C O M P L E X S Y S T E M S A N D B I O M E D I C A L S C I E N C E S

S C I E N T I F I C H I G H L I G H T S

5 8 H I G H L I G H T S 2 0 2 2 I

PRINCIPAL PUBLICATION AND AUTHORS

Lactoferricins impair the cytosolic membrane of Escherichia coli within a few seconds and accumulate inside the cell, E.F. Semeraro (a,b,c), L. Marx (a,b,c), J. Mandl (a,b,c), I. Letofsky-Papst (d), C. Mayrhofer (e), M.P.K. Frewein (a,b,c,f), H.L. Scott (g,h), S. Prévost (f), H. Bergler (a,b,c), K. Lohner (a,b,c), G. Pabst (a,b,c), eLife 11, e72850 (2022); https:/doi.org/10.7554/eLife.72850 (a) University of Graz, Institute of Molecular Biosciences, NAWI Graz, Graz (Austria) (b) BioTechMed Graz, Graz (Austria) (c) Field of Excellence BioHealth University of Graz, Graz (Austria) (d) Institute of Electron Microscopy and Nanoanalysis and Center for Electron Microscopy, Graz University of Technology, NAWI Graz, Graz (Austria) (e) Center for Electron Microscopy, Graz (Austria) (f) Institut Laue- Langevin, Grenoble (France) (g) Center for Environmental Biotechnology, University of Tennessee, Knoxville (USA) (h) Shull Wollan Center, Oak Ridge National Laboratory, Oak Ridge (USA)

REFERENCES

[1] E.F. Semeraro et al., IUCrJ 4, 751-757 (2017). [2] E.F. Semeraro et al., J. Appl. Cryst. 54, 473-485 (2021). [3] L. Marx et al., Front. Med. Tech. 3, 625975 (2021).

i.e., from the sub-micron scales of cell size and density to the nanometre length-scale pertaining to the lipid packing within the outer membrane [1-2] (Figure 47). Data modelling was supported with corresponding transmission electron microscopy (TEM) experiments and combined with an integrated AMP partitioning analysis [3]. That is, the antimicrobial activity was correlated to variations of cell structure and to the AMP distribution within the cells.

The advanced data modelling made it possible to demonstrate that LF11-based AMPs cross bacterial membranes and reach the cytosol within a few seconds. Subsequently, peptides accumulate in all bacterial compartments up to a huge local concentration (~100 mM). Further, the following properties of LF11-based AMPs were identified as key to their high antimicrobial efficacy: (i) a quick translocation through both inner and outer membranes, and (ii) the impairment of various physiological processes due to an overwhelming peptide

concentration into the cytosol. In agreement with previous studies, severe damage of the cell wall was observed (e.g., membrane ruffling, looser lipid lateral-packing, weak cell leakage, etc.). However, this effect also occurred at very low peptide concentrations, where only 1% of the bacteria were inhibited from growing. Moreover, the membrane disruption effect was identical, when quasi all (>99.9%) bacteria were killed. Consequently, the impairment of the cellular membrane can be excluded from being the sole target of AMP activity, as claimed by the majority of studies in the field.

In conclusion, it was found that the membrane activity of the here-studied lactoferricin derivatives is collateral damage: it is necessary to weaken the protective barrier and facilitate AMP internalisation but not sufficient to cause cell death. Recent studies on different compounds are in line with these findings, suggesting that in-depth investigations of AMP activities within cells are needed.

Fig. 47: Overview of combined USAXS/SAXS

and TEM measurements. Scattering curves of bare

E. coli ATCC 25922 (green symbols, data adapted

from [2]) and upon 1-hour incubation with LF11- 324 at the minimum inhibitory

concentration (MIC) (red symbols).