Lactoferricins access the cytosol of Escherichia coli within few seconds

We report the real-time response of E. 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 multi-scale scattering data analysis to biophysical assays for peptide partitioning revealed that the AMPs rapidly saturate the bacterial envelope and reach the cytosol within less than three seconds—much faster than previously considered. Final cytosolic AMP concentrations of ~ 100 mM suggest an efficient shut-down of metabolism as primary cause for bacterial killing. On the other hand, the damage of the cell envelope is 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 reaching cytoplasm and lowest cytosolic peptide concentration.


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Progress in designing antibiotics with novel key-lock mechanisms is not keeping pace with the 29 worldwide growing number of (multi) resistant bacterial strains, encouraging significant research 30 efforts in promising alternatives such as antimicrobial peptides (AMPs) (Lohner, 2001). AMPs are 31 part of the natural innate immune system and provide a first line of defence against pathogens. 32 Their advantage as compared to conventional antibiotics relies on a rapid impairment of the barrier-

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Defining structural reference states of AMP activity in E. coli 90 Unravelling the time-line of structural events occurring in E. coli due to lactoferricin activity by US-  of mainly low-weight molecules from the cytoplasm (Figure 1-Figure Supplement 2B-C). Notably, 134 the observed cell shrinkage of ∼ 5% leads to a decrease of cell surface of approximately 2 × 10 6 135 nm 2 . Apparently this is at least in part compensated by OMV formation, as suggested by their total 136 surface estimate of ∼ (2 − 6) × 10 6 nm 2 from SAS analysis (see Appendix 2).
where max and 0 are, respectively, wavelength and intensity of the emission peak; Γ is the full-  the high-q asymptotic trend of scattering signal from the aggregates (Glatter et al., 1982), where 526 is a scaling factor that depends on the surface properties of the aggregates, and is related to 527 their fractal dimension (Sorensen, 2001) (see Appendix 1).

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Data analysis: bacterial modeling 529 X-ray and neutron scattering data were jointly analyzed with a recently reported analytical scatter-530 ing model (Semeraro et al., 2021). USAXS/SAXS patterns of end-states displayed an excess scat-  (Figure 1), this suggests that the additional scattering contribution in SAXS data could be due 536 to freely diffusing OMVs in the suspension medium.

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All scattering data were fitted with the analytical functions  Table 1). This allowed us to fully describe the scattering-pattern variations upon peptide activity.

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Other parameters, including those accounting for the structure of inner and outer membranes,  Table 2 and Table 3. 562 The scattering intensities of O-LF11-215-aggregates was comparable to that of bacteria in the 563 high q-range (Figure 2-Figure Supplement 1D). While this affected the quality of the ultrastruc-564 tural parameters, it enabled at the same time the investigation of the kinetics of the AMP uptake.

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Indeed, by assuming that O-LF11-215 is primarily forming aggregates in solution, 0 AMP at Δ = 17.5 566 Table 2. List of fixed parameters for the combined analysis of USAXS/SAXS and contrast variation SANS data of E. coli.

Description Fixed parameters Values
Center-to-center distance between the head-group layers in the CM CM (nm) 3.73 Center-to-center distance between the head-group layers in the OM OM (nm) 3.33 Width of the head-group layers for both CM and OM ME (nm) 0.75 Center-to-center distance between the PG layer and the OM Δ PG (nm) 16.7 Width of the PG layer PG (nm) 6.0 Average SLD of the tail group layer in the CM TI (nm −2 ) ×10 −4 8.31 ( ) /0.022 ( ) Average SLD of the tail group layer in the OM TO (nm −2 ) ×10 −4 8.86 ( ) /0.012 ( ) Ratio between major and minor radii 2.0 Effective radius of gyration of each OS core g,OS (nm) 0.45 ( ) X-ray SLDs. ( ) Neutron SLDs. Table 3. List of fixed and D 2 O-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, ME , the SLD of the buffer solution, BF , and the product of the each OS core volume and it contrast relative to the buffer, where kin is the number of O-LF11-215 partitioned within the volume of a single cell that can be Manuscript submitted to eLife cultures, and to S. Keller for the fruitful discussions. Finally, the authors thank the whole staff of 589 ID02 and the D11 for support and availability.

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Competing interests 591 The authors declare that no competing interests exist.

Clusters of acylated peptide O-LF11-215 721
Peptide clusters formed by O-LF11-215 were investigated by Trp fluorescence and USAXS/SAXS. Trp spectra displayed a max ≃ 336 nm and a FWHM of about 67 nm, which can be related to a heterogeneous distribution of Trp with apolar surroundings (Ladokhin et al., 2000). In addition, O-LF11-215 exhibited Trp phosphorescence emission at about 450 nm, which usually is not measurable due to its dynamic quenching by oxygen and impurities in aqueous suspensions (Cioni and Strambini, 2002). Its presence suggests that a significant portion of Trp residues are buried in hydrophobic cores, with no access to the solvent and with a local high viscosity. USAXS/SAXS data in the low q-range ( ∼0.005 nm −1 ) exhibited a featureless decay of intensity with a slope of = 2.4. This slope value is typical for mass fractals, i.e. highly branched objects with high surface-to-volume ratio, while suggests a minimum aggregate size of ∼ 2 ∕ >1 . Furthermore, a Guinier term is needed to fit the shoulder at about = 0.2 nm −1 corresponding to an average radius of gyration ≃ 10 nm. Note that this feature also does not vanish for different choices of scaling constants for background subtraction. Interestingly, this value is way too high to describe O-LF11-215 monomers, whose expected radius of gyration would be < 1 nm. Possibly, peptide monomers create smaller aggregates of mean size ≃ 10 nm, which in turn form an heterogeneous and branched supramolecular structure with the characteristics of a mass fractal. The prefactor of the scattering contribution from extracellular, independent objects used in Equation 6 is 0 OMV = OMV 2 OMV Δ 2 OMV , where OMV is the number of OMVs, OMV is the volume of an OMV and Δ OMV is average the SLD difference to the buffer. This definition of forward scattering (per single cell) is valid for every non-interacting object. Hence, to validate the assumption that this scattering contribution is related to OMVs, it is interesting to calculate possible OMV , OMV and Δ OMV values. Note that even if modelling OMVs as homogeneous spheres appears as a crude first order approximation, OMV can be associated to its radius (within ∼10% confidence). Assuming the same lipid asymmetry as in the outer membrane, the inner leaflet of OMVs can be mimicked by a 3:1 mole mixture of palmitoyl-oleoylphosphatidylethanolamine (POPE) and palmitoyl-oleoyl-phosphatidylglygerol (POPG), respectively (De Siervo, 1969; Lohner et al., 2008; Leber et al., 2018). The SLD membrane profiles of these lipids have been thoroughly investigated (Kučerka et al., 2012, 2015). The outer leaflet might instead be dominated by LPS, whose lipid A possesses about 6 short C14:0 chains (Kim et al., 2016), and the polar region can be approximated as two PG units, in terms of molecular volume and SLD. In addition, LPS inner and outer core volumes and SLDs can be calculated from Heinrichs et al. (1998)   Assuming that the AMP-induced delayed bacterial growth is entirely due to a lower number density of survived cells (Marx et al., 2021b), the inhibited fraction of cells, IG , as a function of peptide and cell concentrations was fitted with a heuristic approach. Specifically, we used the sigmoidal Gompertz function where [ ] is the total peptide molar concentration, and and are related, respectively, to the position and width of the sigmoidal. In addition, the set of and values as a function of cell can be interpolated to obtain, for example, a continuous trend of IC as a function of IG .