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

  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

  • Curated by eLife

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    Evaluation Summary:

    This paper presents groundbreaking data on the effects of antimicrobial peptides on bacterial cells, obtained by time resolved small angle X-ray and neutron scattering experiments coupled to stopped-flow mixing. Application of this approach to cells is highly innovative and provides ms time resolution, and information on multiple length scales (from conformational changes in the cell, to structural changes in the membranes). This is an important extension of the effort of the scientific community to study model membranes. The main result is that the peptides reach the cytosol in a few seconds, accumulating to high concentrations. The data analysis should be improved, and many conclusions are speculative, in particular on the mechanism of entry of the peptides.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

We report the real-time response of Escherichia coli to lactoferricin-derived antimicrobial peptides (AMPs) on length scales bridging microscopic cell sizes to nanoscopic lipid packing using millisecond time-resolved synchrotron small-angle X-ray scattering. Coupling a multiscale scattering data analysis to biophysical assays for peptide partitioning revealed that the AMPs rapidly permeabilize the cytosolic membrane within less than 3 s—much faster than previously considered. Final intracellular AMP concentrations of ∼80–100 mM suggest an efficient obstruction of physiologically important processes as the primary cause of bacterial killing. On the other hand, damage of the cell envelope and leakage occurred also at sublethal peptide concentrations, thus emerging as a collateral effect of AMP activity that does not kill the bacteria. This implies that the impairment of the membrane barrier is a necessary but not sufficient condition for microbial killing by lactoferricins. The most efficient AMP studied exceeds others in both speed of permeabilizing membranes and lowest intracellular peptide concentration needed to inhibit bacterial growth.

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  1. Version published to 10.7554/elife.72850 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    The effects of antimicrobial peptides (AMPs) on bacteria is a major question in biology and is of great importance in medicine. Biophysical studies, such as the one described in the present manuscript, attempt to gain physical insight into the molecular mechanisms behind such effects. These may vary from structural effects of AMPs on the bacterial envelope membranes, to direct effects on metabolism from their presence in the cytosol.

    In this manuscript the authors build upon a recently published seminal paper (Semeraro et al, Acta Crystallographica 2021), where they were able to fit x-ray (USAX and SAXS) and neutron (VSANS and SANS) scattering data from live E.coli cells over four orders of magnitude in length scale. In the present manuscript, they have employed time-resolved USAX/SAXS, using stopped-flow, coupled with contrast variation SANS, transmission electron microscopy, and activity assays, to study the interaction of AMPs with live E.coli cells.

    The shifts in the scattering curve (Figure 1) induced by the AMP appear to be quite subtle, and yet the detailed analysis described here and in the authors' previous paper is able to detect changes in a number of structural parameters (Table 1). How much confidence do the authors have in the estimated errors cited in this table?

    This is indeed a crucial point. Even if the differences in USAXS/SAXS data (reciprocal space) look small at the first glance, this translates in real space into significant effects. Focusing on Figure 1 – Figure supplement 2, and specifically on panel E, the significance of these differences becomes clear even for scattering data. Here, USAXS/SAXS data are scaled to the same sample concentration, allowing a direct comparison of E. coli initial (w/o AMP) and end-states.

    The analytical model, developed in Semeraro et al., J Appl. Cryst. 2021, was fitted to experimental data using a chi-squared-minimization based on a Monte Carlo genetic-selection algorithm, which is a robust method to deal with a high number of adjustable parameters. This yielded distribution functions for all adjustable parameters. We report the mean and standard deviations of these distributions, which yields highly realistic uncertainties. We are thus highly confident in the stated errors. In the revised manuscript we now show these distribution functions (Fig. 2- supplement 5) and also correlation plots between these parameters (Fig. 2- supplement 6). We have revised the manuscript thoroughly to guide the reader better through all aspects of the analysis.

    Reviewer #2 (Public Review):

    This article presents a novel and powerful approach, based on small angle scattering, to study the effects of antimicrobial molecules on bacterial cells in real time, obtaining information at multiple spatial scales (nm-um). As such, it is highly interesting.

    The main result of the present study is that the peptides accumulate in the cytosol within a few seconds. This finding is solid and peptide accumulation inside the cell is in agreement with several previous studies. However, from this observation the authors conclude that blockage of metabolic activity and not membrane perturbation is the mechanism of bacterial killing. In my opinion, this conclusion is not adequately supported by the data, since bacterial killing over time and metabolic activity were not studied, and membrane perturbation took place essentially on the same time-scale of peptide accumulation in the cytosol. Data interpretation should be revised with increased caution

    We thank the reviewer for the highly positive evaluation of our manuscript. We agree that based on our current data, we cannot comment on any blocking of metabolic activity. Our data clearly shows, however, that the vitally detrimental mode action of the presently studied AMPs occurs in the cytosol (without knowing any further detail). We carefully reformulated accordingly.

    Reviewer #3 (Public Review):

    Semeraro et al. present a very interesting work on the impact of antimicrobial peptide Lactoferricin on the structure of bacteria. They use primarily small-angle neutron/X-ray scattering to look for structural hallmarks of the effect of the AMP. Based mainly on SANS/SAXS results they conclude that the peptide enters the cytosol "within seconds" and cause irreversible damage. The work is nicely carried out and well written, but I wonder whether it is a bit too ambitious and bold in its claims. In particular considering the shaky ground of which the SAXS/SANS fit analysis is constructed. I agree that TEM backs it up but the results seem only to clarify structural changes at larger scales / morphological features. The manuscript should be published in some form after carefully taking into account several concerns from the reviewer (listed above).

    We thank the reviewer for the positive evaluation of our manuscript. The claims put forward in our study are based on a solid and thorough data analysis, combining besides neutron/ X-ray scattering and TEM also results obtained from bioscreen, zeta potential, dynamic light scattering and fluorescence spectroscopy experiments. We understand, however, that the SAS data analysis is complex and deserves a better explanation. We originally thought that it is sufficient to refer mainly to our paper, where we give all details about the USAXS/SAXS/VSANS/SANS analysis (Semeraro et al., J. Appl. Cryst, 2021). The revised version of our manuscript now gives some additional details that will help to appreciate the robustness of our statements regarding the activity of LF11-324, LF11-215 and O-LF11-215 in live E. coli.

  3. eLife

    Evaluation Summary:

    This paper presents groundbreaking data on the effects of antimicrobial peptides on bacterial cells, obtained by time resolved small angle X-ray and neutron scattering experiments coupled to stopped-flow mixing. Application of this approach to cells is highly innovative and provides ms time resolution, and information on multiple length scales (from conformational changes in the cell, to structural changes in the membranes). This is an important extension of the effort of the scientific community to study model membranes. The main result is that the peptides reach the cytosol in a few seconds, accumulating to high concentrations. The data analysis should be improved, and many conclusions are speculative, in particular on the mechanism of entry of the peptides.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    The effects of antimicrobial peptides (AMPs) on bacteria is a major question in biology and is of great importance in medicine. Biophysical studies, such as the one described in the present manuscript, attempt to gain physical insight into the molecular mechanisms behind such effects. These may vary from structural effects of AMPs on the bacterial envelope membranes, to direct effects on metabolism from their presence in the cytosol.

    In this manuscript the authors build upon a recently published seminal paper (Semeraro et al, Acta Crystallographica 2021), where they were able to fit x-ray (USAX and SAXS) and neutron (VSANS and SANS) scattering data from live E.coli cells over four orders of magnitude in length scale. In the present manuscript, they have employed time-resolved USAX/SAXS, using stopped-flow, coupled with contrast variation SANS, transmission electron microscopy, and activity assays, to study the interaction of AMPs with live E.coli cells.

    The shifts in the scattering curve (Figure 1) induced by the AMP appear to be quite subtle, and yet the detailed analysis described here and in the authors' previous paper is able to detect changes in a number of structural parameters (Table 1). How much confidence do the authors have in the estimated errors cited in this table?

    I think the approach outlined in this manuscript does help to answer important questions on the sequence of events, and their timescales, following the initial binding of AMPs to the outer membrane, with subsequent translocation to the cytosol, where it can interfere with DNA and other metabolic functions.

    I am generally speaking confident in the modelling of the scattering developed by the authors, even though this has evolved somewhat from the approach taken in their 2017 paper. This group know how to analyse scattering curves, and while some workers in the field might be somewhat skeptical about the value of the approach, to my mind it is something of a tour-de-force, and does yield some valuable and interesting results.

  5. eLife

    Reviewer #2 (Public Review):

    This article presents a novel and powerful approach, based on small angle scattering, to study the effects of antimicrobial molecules on bacterial cells in real time, obtaining information at multiple spatial scales (nm-um). As such, it is highly interesting.

    The main result of the present study is that the peptides accumulate in the cytosol within a few seconds. This finding is solid and peptide accumulation inside the cell is in agreement with several previous studies. However, from this observation the authors conclude that blockage of metabolic activity and not membrane perturbation is the mechanism of bacterial killing. In my opinion, this conclusion is not adequately supported by the data, since bacterial killing over time and metabolic activity were not studied, and membrane perturbation took place essentially on the same time-scale of peptide accumulation in the cytosol. Data interpretation should be revised with increased caution.

  6. eLife

    Reviewer #3 (Public Review):

    Semeraro et al. present a very interesting work on the impact of antimicrobial peptide Lactoferricin on the structure of bacteria. They use primarily small-angle neutron/X-ray scattering to look for structural hallmarks of the effect of the AMP. Based mainly on SANS/SAXS results they conclude that the peptide enters the cytosol "within seconds" and cause irreversible damage. The work is nicely carried out and well written, but I wonder whether it is a bit too ambitious and bold in its claims. In particular considering the shaky ground of which the SAXS/SANS fit analysis is constructed. I agree that TEM backs it up but the results seem only to clarify structural changes at larger scales / morphological features.

  7. Version published to 10.1101/2021.09.24.461681v1 on bioRxiv