The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of <named-content content-type="genus-species">Escherichia coli</named-content>

ABSTRACT The myxobacterial secondary metabolite carolacton inhibits growth of Streptococcus pneumoniae and kills biofilm cells of the caries- and endocarditis-associated pathogen Streptococcus mutans at nanomolar concentrations. Here, we studied the response to carolacton of an Escherichia coli stra...

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Autores principales:	Jannik Donner, Michael Reck, Boyke Bunk, Michael Jarek, Constantin Benjamin App, Jan P. Meier-Kolthoff, Jörg Overmann, Rolf Müller, Andreas Kirschning, Irene Wagner-Döbler
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	Gram-negative bacteria antimicrobial activity antimicrobial agents carolacton drug efflux drug resistance mechanisms Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/11b8e6d6ef754cf5a0d7a8bd838a3564
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spelling	oai:doaj.org-article:11b8e6d6ef754cf5a0d7a8bd838a35642021-11-15T15:22:05ZThe Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of <named-content content-type="genus-species">Escherichia coli</named-content>10.1128/mSphereDirect.00375-172379-5042https://doaj.org/article/11b8e6d6ef754cf5a0d7a8bd838a35642017-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphereDirect.00375-17https://doaj.org/toc/2379-5042ABSTRACT The myxobacterial secondary metabolite carolacton inhibits growth of Streptococcus pneumoniae and kills biofilm cells of the caries- and endocarditis-associated pathogen Streptococcus mutans at nanomolar concentrations. Here, we studied the response to carolacton of an Escherichia coli strain that lacked the outer membrane protein TolC. Whole-genome sequencing of the laboratory E. coli strain TolC revealed the integration of an insertion element, IS5, at the tolC locus and a close phylogenetic relationship to the ancient E. coli K-12. We demonstrated via transcriptome sequencing (RNA-seq) and determination of MIC values that carolacton penetrates the phospholipid bilayer of the Gram-negative cell envelope and inhibits growth of E. coli TolC at similar concentrations as for streptococci. This inhibition is completely lost for a C-9 (R) epimer of carolacton, a derivative with an inverted stereocenter at carbon atom 9 [(S) → (R)] as the sole difference from the native molecule, which is also inactive in S. pneumoniae and S. mutans, suggesting a specific interaction of native carolacton with a conserved cellular target present in bacterial phyla as distantly related as Firmicutes and Proteobacteria. The efflux pump inhibitor (EPI) phenylalanine arginine β-naphthylamide (PAβN), which specifically inhibits AcrAB-TolC, renders E. coli susceptible to carolacton. Our data indicate that carolacton has potential for use in antimicrobial chemotherapy against Gram-negative bacteria, as a single drug or in combination with EPIs. Strain E. coli TolC has been deposited at the DSMZ; together with the associated RNA-seq data and MIC values, it can be used as a reference during future screenings for novel bioactive compounds. IMPORTANCE The emergence of pathogens resistant against most or all of the antibiotics currently used in human therapy is a global threat, and therefore the search for antimicrobials with novel targets and modes of action is of utmost importance. The myxobacterial secondary metabolite carolacton had previously been shown to inhibit biofilm formation and growth of streptococci. Here, we investigated if carolacton could act against Gram-negative bacteria, which are difficult targets because of their double-layered cytoplasmic envelope. We found that the model organism Escherichia coli is susceptible to carolacton, similar to the Gram-positive Streptococcus pneumoniae, if its multidrug efflux system AcrAB-TolC is either inactivated genetically, by disruption of the tolC gene, or physiologically by coadministering an efflux pump inhibitor. A carolacton epimer that has a different steric configuration at carbon atom 9 is completely inactive, suggesting that carolacton may interact with the same molecular target in both Gram-positive and Gram-negative bacteria.Jannik DonnerMichael ReckBoyke BunkMichael JarekConstantin Benjamin AppJan P. Meier-KolthoffJörg OvermannRolf MüllerAndreas KirschningIrene Wagner-DöblerAmerican Society for MicrobiologyarticleGram-negative bacteriaantimicrobial activityantimicrobial agentscarolactondrug effluxdrug resistance mechanismsMicrobiologyQR1-502ENmSphere, Vol 2, Iss 5 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Gram-negative bacteria antimicrobial activity antimicrobial agents carolacton drug efflux drug resistance mechanisms Microbiology QR1-502
spellingShingle	Gram-negative bacteria antimicrobial activity antimicrobial agents carolacton drug efflux drug resistance mechanisms Microbiology QR1-502 Jannik Donner Michael Reck Boyke Bunk Michael Jarek Constantin Benjamin App Jan P. Meier-Kolthoff Jörg Overmann Rolf Müller Andreas Kirschning Irene Wagner-Döbler The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of <named-content content-type="genus-species">Escherichia coli</named-content>
description	ABSTRACT The myxobacterial secondary metabolite carolacton inhibits growth of Streptococcus pneumoniae and kills biofilm cells of the caries- and endocarditis-associated pathogen Streptococcus mutans at nanomolar concentrations. Here, we studied the response to carolacton of an Escherichia coli strain that lacked the outer membrane protein TolC. Whole-genome sequencing of the laboratory E. coli strain TolC revealed the integration of an insertion element, IS5, at the tolC locus and a close phylogenetic relationship to the ancient E. coli K-12. We demonstrated via transcriptome sequencing (RNA-seq) and determination of MIC values that carolacton penetrates the phospholipid bilayer of the Gram-negative cell envelope and inhibits growth of E. coli TolC at similar concentrations as for streptococci. This inhibition is completely lost for a C-9 (R) epimer of carolacton, a derivative with an inverted stereocenter at carbon atom 9 [(S) → (R)] as the sole difference from the native molecule, which is also inactive in S. pneumoniae and S. mutans, suggesting a specific interaction of native carolacton with a conserved cellular target present in bacterial phyla as distantly related as Firmicutes and Proteobacteria. The efflux pump inhibitor (EPI) phenylalanine arginine β-naphthylamide (PAβN), which specifically inhibits AcrAB-TolC, renders E. coli susceptible to carolacton. Our data indicate that carolacton has potential for use in antimicrobial chemotherapy against Gram-negative bacteria, as a single drug or in combination with EPIs. Strain E. coli TolC has been deposited at the DSMZ; together with the associated RNA-seq data and MIC values, it can be used as a reference during future screenings for novel bioactive compounds. IMPORTANCE The emergence of pathogens resistant against most or all of the antibiotics currently used in human therapy is a global threat, and therefore the search for antimicrobials with novel targets and modes of action is of utmost importance. The myxobacterial secondary metabolite carolacton had previously been shown to inhibit biofilm formation and growth of streptococci. Here, we investigated if carolacton could act against Gram-negative bacteria, which are difficult targets because of their double-layered cytoplasmic envelope. We found that the model organism Escherichia coli is susceptible to carolacton, similar to the Gram-positive Streptococcus pneumoniae, if its multidrug efflux system AcrAB-TolC is either inactivated genetically, by disruption of the tolC gene, or physiologically by coadministering an efflux pump inhibitor. A carolacton epimer that has a different steric configuration at carbon atom 9 is completely inactive, suggesting that carolacton may interact with the same molecular target in both Gram-positive and Gram-negative bacteria.
format	article
author	Jannik Donner Michael Reck Boyke Bunk Michael Jarek Constantin Benjamin App Jan P. Meier-Kolthoff Jörg Overmann Rolf Müller Andreas Kirschning Irene Wagner-Döbler
author_facet	Jannik Donner Michael Reck Boyke Bunk Michael Jarek Constantin Benjamin App Jan P. Meier-Kolthoff Jörg Overmann Rolf Müller Andreas Kirschning Irene Wagner-Döbler
author_sort	Jannik Donner
title	The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of <named-content content-type="genus-species">Escherichia coli</named-content>
title_short	The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of <named-content content-type="genus-species">Escherichia coli</named-content>
title_full	The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of <named-content content-type="genus-species">Escherichia coli</named-content>
title_fullStr	The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of <named-content content-type="genus-species">Escherichia coli</named-content>
title_full_unstemmed	The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of <named-content content-type="genus-species">Escherichia coli</named-content>
title_sort	biofilm inhibitor carolacton enters gram-negative cells: studies using a tolc-deficient strain of <named-content content-type="genus-species">escherichia coli</named-content>
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/11b8e6d6ef754cf5a0d7a8bd838a3564
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The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of <named-content content-type="genus-species">Escherichia coli</named-content>

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