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Thanatin targets the intermembrane protein complex required for lipopolysaccharide transport in Escherichia coli

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Science Advances  14 Nov 2018:
Vol. 4, no. 11, eaau2634
DOI: 10.1126/sciadv.aau2634
  • Fig. 1 LPS transport pathway and thanatin-based probes.

    (A) The LPS transport apparatus in Gram-negative bacteria comprises the seven proteins LptA to LptG, which form a macromolecular complex spanning the IM and OM. LPS transport across the periplasm occurs over a bridge formed by one or more copies of LptA. ADP, adenosine 5′-diphosphate. (B) Structures of thanatin and the photoprobe thanatin-PAL5 and fluorescence probe thanatin-BDP-FL.

  • Fig. 2 Electron and fluorescence microscopy studies.

    (A and B) TEM studies of E. coli ATCC 25922, before (A) and after (B) thanatin treatment (1.5 μg/ml), showing internal accumulations of membrane-like material. Scale bars, 500 nm. (C and D) Super-resolution fluorescence microscopy of E. coli ATCC 25922 without (C) or with thanatin (5 μg/ml) (D) and stained with FM4-64, SYTOX Green, or DAPI. Top: The FM4-64 channel (red staining). Bottom: Superimposition of all three channels [with DAPI (blue) and SYTOX Green (nondetected)]. (E) E. coli staining with thanatin-BDP-FL (8 μg/ml) for 2 hours at 30°C (both pictures). Cells were analyzed using a Leica CLSM SP8 gSTED microscope. Scale bars, 4 or 10 μm (bottom right). For experimental details, see section S5.

  • Fig. 3 Photolabeling of E. coli ATCC 25922 with thanatin-PAL5.

    (A) Western blot (biotin detection) and corresponding SDS–polyacrylamide gel electrophoresis (SDS-PAGE) (Coomassie blue staining) of membrane protein fraction from: lane 1, control unlabeled cells; lanes 2 and 3, cells photolabeled with thanatin-PAL5 (10 and 2 μg/ml); and lane 4, cells photolabeled with thanatin-PAL5 (10 μg/ml) + competitor thanatin (200 μg/ml). (B) Western blot and SDS-PAGE after photolabeling with thanatin-PAL5 (2 μg/ml) with (+) or without (−) reduction of extracted membrane proteins with dithiothreitol (DTT). (C) Volcano plot showing relative abundance of E. coli proteins in thanatin-PAL5 labeled versus unlabeled control sample after streptavidin pulldown detected by MS-based proteomic analysis. Significantly enriched proteins (right/above dashed lines) are highlighted in green and represent PAL5-labeled proteins.

  • Fig. 4 The solution structure of the LptAm-thanatin complex.

    (A) Ribbon representation of a single NMR LptAm-thanatin complex in two different orientations. LptAm and thanatin are in green and orange, respectively. The flexible C terminus of LptAm encompassing residues 144 to 159 and the His tag are not shown. (B) Structurally relevant intermolecular NOEs between backbone atoms of the first β-strands of LptA and thanatin are indicated with dashed arrows, and HN-HN and HN-Hα NOEs are colored in blue and green, respectively. (C) Ribbon model of the LptAm-thanatin complex. Residues involved in the protein-peptide hydrophobic interface (left) and in hydrogen bonding and electrostatic interactions (right) are indicated by ball-and-stick representation. (D) Superposition of the LptA dimer (PDB 2R1A; chains B (light blue) and C (violet) with the LptAm-thanatin complex (green/orange). Thanatin occupies a binding site on LptAm which is used to mediate LptA-LptA interactions needed to form the periplasmic bridge connecting IM and OM for LPS transport.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/11/eaau2634/DC1

    Section S1. Bacterial strains and plasmids used in this study

    Section S2. Peptide synthesis

    Section S3. Permeabilization of the E. coli cell envelope

    Section S4. Macromolecular synthesis assays

    Section S5. Photoaffinity labeling

    Section S6. Thanatin-resistant E. coli mutants

    Section S7. Production of LptA-His6, LptAm, LptD/E, and Thanatin in E. coli

    Section S8. Binding assays with LptA by FP and thermophoresis

    Section S9. Binding assays with LptD/E by FP and thermophoresis

    Section S10. NMR studies and structure determination of thanatin-LptAm complex

    Scheme S1. Synthesis of thanatin-PAL5.

    Scheme S2. Synthesis of thanatin-BDP-FL.

    Scheme S3. Structure of thanatin-Cy3.

    Fig. S1. Membrane permeabilization monitored by uptake (or absence thereof) of SYTOX Green.

    Fig. S2. Assays for release of ß-lactamase and of ß-galactosidase.

    Fig. S3. Relative incorporations of 3H label from labeled precursor over 20 min, at 37°C, performed in triplicate.

    Fig. S4. SDS-PAGE of purified LptD/EHis complex from E. coli.

    Fig. S5. Binding assays with LptA by FP.

    Fig. S6. Binding assays with LptA by thermophoresis.

    Fig. S7. Binding assays with LptD/E.

    Fig. S8. HSQC spectra of 15N-labeled LptA.

    Fig. S9. HSQC spectra of 15N-labeled thanatin in free form and bound to LptAm.

    Fig. S10. Weighted 15N,1H chemical shift changes (Δδ) between the free and thanatin-bound LptAm as a function of residue number.

    Fig. S11. Ribbon representation of the 20 lowest energy NMR conformers in two different orientations.

    Fig. S12. Sequence and structure comparisons of the N-terminal regions of LptA and LptD.

    Table S1. Bacterial strains and plasmids used in this study.

    Table S2. ThanR isolates from two independent passaging experiments, with MICs against thanatin after four generations without selection pressure, together with point mutations detected by lptA sequencing.

    Table S3. MIC values (μg/ml) of three selected mutants (ThanR-2, ThanR-4, and ThanR-8) and WT against thanatin and seven standard antibiotics (colistin, erythromycin, gentamicin, vancomycin, rifampicin, ampicillin, and ciprofloxaxin).

    Table S4. Genes mutated in the mutant strain compared to WT (+, indicates a mutation; −, indicates no mutation in the selected gene).

    Table S5. Antimicrobial activities of thanatin (MIC, μg/ml) against E. coli WT strains and strains containing plasmids shown.

    Table S6. Sequences of primers used for cloning experiments.

    Table S7. Statistics from the NMR structure calculations for LptAm-thanatin.

    References (3746)

  • Supplementary Materials

    This PDF file includes:

    • Section S1. Bacterial strains and plasmids used in this study
    • Section S2. Peptide synthesis
    • Section S3. Permeabilization of the E. coli cell envelope
    • Section S4. Macromolecular synthesis assays
    • Section S5. Photoaffinity labeling
    • Section S6. Thanatin-resistant E. coli mutants
    • Section S7. Production of LptA-His6, LptAm, LptD/E, and Thanatin in E. coli
    • Section S8. Binding assays with LptA by FP and thermophoresis
    • Section S9. Binding assays with LptD/E by FP and thermophoresis
    • Section S10. NMR studies and structure determination of thanatin-LptAm complex
    • Scheme S1. Synthesis of thanatin-PAL5.
    • Scheme S2. Synthesis of thanatin-BDP-FL.
    • Scheme S3. Structure of thanatin-Cy3.
    • Fig. S1. Membrane permeabilization monitored by uptake (or absence thereof) of SYTOX Green.
    • Fig. S2. Assays for release of ß-lactamase and of ß-galactosidase.
    • Fig. S3. Relative incorporations of 3H label from labeled precursor over 20 min, at 37°C, performed in triplicate.
    • Fig. S4. SDS-PAGE of purified LptD/EHis complex from E. coli.
    • Fig. S5. Binding assays with LptA by FP.
    • Fig. S6. Binding assays with LptA by thermophoresis.
    • Fig. S7. Binding assays with LptD/E.
    • Fig. S8. HSQC spectra of 15N-labeled LptA.
    • Fig. S9. HSQC spectra of 15N-labeled thanatin in free form and bound to LptAm.
    • Fig. S10. Weighted 15N,1H chemical shift changes (Δδ) between the free and thanatin-bound LptAm as a function of residue number.
    • Fig. S11. Ribbon representation of the 20 lowest energy NMR conformers in two different orientations.
    • Fig. S12. Sequence and structure comparisons of the N-terminal regions of LptA and LptD.
    • Table S1. Bacterial strains and plasmids used in this study.
    • Table S2. ThanR isolates from two independent passaging experiments, with MICs against thanatin after four generations without selection pressure, together with point mutations detected by lptA sequencing.
    • Table S3. MIC values (μg/ml) of three selected mutants (ThanR-2, ThanR-4, and ThanR-8) and WT against thanatin and seven standard antibiotics (colistin, erythromycin, gentamicin, vancomycin, rifampicin, ampicillin, and ciprofloxaxin).
    • Table S4. Genes mutated in the mutant strain compared to WT (+, indicates a mutation; −, indicates no mutation in the selected gene).
    • Table S5. Antimicrobial activities of thanatin (MIC, μg/ml) against E. coli WT strains and strains containing plasmids shown.
    • Table S6. Sequences of primers used for cloning experiments.
    • Table S7. Statistics from the NMR structure calculations for LptAm-thanatin.
    • References (3746

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