Genetic Basis of Persister Tolerance to Aminoglycosides in <named-content content-type="genus-species">Escherichia coli</named-content>

ABSTRACT Persisters are dormant variants that form a subpopulation of drug-tolerant cells largely responsible for the recalcitrance of chronic infections. However, our understanding of the genetic basis of antibiotic tolerance remains incomplete. In this study, we applied transposon sequencing (Tn-S...

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Autores principales:	Yue Shan, David Lazinski, Sarah Rowe, Andrew Camilli, Kim Lewis
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/dbabb562f2e443188d6a7a437fb65090
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spelling	oai:doaj.org-article:dbabb562f2e443188d6a7a437fb650902021-11-15T15:41:34ZGenetic Basis of Persister Tolerance to Aminoglycosides in <named-content content-type="genus-species">Escherichia coli</named-content>10.1128/mBio.00078-152150-7511https://doaj.org/article/dbabb562f2e443188d6a7a437fb650902015-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00078-15https://doaj.org/toc/2150-7511ABSTRACT Persisters are dormant variants that form a subpopulation of drug-tolerant cells largely responsible for the recalcitrance of chronic infections. However, our understanding of the genetic basis of antibiotic tolerance remains incomplete. In this study, we applied transposon sequencing (Tn-Seq) to systematically investigate the mechanism of aminoglycoside tolerance in Escherichia coli. We constructed a highly saturated transposon library that covered the majority of E. coli genes and promoter regions and exposed a stationary-phase culture to a lethal dose of gentamicin. Tn-Seq was performed to evaluate the survival of each mutant to gentamicin exposure. We found that the disruption of several distinct pathways affected gentamicin tolerance. We identified 105 disrupted gene/promoter regions with a more than 5-fold reduction in gentamicin tolerance and 37 genes with a more than 5-fold increased tolerance. Functional cluster analysis suggests that deficiency in motility and amino acid synthesis significantly diminished persisters tolerant to gentamicin, without changing the MIC. Amino acid auxotrophs, including serine, threonine, glutamine, and tryptophan auxotrophs, exhibit strongly decreased tolerance to gentamicin, which cannot be restored by supplying the corresponding amino acids to the culture. Interestingly, supplying these amino acids to wild-type E. coli sensitizes stationary-phase cells to gentamicin, possibly through the inhibition of amino acid synthesis. In addition, we found that the deletion of amino acid synthesis genes significantly increases gentamicin uptake in stationary phase, while the deletion of flagellar genes does not affect gentamicin uptake. We conclude that activation of motility and amino acid biosynthesis contributes to the formation of persisters tolerant to gentamicin. IMPORTANCE Persisters are responsible for the recalcitrance of chronic infections to antibiotics. The pathways of persister formation in E. coli are redundant, and our understanding of the mechanism of persister formation is incomplete. Using a highly saturated transposon insertion library, we systematically analyzed the contribution of different cellular processes to the formation of persisters tolerant to aminoglycosides. Unexpectedly, we found that activation of amino acid synthesis and motility strongly contributes to persister formation. The approach used in this study leads to an understanding of aminoglycoside tolerance and provides a general method to identify genes affecting persister formation.Yue ShanDavid LazinskiSarah RoweAndrew CamilliKim LewisAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 2 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Yue Shan David Lazinski Sarah Rowe Andrew Camilli Kim Lewis Genetic Basis of Persister Tolerance to Aminoglycosides in <named-content content-type="genus-species">Escherichia coli</named-content>
description	ABSTRACT Persisters are dormant variants that form a subpopulation of drug-tolerant cells largely responsible for the recalcitrance of chronic infections. However, our understanding of the genetic basis of antibiotic tolerance remains incomplete. In this study, we applied transposon sequencing (Tn-Seq) to systematically investigate the mechanism of aminoglycoside tolerance in Escherichia coli. We constructed a highly saturated transposon library that covered the majority of E. coli genes and promoter regions and exposed a stationary-phase culture to a lethal dose of gentamicin. Tn-Seq was performed to evaluate the survival of each mutant to gentamicin exposure. We found that the disruption of several distinct pathways affected gentamicin tolerance. We identified 105 disrupted gene/promoter regions with a more than 5-fold reduction in gentamicin tolerance and 37 genes with a more than 5-fold increased tolerance. Functional cluster analysis suggests that deficiency in motility and amino acid synthesis significantly diminished persisters tolerant to gentamicin, without changing the MIC. Amino acid auxotrophs, including serine, threonine, glutamine, and tryptophan auxotrophs, exhibit strongly decreased tolerance to gentamicin, which cannot be restored by supplying the corresponding amino acids to the culture. Interestingly, supplying these amino acids to wild-type E. coli sensitizes stationary-phase cells to gentamicin, possibly through the inhibition of amino acid synthesis. In addition, we found that the deletion of amino acid synthesis genes significantly increases gentamicin uptake in stationary phase, while the deletion of flagellar genes does not affect gentamicin uptake. We conclude that activation of motility and amino acid biosynthesis contributes to the formation of persisters tolerant to gentamicin. IMPORTANCE Persisters are responsible for the recalcitrance of chronic infections to antibiotics. The pathways of persister formation in E. coli are redundant, and our understanding of the mechanism of persister formation is incomplete. Using a highly saturated transposon insertion library, we systematically analyzed the contribution of different cellular processes to the formation of persisters tolerant to aminoglycosides. Unexpectedly, we found that activation of amino acid synthesis and motility strongly contributes to persister formation. The approach used in this study leads to an understanding of aminoglycoside tolerance and provides a general method to identify genes affecting persister formation.
format	article
author	Yue Shan David Lazinski Sarah Rowe Andrew Camilli Kim Lewis
author_facet	Yue Shan David Lazinski Sarah Rowe Andrew Camilli Kim Lewis
author_sort	Yue Shan
title	Genetic Basis of Persister Tolerance to Aminoglycosides in <named-content content-type="genus-species">Escherichia coli</named-content>
title_short	Genetic Basis of Persister Tolerance to Aminoglycosides in <named-content content-type="genus-species">Escherichia coli</named-content>
title_full	Genetic Basis of Persister Tolerance to Aminoglycosides in <named-content content-type="genus-species">Escherichia coli</named-content>
title_fullStr	Genetic Basis of Persister Tolerance to Aminoglycosides in <named-content content-type="genus-species">Escherichia coli</named-content>
title_full_unstemmed	Genetic Basis of Persister Tolerance to Aminoglycosides in <named-content content-type="genus-species">Escherichia coli</named-content>
title_sort	genetic basis of persister tolerance to aminoglycosides in <named-content content-type="genus-species">escherichia coli</named-content>
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/dbabb562f2e443188d6a7a437fb65090
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Genetic Basis of Persister Tolerance to Aminoglycosides in <named-content content-type="genus-species">Escherichia coli</named-content>

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