aminoglycosides, and this sensitivity was blocked by inhibition of transcription. Thus, in MazF model persisters, futile cycles of RNA synthesis and degradation result in both significant metabolic demands and aminoglycoside sensitivity. IMPORTANCE Metabolism plays a critical role in controlling each stage of bacterial persistence (shutdown, stasis, and reawakening). In this work, we generated an E. coli strain in which the MazE antitoxin and MazF toxin were artificially and independently inducible, and we used this strain to generate model persisters and study their metabolism. We found that even though growth of the model persisters was inhibited, they remained highly metabolically active. We further uncovered a futile cycle driven by continued transcription and MazF-mediated transcript degradation that dissipated the energy derived from carbon catabolism. Interestingly, the existence of this futile cycle acted as an Achilles’ heel for MazF model persisters, rendering them vulnerable to killing by aminoglycosides.Wendy W. K. MokJunyoung O. ParkJoshua D. RabinowitzMark P. BrynildsenAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 6 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Wendy W. K. Mok Junyoung O. Park Joshua D. Rabinowitz Mark P. Brynildsen RNA Futile Cycling in Model Persisters Derived from MazF Accumulation
description	ABSTRACT Metabolism plays an important role in the persister phenotype, as evidenced by the number of strategies that perturb metabolism to sabotage this troublesome subpopulation. However, the absence of techniques to isolate high-purity populations of native persisters has precluded direct measurement of persister metabolism. To address this technical challenge, we studied Escherichia coli populations whose growth had been inhibited by the accumulation of the MazF toxin, which catalyzes RNA cleavage, as a model system for persistence. Using chromosomally integrated, orthogonally inducible promoters to express MazF and its antitoxin MazE, bacterial populations that were almost entirely tolerant to fluoroquinolone and β-lactam antibiotics were obtained upon MazF accumulation, and these were subjected to direct metabolic measurements. While MazF model persisters were nonreplicative, they maintained substantial oxygen and glucose consumption. Metabolomic analysis revealed accumulation of all four ribonucleotide monophosphates (NMPs). These results are consistent with a MazF-catalyzed RNA futile cycle, where the energy derived from catabolism is dissipated through continuous transcription and MazF-mediated RNA degradation. When transcription was inhibited, oxygen consumption and glucose uptake decreased, and nucleotide triphosphates (NTPs) and NTP/NMP ratios increased. Interestingly, the MazF-inhibited cells were sensitive to aminoglycosides, and this sensitivity was blocked by inhibition of transcription. Thus, in MazF model persisters, futile cycles of RNA synthesis and degradation result in both significant metabolic demands and aminoglycoside sensitivity. IMPORTANCE Metabolism plays a critical role in controlling each stage of bacterial persistence (shutdown, stasis, and reawakening). In this work, we generated an E. coli strain in which the MazE antitoxin and MazF toxin were artificially and independently inducible, and we used this strain to generate model persisters and study their metabolism. We found that even though growth of the model persisters was inhibited, they remained highly metabolically active. We further uncovered a futile cycle driven by continued transcription and MazF-mediated transcript degradation that dissipated the energy derived from carbon catabolism. Interestingly, the existence of this futile cycle acted as an Achilles’ heel for MazF model persisters, rendering them vulnerable to killing by aminoglycosides.
format	article
author	Wendy W. K. Mok Junyoung O. Park Joshua D. Rabinowitz Mark P. Brynildsen
author_facet	Wendy W. K. Mok Junyoung O. Park Joshua D. Rabinowitz Mark P. Brynildsen
author_sort	Wendy W. K. Mok
title	RNA Futile Cycling in Model Persisters Derived from MazF Accumulation
title_short	RNA Futile Cycling in Model Persisters Derived from MazF Accumulation
title_full	RNA Futile Cycling in Model Persisters Derived from MazF Accumulation
title_fullStr	RNA Futile Cycling in Model Persisters Derived from MazF Accumulation
title_full_unstemmed	RNA Futile Cycling in Model Persisters Derived from MazF Accumulation
title_sort	rna futile cycling in model persisters derived from mazf accumulation
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/9ed2f20e632b491d90cb66dbe686cafe
work_keys_str_mv	AT wendywkmok rnafutilecyclinginmodelpersistersderivedfrommazfaccumulation AT junyoungopark rnafutilecyclinginmodelpersistersderivedfrommazfaccumulation AT joshuadrabinowitz rnafutilecyclinginmodelpersistersderivedfrommazfaccumulation AT markpbrynildsen rnafutilecyclinginmodelpersistersderivedfrommazfaccumulation
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RNA Futile Cycling in Model Persisters Derived from MazF Accumulation

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