Article Figures & Data
Figures
- 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
Pie charts showing the distribution of gene ontologies up- and downregulated in response to teixobactin. recomb., recombination.
- 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
- TABLE 2
The 20 most up- and downregulated genes in response to teixobactin in E. faecalis V583 and JH2-2
Gene regulation Gene in E. faecalis: Gene name F/Ca Function V583 JH2-2 Upregulated EF1518 1316 9.5 Soluble lytic murein transglycosylase EF0443 2523 8.7 Endopeptidase EF1533 1329 7.9 Conserved hypothetical protein EF0802 545 7.8 DUF3955 domain-containing protein EF1665 1454 7.6 Conjugal transfer protein TraX EF1231 1016 7.6 Metallophosphoesterase EF2896 2418 7.2 DUF3955 domain-containing protein EF0932 663 7.1 Hypothetical protein EF2986 287 6.8 ABC transporter ATP-binding protein EF2050 1817 6.3 Peptide ABC transporter: ATP-binding protein EF2987 286 6.3 RND transporter EF1532 1328 6.2 Hypothetical protein EF0038 2863 proB 6.2 Glutamate-5-kinase EF2771 2347 6.0 TraX family protein EF1258 1042 6.0 Hypothetical protein EF2214 1918 5.8 VOC family protein EF1198 982 5.7 ABC transporter permease EF0737 484 5.5 Amidase EF2211 1915 5.4 YxeA family protein EF0680 422 5.4 Penicillin binding protein 1A Downregulated EF0411 2555 −11.6 PTS mannitol transporter subunit IICB EF3139 138 −10.9 PTS sugar transporter subunit IIC EF0412 2554 −10.2 PTS mannitol transporter subunit IIA EF3141 136 −9.9 2-Hydroxyacid dehydrogenase EF2965 2466 −9.9 PTS sugar transporter subunit IIB EF3142 135 −9.7 6-Phosphogluconate dehydrogenase EF0413 2553 −9.7 Mannitol-1-phosphate 5-dehydrogenase EF3213 69 −9.6 PTS mannose transporter subunit IID EF3140 137 −9.4 Oxidoreductase EF3211 71 −9.4 PTS mannose/fructose/sorbose/N-acetylglucosamine subunit IIB EF2966 2467 −9.3 MltR-like mannitol-operon transcriptional regulator EF2582 2168 −9.0 Chlorohydrolase/aminohydrolase EF3138 139 −9.0 PTS mannose transporter subunit IID EF2964 2465 ulaA −8.9 PTS ascorbate transporter subunit IIC EF2223 1927 −8.8 ABC transporter family EF3212 70 −8.7 PTS mannose/fructose/sorbose/N-acetylglucosamine subunit IIC EF3327 2909 −8.7 Citrate transporter EF3210 72 −8.6 PTS mannose/fructose/sorbose/N-acetylglucosamine subunit IIA EF1031 742 −8.4 PTS sugar transporter subunit IIC EF1207 991 maeP −8.4 l-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
Antimicrobial WT ΔcroRS mutant MIC (μg ml−1) MBC (μg ml−1) MIC (μg ml−1) MBC (μg ml−1) Teixobactin 1 16 1 1 Vancomycin 1 >128 1 1–2 Bacitracin 32 64 16 16 Ampicillin 0.5 2 0.5 2 Gentamicin 32 32 8 8 ↵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.
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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
