TY - JOUR

T1 - The biofilm inhibitor carolacton enters Gram-negative cells

T2 - Studies using a Tol-Cdeficient strain of Escherichia coli

AU - Donner, Jannik

AU - Reck, Michael

AU - Bunk, Boyke

AU - Jarek, Michael

AU - App, Constantin Benjamin

AU - Meier-Kolthoff, Jan P.

AU - Overmann, Jörg

AU - Müller, Rolf

AU - Kirschning, Andreas

AU - Wagner-Döbler, Irene

N1 - Funding information: We are thankful to Bettina Elxnat for excellent technical assistance. Furthermore, we thank Cathrin Spröer, Simone Severitt, and Nicole Heyer for PacBio DNA library construction and Isabel Schober for genome finishing. We are grateful to Rolf Jansen for providing sorangicin and corallopyronin, and we thank Mark Brönstrup for his suggestion to test the synergistic activity of carolacton with efflux pump inhibitors. This work was carried out as an integral part of the BIOFABRICATION FOR NIFE Initiative, which is financially supported by the ministry of Lower Saxony and the Volkswagen Stiftung (NIFE is the Lower Saxony Center for Biomedical Engineering, Implant Research and Development, a joint translational research center of the Hannover Medical School, the Leibniz University Hannover, the University of Veterinary Medicine Hannover, and the Laser Center Hannover). This work was funded by the German Ministry for Research and Technology (BMBF) in the program e:bio (grant number 031 A299) and by the President's Initiative and Networking Fund of the Helmholtz Association of German Research Centres (HGF) under contract number VH-GS-202. We declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

PY - 2017/9/1

Y1 - 2017/9/1

N2 - The myxobacterial secondary metabolite carolacton inhibits growth of Streptococcus pneumoniae and kills biofilm cells of the caries- and endocarditisassociated 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.

AB - The myxobacterial secondary metabolite carolacton inhibits growth of Streptococcus pneumoniae and kills biofilm cells of the caries- and endocarditisassociated 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.

KW - Antimicrobial activity

KW - Antimicrobial agents

KW - Carolacton

KW - Drug efflux

KW - Drug resistance mechanisms

KW - Efflux pumps

KW - Gene sequencing

KW - Genome analysis

KW - Gram-negative bacteria

UR - http://www.scopus.com/inward/record.url?scp=85041503813&partnerID=8YFLogxK

U2 - 10.1128/mspheredirect.00375-17

DO - 10.1128/mspheredirect.00375-17

M3 - Article

AN - SCOPUS:85041503813

VL - 2

JO - mSphere

JF - mSphere

SN - 2379-5042

IS - 5

M1 - e00375-17

ER -