1. Physics of Living Systems
  2. Structural Biology and Molecular Biophysics

Lactoferricins impair the cytosolic membrane of Escherichia coli within a few seconds and accumulate inside the cell

  1. Enrico F Semeraro  Is a corresponding author
  2. Lisa Marx
  3. Johannes Mandl
  4. Ilse Letofsky-Papst
  5. Claudia Mayrhofer
  6. Moritz PK Frewein
  7. Haden L Scott
  8. Sylvain Prévost
  9. Helmut Bergler
  10. Karl Lohner
  11. Georg Pabst  Is a corresponding author
  1. University of Graz, Institute of Molecular Biosciences, NAWI Graz, Austria
  2. BioTechMed Graz, Austria
  3. Field of Excellence BioHealth – University of Graz, Austria
  4. Institute of Electron Microscopy and Nanoanalysis and Center for Electron Microscopy, Graz University of Technology, NAWI Graz, Austria
  5. Center for Electron Microscopy, Austria
  6. Institut Laue-Langevin, France
  7. Center for Environmental Biotechnology, University of Tennessee, United States
  8. Shull Wollan Center, Oak Ridge National Laboratory, United States
https://doi.org/10.7554/eLife.72850
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Cite this article
  1. Enrico F Semeraro
  2. Lisa Marx
  3. Johannes Mandl
  4. Ilse Letofsky-Papst
  5. Claudia Mayrhofer
  6. Moritz PK Frewein
  7. Haden L Scott
  8. Sylvain Prévost
  9. Helmut Bergler
  10. Karl Lohner
  11. Georg Pabst
(2022)
Lactoferricins impair the cytosolic membrane of Escherichia coli within a few seconds and accumulate inside the cell
eLife 11:e72850.
https://doi.org/10.7554/eLife.72850
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9 figures, 3 tables and 1 additional file

Figures

Figure 1 with 4 supplements
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Overview of combined X-ray scattering and electron-microscopy measurements.

(A) Mapping the main structural changes in E. coli ATCC 25922 (green symbols) upon 1 hr incubation with LF11-324 (red symbols) as observed by (ultra) small-angle X-ray scattering (USAXS/SAXS) and …

Figure 1—figure supplement 1
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Cell number-dependent minimum inhibitory concentration (MIC) plots for different peptides.

MIC values as a function of ncell for LF11-215 (green squares), LF11-324 (red dots), and O-LF11-215 (blue triangles) and best fits using Equation 1 and Equation 2. Error bars represent the absolute …

Figure 1—figure supplement 2
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Comparison between (ultra) small-angle X-ray scattering (USAXS/SAXS) and contrast-variation small-angle neutron scattering (SANS) data and details of the scattering data analysis.

(A) X-ray and neutron scattering data of bacterial systems after 1 hr incubation with LF11-324 (end states) at the minimum inhibitory concentration (MIC) (SANS) and 1.2× MIC (SAXS). Lines are the …

Figure 1—figure supplement 3
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Transmission electron microscopy (TEM) observations for LF11-215, LF11-324, and O-LF11-215 at the minimum inhibitory concentrations (MICs) and sub-MICs.

TEM images of E. coli ATCC (A) and end states in the presence of LF11-215 (B, E), LF11-324 (C, F), and O-LF11-215 (D, G). All systems were probed at the MICs (B–D) and half the MICs (E–G).

Figure 1—figure supplement 4
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Graphical scheme of the adjustable parameters.
Figure 2 with 6 supplements
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Kinetics of the bacterial structural response upon addition of peptide.

(A–F) Kinetics of the bacterial structural response to attack by LF11-324; results for three different peptide concentrations are shown. Lipopolysaccharide (LPS) packing (A); cytoplasm and periplasm …

Figure 2—figure supplement 1
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Schematic of the stopped-flow rapid mixing (ultra) small-angle X-ray scattering (USAXS/SAXS) experiments, including selected scattering patterns.

Schematic of the stopped-flow rapid mixing setup used for USAXS/SAXS experiments and kinetic changes of USAXS/SAXS curves of bacterial samples upon mixing with LF11-324 at minimum inhibitory …

Figure 2—figure supplement 2
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Kinetics of the adjustable parameters for LF11-215 and O-LF11-215 systems.

(A–C) Kinetics of the adjustable parameters upon mixing with two concentrations of LF11-215. These are the intermembrane distance (~ periplasm thickness) (A), its deviation from the average value (B)…

Figure 2—figure supplement 3
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Kinetics of the adjustable parameters for LF11-215 systems.

(A–C) Kinetics of the adjustable parameters upon mixing with two concentrations of LF11-215. These parameters are the lipopolysaccharide (LPS) packing (A); the cytoplasm and periplasm scattering …

Figure 2—figure supplement 4
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Representative scattering patterns including errors and fitted curves.

(A) Representative (ultra) small-angle X-ray scattering (USAXS/SAXS) curves of bacterial samples upon mixing with LF11-324 at minimum inhibitory concentration (MIC) ×1.2, highlighting intensity …

Figure 2—figure supplement 5
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Representative distributions of the adjustable parameters at different time points.

Exemplary distributions obtained from our data analysis for adjustable parameters for LF11-324 system at minimum inhibitory concentration (MIC) ×1.2. Results refer to 0.02 s after mixing (light …

Figure 2—figure supplement 6
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Representative series of correlation plots and coefficients of the adjustable parameters.

Selected example of the list of correlation plots and associated correlation coefficients (defined in the range 0–1). The adjustable parameters refer to LF11-324 system at minimum inhibitory …

Figure 3
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Kinetics of outer membrane vesicle formation and O-LF11-215 absorption.

(A) Kinetics of the forward intensity of outer membrane vesicle (OMV) scattering for different concentrations of LF11-324 and LF11-215. The dashed horizontal line represents the …

Figure 4 with 1 supplement
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Full partitioning maps for LF11-324 and LF11-215.

Amount of LF11-324 or LF11-215 required to attain growth-inhibited fractions of either 0.999 (minimum inhibitory concentration [MIC], up triangles), 0.5 (circles), or 0.01 (down triangles) in E. coli…

Figure 4—figure supplement 1
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Inhibitory concentration (ICx) as a function of ϕIG (inverse cumulative distribution function [CDF]).

(A–C) ICx as a function of inhibited fractions ϕIG [inverse CDF, F-1⁢(x;b,c)] for different peptide and cell concentrations. Low ϕIG values for O-LF11-215 were not accessible due to the high noise-to-signal …

Figure 5 with 2 supplements
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Partitioning parameters as a function of cell-growth inhibition.

(A, B) NB and Ke⁢f⁢f values as a function of inhibited fraction. In the case of O-LF11-215, NB≡NBe⁢f⁢f. (C) Ratio between the maximum number of peptides on the outer leaflet and total number of partitioned …

Figure 5—figure supplement 1
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ζ-potential and size measurements of LF11-215 and LF11-324.

ζ-potential (A) and size (B) measurements as a function of peptide concentration (normalized by the respective minimum inhibitory concentrations (MICs) of LF11-215 and LF11-324 peptides). Lines are …

Figure 5—figure supplement 2
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ζ-potential and size measurements of O-LF11-215.

Comparison between ζ-potential (A, C) and size (B, D) measurements of O-LF11-215 antimicrobial peptide (AMP) alone and mixed with E. coli as a function of peptide concentration (normalized by the …

Figure 6
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Simplified time sequence of LF11-215 and LF11-324 mode of action.

The measured time onsets and boundaries refers to LF11-324 at minimum inhibitory concentration (MIC) ×0.3 and MIC ×1.2, which correspond to measured inhibited fractions of ~1 and > 99.9%, …

Appendix 1—figure 1
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Trp-fluorescence and SAXS analyis of O-LF11-215 clustering.

(A) Trp fluorescence data of LF11-215 (green squares) and O-LF11-215 (blue triangles) at 100 µg/ml (LF11-324 are not shown to avoid redundancy). Data were fitted with Equation 4. Arrows mark the …

Appendix 3—figure 1
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Statistical analysis of the AMP-induced bactericidal events.

(A) Selected ϕIG data for LF11-324 and corresponding fits with the Gompertz function. (B) Corresponding probability density functions (PDFs).

Appendix 4—figure 1
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AMP-partitioning study in live bacteria based on Trp-fluorescence.

(A) Example of Trp fluorescence analysis in LF11-324 systems. The solid line is the best fit, and the dotted and dashed lines represent the Trp emissions from peptide in bulk and cell-associated, …

Tables

Table 1
Change of E. coli structure due to LF11-324 ([P]∼MIC) as observed from USAXS/SAXS/SANS data analysis.

Values are the difference between end- and initial state. See a graphical scheme of the adjustable parameters in Figure 1—figure supplement 4, and Semeraro et al., 2021b for a more detailed schematic.

ParametersValuesDescription
ΔρCP×10−4(nm−2)–0.17 ± 0.02*; –0.14 ± 0.05†ρCP → SLD of cytoplasmic space
ΔρPP×10−4(nm−2)0.18 ± 0.06*; 0.12 ± 0.04†ρPP → SLD of periplasmic space
ΔΔOM(nm)6 ± 3ΔOM → inter-membranes distance
ΔσOM(nm)3.4 ± 1.7σOM→ SD around ΔOM
ΔρPG×10−4(nm−2)–0.27 ± 0.07*; –0.59 ± 0.14†ρPG→ SLD of peptidoglycan layer
Δ⁢pLPS–0.44 ± 0.08*; –0.26 ± 0.11†pLPS → LPS packing parameter
ΔR(nm)–27 ± 7R → minor radius of the cytoplasmic space

  1. Differences in X-ray and neutron SLDs are due to different physical interactions with matter. In case of pLPS, this originates from a biological variation of different bacterial cultures.

  2. USAXS/SAXS: (ultra) small-angle X-ray scattering; SANS: small-angle neutron scattering; SLD: scattering length density; LPS: lipopolysaccharide.

  3. *

    From SAXS.

  4. †

    From SANS (SLDs were obtained by extrapolating to 0 wt% D2O); see Figure 1—figure supplement 2B–D.

Table 2
List of fixed parameters for the combined analysis of USAXS/SAXS and contrast variation SANS data of E. coli.
DescriptionFixed parametersValues
Center-to-center distance between the head-group layers in the CMDCM(nm)3.73
Center-to-center distance between the head-group layers in the OMDOM(nm)3.33
Width of the head-group layers for both CM and OMWME(nm)0.75
Center-to-center distance between the PG layer and the OMΔPG(nm)16.7
Width of the PG layerWPG(nm)6.0
Average SLD of the tail group layer in the CMρTI(nm−2)×10−48.31*/0.022†
Average SLD of the tail group layer in the OMρTO(nm−2)×10−48.86*/0.012†
Ratio between major and minor radiiϵ2.0
Effective radius of gyration of each OS coreRg,OS(nm)0.45

  1. USAXS/SAXS: (ultra) small-angle X-ray scattering; SANS: small-angle neutron scattering; SLD: scattering length density.

  2. CM: Cytoplasmic membrane; OM: Outer membrane; PG: peptidoglycan; OS: Oligosaccharides.

  3. *

    X-ray SLDs.

  4. †

    Neutron SLDs.

Table 3
List of fixed and D2O-dependent parameters for the combined analysis of USAXS/SAXS and contrast variation SANS data of E. coli.

The average SLD of both CM and OM head-group layers, ρME, the SLD of the buffer solution, ρBF, and the product of the each OS core volume and its contrast relative to the buffer, βOS=VOSΔρOS.

Fixed parametersValues
Neutrons (wt% D2O)
X-rays1030405090
ρME(nm−2)×10−412.91.562.202.522.844.11
ρBF(nm−2)×10−49.4760.1351.542.202.815.54
βOS(nm)×10−410.73.832.321.680.69–2.44

  1. USAXS/SAXS: (ultra) small-angle X-ray scattering; SANS: small-angle neutron scattering; SLD: scattering length density.

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