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Research Article | Molecular Biology and Physiology

Genomewide Profiling of the Enterococcus faecalis Transcriptional Response to Teixobactin Reveals CroRS as an Essential Regulator of Antimicrobial Tolerance

Rachel L. Darnell, Melanie K. Knottenbelt, Francesca O. Todd Rose, Ian R. Monk, Timothy P. Stinear, Gregory M. Cook
Paul D. Fey, Editor
Rachel L. Darnell
aDepartment of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
bMaurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand
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Melanie K. Knottenbelt
aDepartment of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
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Francesca O. Todd Rose
aDepartment of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
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Ian R. Monk
cDepartment of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Australia
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Timothy P. Stinear
cDepartment of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Australia
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Gregory M. Cook
aDepartment of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
bMaurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand
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Paul D. Fey
University of Nebraska Medical Center
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DOI: 10.1128/mSphere.00228-19
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  • FIG 1
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    FIG 1

    Time-dependent kill kinetic assay of S. aureus and E. faecalis upon teixobactin challenge. Strains were grown to mid-exponential phase (5 × 108 CFU ml−1) and untreated or challenged with 50× MIC of teixobactin. Cell survival (number of CFU ml−1) was measured at time zero and 1, 2, 3, 4, 6, and 24 h postchallenge. Results are the mean ± SD (data are for biological triplicates).

  • FIG 2
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    FIG 2

    Pie charts showing the distribution of gene ontologies up- and downregulated in response to teixobactin. recomb., recombination.

  • FIG 3
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    FIG 3

    Time-dependent kill kinetic assay of the E. faecalis JH2-2 wild type and ΔcroRS mutant. Strains were grown to mid-exponential phase (5 × 108 CFU ml−1) and untreated or challenged with 25× or 50× MIC of teixobactin (Tx) and vancomycin (Van), respectively. Cell survival (number of CFU ml−1) was measured at time zero and 1, 2, 3, 4, 6, 8, and 24 h postchallenge. Results are the mean ± SD (data are for biological triplicates).

Tables

  • Figures
  • Supplemental Material
  • TABLE 1

    Teixobactin MICs and MBCs for S. aureus and E. faecalisa

    StrainbAntimicrobialMIC (μg ml−1)MBC (μg ml−1)
    S. aureusTeixobactin22
    E. faecalisTeixobactin216
    E. faecalisVancomycin1>128
    E. faecalisBacitracin3264
    E. faecalisAmpicillin0.52
    E. faecalisPenicillin G22
    E. faecalisDaptomycin24
    • ↵a Mean MICs and MBCs for at least three biological replicates are reported.

    • ↵b S. aureus strain ATCC 6538 and E. faecalis strain JH2-2.

  • TABLE 2

    The 20 most up- and downregulated genes in response to teixobactin in E. faecalis V583 and JH2-2

    Gene regulationGene in E. faecalis:Gene nameF/CaFunction
    V583JH2-2
    UpregulatedEF151813169.5Soluble lytic murein transglycosylase
    EF044325238.7Endopeptidase
    EF153313297.9Conserved hypothetical protein
    EF08025457.8DUF3955 domain-containing protein
    EF166514547.6Conjugal transfer protein TraX
    EF123110167.6Metallophosphoesterase
    EF289624187.2DUF3955 domain-containing protein
    EF09326637.1Hypothetical protein
    EF29862876.8ABC transporter ATP-binding protein
    EF205018176.3Peptide ABC transporter: ATP-binding protein
    EF29872866.3RND transporter
    EF153213286.2Hypothetical protein
    EF00382863proB6.2Glutamate-5-kinase
    EF277123476.0TraX family protein
    EF125810426.0Hypothetical protein
    EF221419185.8VOC family protein
    EF11989825.7ABC transporter permease
    EF07374845.5Amidase
    EF221119155.4YxeA family protein
    EF06804225.4Penicillin binding protein 1A
    DownregulatedEF04112555−11.6PTS mannitol transporter subunit IICB
    EF3139138−10.9PTS sugar transporter subunit IIC
    EF04122554−10.2PTS mannitol transporter subunit IIA
    EF3141136−9.92-Hydroxyacid dehydrogenase
    EF29652466−9.9PTS sugar transporter subunit IIB
    EF3142135−9.76-Phosphogluconate dehydrogenase
    EF04132553−9.7Mannitol-1-phosphate 5-dehydrogenase
    EF321369−9.6PTS mannose transporter subunit IID
    EF3140137−9.4Oxidoreductase
    EF321171−9.4PTS mannose/fructose/sorbose/N-acetylglucosamine subunit IIB
    EF29662467−9.3MltR-like mannitol-operon transcriptional regulator
    EF25822168−9.0Chlorohydrolase/aminohydrolase
    EF3138139−9.0PTS mannose transporter subunit IID
    EF29642465ulaA−8.9PTS ascorbate transporter subunit IIC
    EF22231927−8.8ABC transporter family
    EF321270−8.7PTS mannose/fructose/sorbose/N-acetylglucosamine subunit IIC
    EF33272909−8.7Citrate transporter
    EF321072−8.6PTS mannose/fructose/sorbose/N-acetylglucosamine subunit IIA
    EF1031742−8.4PTS sugar transporter subunit IIC
    EF1207991maeP−8.4l-Malate permease
    • ↵a F/C, log2 fold change.

  • TABLE 3

    Cell antimicrobial MICs and MBCs for the E. faecalis JH2-2 wild type and ΔcroRS mutanta

    AntimicrobialWTΔcroRS mutant
    MIC (μg ml−1)MBC (μg ml−1)MIC (μg ml−1)MBC (μg ml−1)
    Teixobactin11611
    Vancomycin1>12811–2
    Bacitracin32641616
    Ampicillin0.520.52
    Gentamicin323288
    • ↵a Mean MICs and MBCs for at least three biological replicates are reported. Where the MBC values for the biological replicates differed, the MBC ranges are shown. WT, wild type.

Supplemental Material

  • Figures
  • Tables
  • TABLE S1

    Genes upregulated in response to teixobactin in Enterococcus faecalis JH2-2. #, log2 fold change (2-fold log2 minimum threshold). Download Table S1, DOCX file, 0.07 MB.

    Copyright © 2019 Darnell et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S2

    Genes downregulated in response to teixobactin in Enterococcus faecalis JH2-2. #, log2 fold change (2-fold log2 minimum threshold). Download Table S2, DOCX file, 0.06 MB.

    Copyright © 2019 Darnell et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S3

    A comparison of the differential changes in gene expression for 10 genes in response to teixobactin using qRT-PCR and RNA-seq. #, not detected (ND; below the 2-fold log2 threshold in RNA-seq analysis). Download Table S3, DOCX file, 0.01 MB.

    Copyright © 2019 Darnell et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S1

    Optimization of teixobactin concentration for RNA sequencing. E. faecalis JH2-2 was grown to mid-exponential phase (OD600, 0.5) and challenged with 0, 0.2, 0.5, and 1 μg ml−1 of teixobactin or DMSO at time zero. Growth was measured by determination of the cell density (OD600) for 3 h postchallenge. RNA was extracted at 1 h postchallenge. Results are the mean ± SD (data are for technical duplicates). Download FIG S1, TIF file, 0.2 MB.

    Copyright © 2019 Darnell et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S2

    qRT-PCR of E. faecalis JH2-2 gene expression in response to teixobactin. Quantitative real-time-PCR was carried out for 10 genes using E. faecalis JH2-2 cDNA to validate the changes in gene expression observed in the RNA sequencing data. Fold change is represented as a ratio of the mean CT values normalized to the CT value for the constitutively expressed EF0013. Results are the mean ± SD (data are for technical triplicates). Download FIG S2, TIF file, 0.1 MB.

    Copyright © 2019 Darnell et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S4

    Comparison of differential gene expression in response to cell wall-targeting antimicrobials teixobactin (Teix), bacitracin (Bac), vancomycin (Van), and ampicillin (Amp) in Enterococcus faecalis. #, 2-fold log2 minimum threshold; †, differential gene expression in E. faecalis JH2-2 (this study); ‡, differential gene expression in E. faecalis O1GRF (10). Download Table S4, DOCX file, 0.04 MB.

    Copyright © 2019 Darnell et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S3

    Glycine assay of the E. faecalis JH2-2 wild type (WT) and ΔcroRS mutant. SM17 broth (0.5 M sucrose plus M17 medium) containing a range of glycine concentrations was inoculated with an overnight culture of the E. faecalis JH2-2 WT or ΔcroRS mutant. Cultures were grown overnight at 37°C with no aeration, and growth was measured by determination of the OD600. Results are the mean ± SD (data are for biological triplicates). Download FIG S3, TIF file, 0.2 MB.

    Copyright © 2019 Darnell et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S5

    Teixobactin MIC and MBC for E. faecalis JH2-2 Δef0443 compared to those for its isogenic wild type (WT). Mean MICs for three biological replicates are reported. †, MIC; minimum bactericidal concentration. Download Table S5, DOCX file, 0.01 MB.

    Copyright © 2019 Darnell et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S6

    List of primer sequences used in this study. Download Table S6, DOCX file, 0.01 MB.

    Copyright © 2019 Darnell et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

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Genomewide Profiling of the Enterococcus faecalis Transcriptional Response to Teixobactin Reveals CroRS as an Essential Regulator of Antimicrobial Tolerance
Rachel L. Darnell, Melanie K. Knottenbelt, Francesca O. Todd Rose, Ian R. Monk, Timothy P. Stinear, Gregory M. Cook
mSphere May 2019, 4 (3) e00228-19; DOI: 10.1128/mSphere.00228-19

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Genomewide Profiling of the Enterococcus faecalis Transcriptional Response to Teixobactin Reveals CroRS as an Essential Regulator of Antimicrobial Tolerance
Rachel L. Darnell, Melanie K. Knottenbelt, Francesca O. Todd Rose, Ian R. Monk, Timothy P. Stinear, Gregory M. Cook
mSphere May 2019, 4 (3) e00228-19; DOI: 10.1128/mSphere.00228-19
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    • ABSTRACT
    • INTRODUCTION
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KEYWORDS

CroRS
Enterococcus
RNA sequencing
teixobactin
antimicrobial resistance
antimicrobial tolerance
mechanisms of resistance

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