# Cathelicidin-BF: A Potent Antimicrobial Peptide Leveraging Charge and Phospholipid Recruitment against Multidrug-Resistant Clinical Bacterial Isolates

Evgeniy Salnikov, Morgane Adélaïde, Francisco Ramos-Martín, Ahmad Saad, Jennifer Schauer, Martina Cremanns, Mariam Rima, Christopher Aisenbrey, Saoussen Oueslati, Thierry Naas, et al.

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To cite this version:

Evgeniy Salnikov, Morgane Adélaïde, Francisco Ramos-Martín, Ahmad Saad, Jennifer Schauer, et al. "Cathelicidin-BF: A Potent Antimicrobial Peptide Leveraging Charge and Phospholipid Recruitment against Multidrug-Resistant Clinical Bacterial Isolates." Journal of the American Chemical Society, 2025, 147 (13), pp.11199-11215. 10.1021/jacs.4c17821. hal-05006267

HAL Id: hal-05006267

https://u-picardie.hal.science/hal-05006267v1

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# Cathelicidin-BF: A Potent Antimicrobial Peptide Leveraging Charge and Phospholipid Recruitment against Multidrug-Resistant Clinical Bacterial Isolates

Evgeniy Salnikov<sup>a,b,†</sup>, Morgane Adélaïde<sup>a,†</sup>, Francisco Ramos-Martín<sup>a,*</sup>, Ahmad Saad<sup>b</sup>, Jennifer Schauer<sup>c</sup>, Martina Cremanns<sup>c</sup>, Mariam Rima<sup>d</sup>, Christopher Aisenbrey<sup>b</sup>, Saoussen Oueslati<sup>d</sup>, Thierry Naas<sup>d</sup>, Niels Pfennigwerth<sup>c</sup>, Söeren Gatermann<sup>c</sup>, Catherine Sarazin<sup>a</sup>, Burkhard Bechinger<sup>b,e</sup>, and Nicola D'Amelio<sup>a,*</sup>

**Abstract:** Cathelicidin-BF (CatBF) is an antimicrobial peptide (AMP) isolated from *Bungarus fasciatus* with an exceptional portfolio of antimicrobial, antiviral, antifungal, and anticancer activities. Contrary to many AMPs, it showed a good pharmacological profile with a half-life of at least one hour in serum, and efficacy against bacterial infections in mice. To evaluate its potential against resistant nosocomial infections, we assessed its activity against 81 clinically relevant resistant bacterial isolates. CatBF exhibited minimum inhibitory concentrations (MICs) as low as 0.5 µM against carbapenem-resistant *Acinetobacter baumannii*, *Klebsiella pneumoniae*, and *Escherichia coli*. Independence of resistance mechanism and Gram phenotype, prompted us to elucidate its molecular mode of action. NMR spectroscopy, paramagnetic probes and molecular dynamics (MD) simulations were employed to define its structure, penetration depth and orientation in various membrane models, including micelles, bicelles, oriented bilayers, and vesicles. We found that CatBF's potent activity relies on its strong charge, allowing membrane neutralization at low peptide/lipid ratios and selective recruitment of charged phospholipids. At higher concentrations, a change in peptide orientation reveals membrane invagination, a possible prelude to its rupture, with consequent bacterial death. Our findings highlight the potential of CatBF as a model for developing resistance-independent agents to combat MDR bacterial infections.

## Introduction

The emergence of multidrug-resistant (MDR) pathogens poses a significant challenge in healthcare settings, contributing to tens of thousands of fatalities annually ¹,². These MDR microbes are particularly concerning in the context of nosocomial infections, frequently afflicting patients within healthcare facilities ¹,³,⁴. Notably, MDR pathogens exhibit a propensity for colonizing various medical devices, including catheters, implants, stents, contact lenses, and ventilators ¹,⁵. Disinfection of these

colonized surfaces becomes particularly challenging for highly resistant pathogens due to their ability to form spores or biofilms ¹,⁵.

Antimicrobial peptides (AMPs) constitute a critical component of the innate immune system in a wide range of organisms ⁶,⁷. These peptides are typically short, positively charged molecules enriched in hydrophobic residues. This unique composition enables AMPs to selectively target the microbial membrane ⁸, which typically exposes negatively charged lipids on its outer leaflet while remaining hydrophobic at its core ⁹,¹⁰. AMPs are known for their reduced tendency to induce resistance in their target ¹¹⁻¹⁴, due to their high killing rate (higher than common antibiotics), which do not allow microbial replication ¹⁵⁻¹⁷. Furthermore, resistance often implies remodeling of the membrane, a process that requires more mutational effort compared to the modification of a single molecular target (often shared among different molecules, developing cross-resistance), as in the case of many antibiotics ¹⁴. The application of AMPs in coatings for medical devices such as implants or catheters holds significant promise for combating and preventing nosocomial infections, including metallic devices ¹,⁴,⁵,¹⁸,¹⁹. Notably, immobilizing AMPs on these surfaces can mitigate some of their inherent limitations, such as their susceptibility to degradation by proteases, which shortens their half-life in biological fluids ²⁰.

Cathelicidins are a family of AMPs produced by many vertebrates. They are released by proteolytic cleavage from their inactive precursors upon leukocyte activation (usually neutrophils and macrophages) ⁸,²¹. Their name is due to "cathelin", a relatively well conserved domain, at their N-termini. The homology is higher in the precursor form, while mature forms show different amino acid sequences, structures, and lengths ⁸ especially at the level of the C-terminus ⁸,²²,²³. Cathelicidins display bactericidal but also antiviral, antifungal, or anticancer activities. Additionally, they play important immunomodulatory roles and regulate inflammation processes. They usually exert their action by affecting membrane architecture, but they can modulate the immune response by targeting receptors ⁸,²¹,²⁴ or act as cell

<footnote><sup>a</sup> Unité de Génie Enzymatique et Cellulaire, UMR 7025 CNRS, Université de Picardie Jules Verne, Amiens, 80039, France</footnote>

<footnote><sup>b</sup> Université de Strasbourg/CNRS, UMR7177, Institut de Chimie, Strasbourg, France</footnote>

<footnote><sup>c</sup> Department of Medical Microbiology, Ruhr-University, Bochum, Germany</footnote>

<footnote><sup>d</sup> Université Paris Sud Faculté de Médecine Hôpital de Bicêtre Service de Bactériologie Bâtiment Broca, 3ème étage, 78 rue du Gal Leclerc, Paris, 94270, France</footnote>

<footnote><sup>e</sup> Institut universitaire de France, 75005 Paris, France</footnote>

<footnote>† These authors contributed equally to this work.</footnote>

<footnote>* Correspondence: nicola.damelio@u-picardie.fr; francisco.ramos@u-picardie.fr; Tel.: +33 3 22 82 74 73</footnote>

<footnote>Supporting information for this article is given via a link at the end of the document.</footnote>
penetrating peptides<sup>25-29</sup> that pass through the external membrane without causing damage <sup>30</sup>.

Cathelicidin-BF (CatBF) is a peptide isolated from the venom of the snake *Bungarus fasciatus* <sup>31</sup> with antibacterial <sup>31-33</sup>, antifungal <sup>31,34,35</sup>, anticancer <sup>25-27,36</sup>, and immunomodulatory <sup>37-40</sup> properties. It has been successfully used against *Staphylococcus aureus*, *Propionibacterium acnes*, *Salmonella typhi*, *Shigella dysenteriae*, and *Escherichia coli* <sup>41-43</sup>. Despite exhibiting degradation in simulated intestinal fluid, CatBF demonstrates a favorable stability profile in serum and simulated gastric fluid, with a half-life exceeding one hour. Moreover, in murine models, CatBF has shown efficacy against both *Pseudomonas aeruginosa* and *Staphylococcus aureus* <sup>28,44,45</sup>.

In this work, CatBF demonstrated potent antimicrobial activity against a diverse panel of 81 drug-resistant clinical isolates, including carbapenem-resistant *Acinetobacter baumannii*. By means of solid and solution NMR and atomistic molecular dynamics (MD) simulations, we show how CatBF selectively disrupts bacterial membranes.

## Results and Discussion

### CatBF displays sub-micromolar activity on several multiresistant clinical isolates

The performance of CatBF was assessed in vitro on 81 clinical bacterial isolates collected from two National Reference Centers for antimicrobial resistance from France and Germany (Table 1). This panel included 58 Enterobacterales, 21 non-fermenters (*A. baumannii*, *P. aeruginosa*, *Stenotrophomonas maltophilia*, and *Burkholderia cepacia*), and 2 Gram-positive isolates (methicillin-resistant *S. aureus* and *Enterococcus faecalis*). The choice of these isolates was based on their clinical relevance, their multi- and even pan-drug resistance to antibiotics, and the difficulty of finding effective therapies in cases of infection <sup>2</sup>.

Our results showed that the CatBF MIC values of all the strains were 2 mg/L or higher, with a MIC<sub>50</sub> of 4 mg/L and a MIC<sub>90</sub> of 16 mg/L. The MIC values for 18 *P. aeruginosa* and *A. baumannii* isolates were between 2 mg/L and 16 mg/L, with an MIC<sub>50</sub> of 6 mg/L. Interestingly, the MIC values obtained in the cation-adjusted medium (containing higher amounts of Ca<sup>2+</sup> and Mg<sup>2+</sup>) were significantly higher than in the regular medium. Indeed, some cationic amphipathic AMPs need to displace Ca<sup>2+</sup> and Mg<sup>2+</sup> ions, which bridge lipopolysaccharide (LPS) molecules, in order to disrupt the outer membrane of Gram-negative bacteria <sup>46-50</sup>.

CatBF demonstrates efficacy against both Gram-negative and Gram-positive bacteria (Table 1, S1, S2), likely targeting a common molecular site: the inner and outer membranes of Gram-negative and Gram-positive bacteria, respectively. This explains why MIC values were not correlated with specific antibiotic resistance mechanisms and also why *S. maltophilia* and *B. cepacia*, intrinsically resistant to many antibiotics and challenging to treat in immunocompromised patients <sup>51-53</sup> exhibited significant resistance to CatBF. Indeed, these bacteria display unique inner membrane phospholipid and LPS compositions <sup>51-53</sup>. Finally, differences in phospholipid

composition between their outer membrane and the inner membrane of Gram-negative bacteria (9) may explain the slightly reduced sensitivity observed for Gram-positive strains such as *S. aureus* and *E. faecalis*. To delve deeper into its mechanism of action, we investigated CatBF's structural interactions with bacterial membrane models.

### Interaction of CatBF with model membranes

#### CatBF is largely unstructured in solution.

The sequence of CatBF (KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF) is compatible with the formation of an amphipathic α-helix (Figure 1A). Indeed, structure calculations by the I-TASSER web server <sup>54</sup>, based on multiple threading alignment and biological function recognition, predicts an α-helical structure for about two thirds of its sequence (up to Pro21) (Figure 1B), where residues 21–30 fold back to make van der Waals contacts with the apolar face of the helix formed by residues 1–20, thus protecting the C-terminal residues from exposure to the polar solvent.

NMR analysis revealed significant overlap in the amide region of the ¹H NMR spectrum (Figure 1C), indicating a random coil conformation as previously reported <sup>31</sup>. Backbone atom assignments (HN, Hα, Cα) were determined using ¹H,¹H-TOCSY, ¹H,¹H-NOESY, and ¹H,¹³C-HMQC experiments (Table S3). Analysis of chemical shift deviations with respect to the random coil values estimated by POTENCI <sup>55</sup> revealed that CatBF is largely unstructured at the experimental concentrations, with deviations well below the thresholds expected for α-helices or β-strands (0.1 ppm for ¹H and 0.7 ppm for ¹³C, Figure 1D,F) <sup>56</sup>. CD spectra further confirmed the unstructured nature of CatBF, showing a large minimum at around 200 nm (Figure S1).

#### The N-terminal part of CatBF forms a stable α-helix in membrane models

To investigate the interaction of CatBF with biological membranes, we initially employed a simplified model system comprising deuterated dodecylphosphocholine (DPC:d<sub>38</sub>) micelles. These micelles offer several advantages for studying peptides and proteins in solution, including their small size, rapid rotational tumbling, and relatively narrow NMR lines <sup>57</sup>. Although DPC does not form bilayers, its choline headgroup mimics the interface of biological membranes, while the apolar chains create a hydrophobic core.

The ¹H NMR spectrum of CatBF undergoes significant changes in the presence of DPC micelles (Figure 1C). At a peptide-to-DPC molar ratio of 1:20, abrupt spectral changes indicate slow exchange on the NMR timescale, consistent with a sub-micromolar dissociation constant. Backbone atoms (HN, Hα, Cα) of the bound form of CatBF were assigned by ¹H,¹H-TOCSY, ¹H,¹H-NOESY, and ¹H,¹³C-HMQC (Table S3, Figures 1C and S2). Analysis of chemical shift deviations from random coil values (Figure 1D-G) clearly reveals that the peptide adopts a structured conformation in the presence of micelles. The negative and positive deviations of the Hα and Cα chemical shifts (Figure 1E,G), respectively, suggest an α-helical conformation up to Lys20. The C-
terminal tail (residues 21–30), beginning with Pro (a known α-helix disruptor), appears unstructured and highly flexible, as evidenced by the strong intensity of its backbone cross-peaks in two-dimensional spectra (Figure S2). These findings are in excellent agreement with previous observations in SDS micelles and TFE/H₂O mixtures³¹.

To confirm the α-helical structure of the peptide and determine its orientation within the micelle model, we employed 16-DSA (16-doxyl stearic acid), a paramagnetic probe that contains a spin label at the acyl chain terminus that gets inserted into the center of DPC micelles. The proximity of atoms to the spin label affects their NMR signal intensities, with a distance-dependent effect inversely proportional to its sixth power⁵⁸⁻⁶⁰. Although extensive overlap prevented a complete analysis of paramagnetic effects on backbone Hα/HN cross peaks in ¹H,¹H-NOESY spectra and Hα/Cα peaks in ¹H,¹³C-HMQC appear very weak, a clear reduction in intensity of the Hα/Cα peaks of F3, L6, V10, and A14 (Figure 1H and S3A) and of the H/C signal of the F17 aromatic ring was observed (Figure S3B). These effects suggest an orientation of the helix with its apolar chains facing the micelle center, while lysines and arginines point outwards. Despite severe overlap among the NMR signals of the α, β, γ, δ, and ε of the 9 lysine residues and α, β, γ, δ of the 2 arginine residues (each within their atom type), it is evident that these groups of signals are affected by 16-DSA in the order α > β > γ > δ > ε, as expected for a chain pointing away from the micelle core. Furthermore, Hα/Cα peaks of residues in the C-terminus are not significantly affected (Figure 1H), indicating that the unstructured tail remains far from the hydrophobic core of the micelle. Notably, C-terminal truncated versions of CatBF retain their antibacterial activity⁶¹.

While DPC micelles provide valuable insights into the structure and orientation of CatBF, isotropic bicelles can better model target membranes by incorporating headgroups typically found in bacteria, such as phosphatidylglycerol (PG), and forming a lipid bilayer. To this end, we titrated a CatBF solution with DHPC/DMPC/DMPG isotropic bicelles (Figure S4). Similar to the DPC micelle experiments, the ¹H spectrum changes drastically upon increasing the bicelle concentration, with new peaks appearing and those of the free form gradually disappearing, indicative of slow exchange on the NMR timescale. The larger size of the bicelle system resulted in broadened Cα/Hα peaks beyond detection in ¹H,¹³C-HMQC spectra, preventing assignment. However, a clear increase in peak dispersion in the amide region suggests structuring, while peaks belonging to aromatic and methyl protons shift upfield, likely due to the insertion of their moieties into the apolar core of the bilayer.

The curvature of micelles and the small surface area of bicelles, together with the constraints in the nature and relative amounts of lipid head groups for both systems, limit their capability of modeling biological membranes.

Liposomes better reproduce the curvature of a cell, and their phospholipid composition can be freely varied. Unfortunately, their relatively large size (from 20 nm to tens of µm) and their slow tumbling rate hamper the application of solution NMR techniques. In these cases, important information can be obtained by magic angle spinning solid state NMR (MAS-ssNMR), in which the fast mechanical rotation of the sample at the magic angle of 54.74° with respect to the static magnetic field is able to quench dipolar interactions that otherwise give rise to fast relaxation.

To further confirm our structure in lipid bilayers, we acquired the solid state ¹³C spectrum of CatBF in the presence of POPE/POPG:d₃₁ 3/1 mole/mole vesicles and ¹³C-labeled at Ala14, a residue localized in the center of the helix (Figure S5). These signals are sensitive to the peptide secondary structure and resonate in a region of the spectrum free from the signals from POPG and POPE phospholipids. While at 37°C temperature, signals are weak, probably due to conformational exchange close to coalescence, at lower temperatures, the chemical shifts are clearly compatible with a helical conformation (positive deviations from random coil values of ¹³Cα and ¹³C' (carbonyl) chemical shifts and negative deviations of ¹³Cβ). Interestingly, MD simulations show that, once in close contact with the bilayer, CatBF transiently loses its α-helical structure (Figure S6), which is otherwise well conserved in all 500 ns simulations (Figure S7). This phenomenon could explain the lack of signal at 37°C and CD data (Figure S1), for which spectra show only partially α-helical structures.

<table><thead><tr><th>Species</th><th>Strain</th><th>Acquired resistant genes</th><th>MIC (mg/L)</th><th>MIC (µM)</th></tr></thead><tbody><tr><td rowspan="2">A. baumannii</td><td>NRZ-46913</td><td>GIM-1 carbapenemase</td><td>2</td><td>0,5</td></tr><tr><td>NRZ-47224</td><td>OXA-58 carbapenemase</td><td>2</td><td>0,5</td></tr><tr><td>E. cloacae</td><td>NRZ-11140</td><td>chromosomal AmpC beta-lactamase + porin loss</td><td>2</td><td>0,5</td></tr><tr><td>E. coli</td><td>NRZ-11144</td><td>DHA-type AmpC beta-lactamase</td><td>2</td><td>0,5</td></tr></tbody></table>
<table><caption>Table 1. MIC values ≤ 4 mg/l displayed by CatBF towards clinical isolates (higher MICs in Tables S1 and S2).</caption><tbody><tr><td rowspan="10">K. aerogenes</td><td>NRZ-11721</td><td>OXA-1 beta-lactamase</td><td>2</td><td>0,5</td></tr><tr><td>NRZ-12405</td><td>porin loss</td><td>2</td><td>0,5</td></tr><tr><td>NRZ-12632</td><td>KPC-2 carbapenemase</td><td>2</td><td>0,5</td></tr><tr><td>NRZ-16145</td><td>OXA-181 carbapenemase</td><td>2</td><td>0,5</td></tr><tr><td>NRZ-16424</td><td>NDM-5 metallo-beta-lactamase</td><td>2</td><td>0,5</td></tr><tr><td>NRZ-16329</td><td>NDM-1 metallo-beta-lactamase</td><td>2</td><td>0,5</td></tr><tr><td>NRZ-16577</td><td>VIM-1 metallo-beta-lactamase</td><td>2</td><td>0,5</td></tr><tr><td>NRZ-04093</td><td>VIM-19, CMY-4 AmpC beta-lactamase</td><td>2</td><td>0,5</td></tr><tr><td>NRZ-13101</td><td>ESBL + porin loss</td><td>2</td><td>0,5</td></tr><tr><td>NRZ-15696</td><td>OXA-48 carbapenemase</td><td>2</td><td>0,5</td></tr><tr><td rowspan="7">K. pneumoniae</td><td>NRZ-16790</td><td>OXA-1 beta-lactamase</td><td>2</td><td>0,5</td></tr><tr><td>NRZ-18232</td><td>KPC-3 carbapenemase</td><td>2</td><td>0,5</td></tr><tr><td>O81E9</td><td>CTX-M-15- ESBL</td><td>2</td><td>0,5</td></tr><tr><td>NRZ-00960</td><td>OXA-204 carbapenemase, CTX-M-14 ESBL, CMY-4 AmpC beta-lactamase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-12535</td><td>ESBL + porin loss</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-15694</td><td>KPC-2 carbapenemase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-15699</td><td>OXA-48 carbapenemase</td><td>4</td><td>1,1</td></tr><tr><td rowspan="6">A. baumannii</td><td>NRZ-16654</td><td>GES-5 carbapenemase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-46129</td><td>NDM-1 metallo-beta-lactamase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-46504</td><td>NDM-1, OXA-23 carbapenemase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-47066</td><td>OXA-23 carbapenemase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-47090</td><td>OXA-23 carbapenemase</td><td>4</td><td>1,1</td></tr><tr><td rowspan="5">C. freundii</td><td>NRZ-16672</td><td>VIM-1 metallo-beta-lactamase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-16864</td><td>IMP-1 metallo-beta-lactamase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-25677</td><td>GES-5 carbapenemase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-49994</td><td>GMB-1 metallo-beta-lactamase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-10170</td><td>LMB-1 metallo-beta-lactamase</td><td>4</td><td>1,1</td></tr><tr><td rowspan="10">E. cloacae</td><td>NRZ-12168</td><td>chromosomal AmpC beta-lactamase + porin loss</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-12683</td><td>chromosomal AmpC beta-lactamase + porin loss</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-16583</td><td>GES-5 carbapenemase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-16718</td><td>NDM-1 metallo-beta-lactamase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-17198</td><td>VIM-1 metallo-beta-lactamase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-18157</td><td>GIM-1 carbapenemase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-42093</td><td>NMC-A carbapenemase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-15698</td><td>OXA-48 carbapenemase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-18363</td><td>OXA-232 carbapenemase</td><td>4</td><td>1,1</td></tr><tr><td rowspan="4">E. coli</td><td>NRZ-44622</td><td>CMY-type AmpC beta-lactamase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-47795</td><td>NDM-5 metallo-beta-lactamase</td><td>4</td><td>1,1</td></tr><tr><td>R5A5</td><td>IMP-1 carbapenemase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-13997</td><td>OXA-48 carbapenemase</td><td>4</td><td>1,1</td></tr><tr><td rowspan="3">K. aerogenes</td><td>NRZ-17853</td><td>chromosomal AmpC beta-lactamase + porin loss</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-13047</td><td>KPC-2 carbapenemase</td><td>4</td><td>1,1</td></tr><tr><td>NRZ-13402</td><td>OXA-48 carbapenemase, CTX-M-1</td><td>4</td><td>1,1</td></tr><tr><td>P. aeruginosa</td><td>NRZ-32894</td><td>OXA-198 carbapenemase</td><td>4</td><td>1,1</td></tr></tbody></table>
<figure><img src="image_3.png" /><figcaption>**Figure 1.** (A) Helical wheel for CatBF (created by Netwheels ⁶²). (B) Structure prediction by the I-TASSER web server ⁵⁴. (C) 500 MHz ¹H NMR spectra (amide and aromatic region) of 0.8 mM CatBF in the presence of increasing concentrations of DPC-d38. Ha (D, E) and Ca (F, G) chemical shift deviations from random coil values estimated by POTENCI ⁵⁵ of CatBF in the absence (D, F) and the presence (E, G) of DPC micelles. (H) Percental intensity loss of Ha/Cα ¹H, ¹³C-HSQC peaks of CatBF in the presence of paramagnetic 16-DSA. The expected trend based on the presence of an α-helix from residue 2 to residue 19 is drawn in orange using a cosine function with periodicity 3.6, which corresponds to the number of residues per turn in a classical α-helix.</figcaption></figure>
## CatBF inserts in bacterial model membranes at the level of lipid phosphodiester bonds with its helical axis almost parallel to its surface

Since in an α-helix structure the direction of the HN amide bond is nearly parallel to the helix axis, we used the ¹⁵N chemical shift of selectively labeled peptides to determine the orientation of CatBF's helix in biomembranes in the membrane bilayer. To this aim, we used uniaxially oriented lipid bilayers that were inserted into the NMR coil to align the membrane normal parallel to the magnetic field of the NMR spectrometer. This arrangement assures that all molecules maintain the same angle with respect to the magnetic field even when undergoing lateral or rotational diffusion. In such oriented systems, the chemical shift is a function of the angle between the principal components of the chemical shift tensor and the external magnetic field and can be used to determine the orientation of bonds and molecules in the bilayer ⁶³. A typical ³¹P solid-state NMR spectrum of an oriented membrane encompassing 1% CAT BF is shown in Figure S8A and characterized by a major intensity at 28 ppm, confirming the good macroscopic alignment of the phospholipid bilayer ⁶⁴.

In order to determine the orientation of the helix and of the C terminus in CatBF, we synthesized five selectively labeled samples: ¹⁵N-F17, ¹⁵N-V10, ²H₃-Ala14, ¹⁵N-G25, and ²H₇-V23 CatBF. Figure 2A shows the ¹⁵N spectrum of ¹⁵N-F17 CatBF. The observed ¹⁵N chemical shift of 66 ± 5 ppm for the F17 amide suggests an approximate parallel alignment of the HN vector with the membrane surface. To further refine the helix tilt and pitch angles, a complementary ¹⁵N probe was introduced at V10, another residue within the same helical structure (Figure 2B), and the ²H₃-labeled methyl group of A14 was investigated. The measured ²H quadrupolar coupling of ²H₃-A14 is 20 ± 3 KHz (Figure 2C). In this labeling scheme, the ²H quadrupolar coupling, a function of the A14 methyl group orientation relative to the magnetic field, and the ¹⁵N amide labels provide highly complementary information on helix alignment ⁶³.

The three orientation-dependent parameters (two ¹⁵N chemical shifts and one ²H quadrupolar coupling) obtained from oriented solid-state NMR (ssNMR) were used to determine the pitch and tilt angles that define the orientation of the helix in POPE/POPG 3/1 mole/mole bilayers. Each ssNMR measurement limits the possible values of pitch and tilt angle, with only one set of angles satisfying all the experimental values (Figure 2G). The resulting orientation is shown in Figure 2H, where the helix axis lies almost parallel to the membrane surface and the lysine residues point upward into the aqueous environment. Remarkably, this orientation is perfectly compatible with the one found in micelles and defined by paramagnetic effects (see previous section), and its biological significance is supported by the fact that the same ¹⁵N chemical shift values are obtained in bilayers formed by E. coli total lipid extracts (Figure S8B). A similar procedure was used to confirm the conformation of the C-terminus in oriented POPE/POPG bilayers. The nearly isotropic chemical shift at 100 ± 7 ppm observed from ¹⁵N-G25 CatBF (Figure 2D) and the vanishing of the quadrupolar coupling from ²H₇-V23 residue (Figure 2E) confirmed the high flexibility of the C-terminal portion of the peptide.

Once we determined the orientation of the helix, we investigated the depth of insertion of CatBF in the bilayer by means of paramagnetic effects induced by the presence of Mn²⁺ ions in POPE/POPG:d₃₁ vesicles. Figure 2I shows ¹H lipids to ¹³C peptide diffusion transfer experiments ⁶⁵ in the absence (with peak intensities S₀) and in the presence of Mn²⁺ ions (with peak intensities S). Mn²⁺ binding at the membrane surface induces a depth-dependent dephasing of ¹³C signals, with greater dephasing observed for surface-proximal groups. The intensity ratio S/S₀ (shown in Figure 2J where intensities are normalized with respect to the most unaffected signal, which is typically the methyl carbon, ω) allows the comparison of the paramagnetic effects that localize the alanine of the peptide (and the full helix, given its almost parallel orientation to the membrane surface) just below the lipid phosphodiester groups, in excellent agreement with what was observed by paramagnetic experiments in DPC micelles. Such a localization is further supported by the recurrence of salt bridges between lysine and arginine residues with phosphate groups of phospholipids in MD simulations.

## CatBF at high concentrations reorients onto the target membrane up to a charge-compensating peptide/lipid ratio

At CatBF peptide-to-lipid ratios > 1 mole%, the ³¹P solid-state NMR spectra show additional intensities reaching up to -10 ppm (Figure S8A, bottom). These are compatible with phospholipids not aligned parallel to the surface normal or alternate conformers at the level of the phosphate groups.

Furthermore, the ²H solid-state NMR spectra show additional quadrupolar couplings (of about 30 KHz), indicating an additional pool of peptide orientations (Figure 2F). Simulating a system with coexisting peptide populations, one undergoing well-defined rocking and wobbling motions (as defined in Figure 2H) and another with a less mobile, Gaussian distribution of orientations, successfully reproduces both peptide ¹⁵N and ²H spectra (Figure S9).

Oligomerization of the peptide is also observed in almost all MD simulations (Figure S10). These oriented solid-state NMR data are compatible with a model where oligomerization of the peptide at the bilayer surface results in the invagination of the membrane. The formation of such multimers, where the relative orientation of the helix is variable, is in agreement with a well-defined alignment of the CatBF helix at low concentrations and a distribution of orientations at higher concentrations. Membrane deformation induced by the presence of CatBF is clearly highlighted by the electron density profile (Figure S11), showing significant lipid interdigitation (higher density in the membrane core) and the separation of the maxima related to phosphate groups into two populations on each leaflet.

A selectively labeled peptide where the alanine-14 was uniformly labeled with ¹³C (¹³C₃-A14) was reconstituted into multilamellar vesicles and studied by ¹³C MAS solid-state NMR spectroscopy. The ¹³C chemical shifts are indicative of α-helical conformations at the higher concentrations investigated (Figure S5).
<figure><img src="image_4.png" /><figcaption>**Figure 2.** ssNMR studies of CatBF in lipid bilayers. Proton-decoupled ¹⁵N (A, B, D), and ²H (C, E, F) spectra of 1 mol% (A, C) or 2 mol% (B, D-F) CatBF in uniaxially oriented POPE:POPG 3:1 at 310 K, carrying ¹⁵N label on Phe17 (A), Val10 (B), or Gly25 (D) and ²H label on the methyl group of Ala14 (C, F) or ²H₇ labeled Val23 (E). (G) Restriction analysis and (H) corresponding peptide alignments relative to the membrane normal (only the helical residues 1 to 20 are shown) for 1 mol% CatBF in POPE:POPG 3:1 mole/mole bilayer. The restrictions for the tilt and pitch angle of the helix formed by CatBF are shown in blue for ¹⁵N-Val10 (70.0 ± 4.5 ppm), green for ¹⁵N-Phe17 (65.0 ± 4.5 ppm), and black for ²H₃-Ala14 (20.0 ± 3.0 kHz) (G). The restraints were obtained assuming rocking (18° Gaussian distribution) and wobbling (10°) motions of an ideal helix (φ = -65°, ψ = -45°). In panel H, leucines, isoleucines, phenylalanines, and valines are shown in yellow; alanine and proline in gray; lysines in blue; glutamines in magenta; and arginines and serine in cyan. One possible solution is found for pitch and tilt angles of 1 mol% CatBF, corresponding to the values highlighted by a red circle in panel G. (I) Proton-decoupled ¹³C MAS ssNMR spectra of 9 mol% [¹³C₃-A14]CatBF in POPE/POPG 3:1 mole/mole membranes with (red line) and without (black line) addition of 13.7 mM Mn²⁺ ions. (J) ¹³C dephasing curves</figcaption></figure>
for various lipid segments were obtained by calculating the ratio of peak heights between Mn²⁺-bound and Mn²⁺-free samples. These ratios were normalized to the maximum acyl chain signal (typically the methyl carbon, ω).

However, it should be noted that at 37°C no signal was visible at low peptide/lipid ratio (Figure S12), indicating conformational exchange rather than a well-defined structure as discussed previously. More importantly, at 2.3% mol, the negative charge of the vesicles arising from POPG (present at 25 mole%) is completely compensated by the positive charge of CatBF (nominally bearing 11 positive charges), and this explains why, at a concentration of 4% and beyond, new ¹³C solid-state NMR signals appear at 37° C. These are compatible with random coil conformations (Figure S12) and probably arise from free peptide, not internalized into the membrane.

## CatBF recruits PG lipids in bacterial membrane models, possibly inducing phase separation at high concentrations

Once the orientation and the level of insertion of the peptide were determined, we studied the effect of CatBF on membrane phospholipids by measuring the ²H quadrupolar coupling of deuterated palmitoyl chains in either POPE or POPG in POPE/POPG mixtures (Figure 3). Quadrupolar couplings are not only dependent on the orientation of acyl C-D moieties with respect to the magnetic field but also on their dynamics, which scales their value by a quantity inversionally proportional to the order parameter $S_{CD}$. $S_{CD}$ can range from 1 (perfect order) to 0 (complete disorder). In liposomes, the larger population of molecules at a defined angle with respect to the magnetic field is constituted by the pool of phospholipids whose axis is at 90°, giving rise to the two maximal intensities in the ²H ssNMR 'powder pattern' spectrum. This means that measuring the quadrupolar coupling as the distance of these maxima gives information about the dynamics of the corresponding CD₂ segment.

Figure 3A shows the static ²H solid-state NMR spectra of POPE/POPG-d₃₁ liposomes. Interestingly, the ²H ssNMR spectra of ²H₃₁-POPG in the presence of 1 mole% or 2 mole% peptide are indicative of the existence of two lipid populations, one corresponding to the free liposomes and one affected by the presence of the peptide (Table S4). The latter are well visible when subtracting the spectrum of pure liposomes from the overall spectrum (magenta in Figure 3B and cyan in Figure 3C). Upon addition of 4 mole% CatBF, the population of the unaffected lipids disappears (Table S4), and the ²H ssNMR spectra are indicative of a homogenous population of perturbed ²H-POPG (Figure 3D). This further confirms that the interaction of CatBF is inhibited beyond the point where the charge of the vesicles has been compensated, as discussed in the previous section. The existence of two populations implies prevention of molecular exchange with 'free lipids'. However, as the time scale of the ²H ssNMR are rather long (10⁻⁵ sec), these observations may also suggest a non-homogenous distribution of peptides by preferential accumulation on some vesicles. Indeed, it has been proposed that binding of proteins to one vesicle induces further binding on the same ⁶⁶. Supporting this hypothesis, MD simulations reveal peptide aggregation on the membrane surface, where new peptides interact with pre-existing ones covering the surface. The formation of aggregated peptide domains has been considered key for the membrane permeabilizing activity of cateslytin ⁶⁷.

²H spectra were used to measure the order parameters of all deuterated carbon in the acyl chain as calculated by the ratio of the experimental and theoretical quadrupolar coupling in the absence of dynamics (Figure 3E). Even in the absence of peptide, the order parameters decrease gradually as we move away from the phosphate group along the palmitoyl chain, indicating that lipids are more dynamic in the core of the bilayer. The addition of CatBF affects all order parameters (Figure 3E and Table S4) and especially the palmitoyl chain segments that make up the innermost core of the membrane, as highlighted by the ratio of the order parameters in the presence and absence of peptide (Figure 3F). This observation is in agreement with CatBF intercalating below the phosphate head groups as suggested by the orientation found by paramagnetic effects (Figure 2J), thereby disrupting the lipid packing of the fatty acyl chains in the membrane interior. To get insight into the effect of CatBF, we calculated the order parameter of the palmitoyl chains by MD simulations (Figures 3G and S13-S16). As already observed for other highly charged peptides ⁶⁸⁻⁷⁴, CatBF initially (first 500 ns) has no effect or tends to increase the order parameter of both pure bacterial phospholipids and their mixtures (Figure S13), unless a high peptide concentration is used (Figure S14). While all-atom molecular dynamics simulations allow deciphering the mechanism of action of the peptides at an atomic level, they cannot easily explore the long time scales accessible with enhanced sampling methods ⁷⁵. Because of that, we decided to run longer simulations (4µs) for the PE/PG mixtures. Indeed, such simulations can better reproduce experimental data (Figure S15), especially when six peptides are included in the simulations (Figure 3G and S16).

Intriguingly, the effect on the NMR-derived order parameter is less pronounced when the deuterated palmitoyl chain of POPE is used as a probe of dynamics (Figure S17). This finding suggests that the strong positive charge of CatBF is able to recruit negatively charged POPG phospholipids in its surroundings, effectively acting locally as a phase separator or even influencing the composition of vesicles during their reconstitution process (Figure 3H). Contrary to what is observed in the presence of POPC membranes, CatBF promptly interacts with phospholipids typically found in bacterial membranes (POPE/POPG and POPE/POPG/CL). This is demonstrated by the significant amount of both polar (Figures S18-S21) and apolar contacts found when the MD traces are analyzed in atomic detail (Figures S22-S24). It is interesting to point out that in all systems, CatBF clearly prefers the interaction with POPG (Figure 3I) and CL (Figure S21) over POPE. The preference for negatively charged PG and CL is clear when analyzing the area per lipid of pure lipid bilayers, which reflect the membrane deformation caused by the strong interaction with the peptide (Figure S25). The occurrence of polar contacts in simulations with pure PE, PG, and CL bilayers or their mixtures POPE/POPG/CL shows that this preference follows the order of the global charge: CL> PG> PE (Figures S18-S21). Another interesting feature highlighted by polar contacts is that the peptide tends to approach the membrane with its N-terminus, in agreement with the tilt angle found by ssNMR spectroscopy (Figure 3H). The
selective recruitment of PG disrupts the POPE/POPG network of interactions, thus inducing the deformation discussed before, also due to the accumulation of cone-shaped PE head groups in POPG poor areas.

In order to deepen our understanding, we repeated the measurement with magainin 2, a cationic peptide with a much lower net positive charge (+3 vs. +11). Indeed, in this case, ²H spectra are compatible with a single component even at substoichiometric peptide/lipid ratios (Figure S26A), where the lateral lipid diffusion averages the effects of the presence of the peptide among the affected and unaffected lipids. Moreover, consistent

with what was hypothesized in the previous section, the effect of magainin 2 is observed up to a charge-compensating peptide/lipid ratio of 9.8, which is very close to the theoretical one (33/3=11, Figure S26B). Beyond this concentration, magainin 2 is unable to further affect lipid dynamics. Finally, it should be noted that the effect of CatBF on the order parameter is significantly more pronounced at low concentrations (relative order parameter down to 0.8 at 1% mol vs. 0.99 of magainin 2, Figure S26C), while magainin 2 achieves lower order parameters at higher concentrations (up to 0.46 vs. 0.65, Figure S26C), probably because more peptides can

A

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B

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C

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D

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E

<figure><img src="image_9.png" /></figure>

F

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G

<figure><img src="image_11.png" /></figure>

H

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<figure><img src="image_13.png" /></figure>

I

<figure><img src="image_14.png" /></figure>
**Figure 3.** (A-D) Quadrupolar $^2H ssNMR spectra of chain-deuterated POPG-d31 in the presence or absence of CatBF at increasing concentrations. Spectrum in the presence of 1 mol% of CatBF was found to be the overlap of 25% unaffected lipid membrane and membrane-distorted lipid fraction (B). With 2 mol% of CatBF, 55% of the spectrum integral intensity (int.) of pure lipid membrane is lost (C). Order parameters $S_{CD}$ of lipid palmitoyl chain C-D bonds in the absence (black) and in the presence of CatBF (E). Ratio of $S_{CD}$ in the presence of peptide with $S_{CD}$ in its absence (F). (G) Order parameter for POPE/POPG (in red) and without (in black) CatBF peptide as calculated by MD. (H) Schematic representation of CatBF-induced recruitment of PG lipids. (I) Polar contact recurrence (hydrogen bonds and salt bridges) between nitrogen and oxygen atoms of CatBF and those of POPG (violet) and POPE (green) in molecular dynamic simulations.

penetrate the membrane before its negative charge is balanced (thus justifying a more disruptive penetration depth of this peptide). In conclusion, a strong membrane perturbing effect is already achieved at relatively low concentrations of CatBF when compared to magainin 2 (9.8 vs. 4%, Figure S26C), although at high concentrations the latter has a more profound effect on membrane lipid order.

## Molecular dynamic simulations of CatBF interacting with other phospholipid bilayers

Molecular dynamics (MD) simulations have been widely used to get insight into the membrane-destabilizing action of several AMPs. In this study, we have explored the interaction of CatBF with models mimicking bacterial and mammalian cell membranes ⁹, the latter used for ascertaining potential cytotoxicity. POPC exposes the phosphatidylcholine (PC) moiety, which is the most common headgroup found in mammalian bilayers. On the contrary, bacterial membranes expose phosphatidylethanolamine (PE), PG, and possibly cardiolipin (CL) ⁹. Besides its antibacterial activity ³¹⁻³³,⁷⁶, CatBF also displays antifungal ³¹,³⁴ and anticancer ²⁵⁻²⁷,³⁶ properties, whose study must take into account the exposition of PS (cancer cells and fungi) and PE (fungi) on the target membrane ⁹. For all these activities, membranes formed by pure components (PE, PG, CL, and PS) were also examined to ascertain the contribution of each lipid to the interaction with CatBF.

### CatBF poorly interacts with mammalian model membranes.

In MD simulations, CatBF rarely interacts with POPC membranes. Polar (salt bridges and H-bonds) and van der Waals contacts were estimated by the radial distribution function ⁷⁷ at the relevant distances. In the case of CatBF, few superficial contacts are established (Figure S18), indicating infrequent interactions. No internalization is observed, as demonstrated by the lack of van der Waals contacts (Figure S22). The electron density profile along the bilayer normal provides a clear view of the membrane thickness and the localization of phosphate groups during the simulation. Such profiles clearly show the absence of alterations (including lack of lipid interdigation) induced by the presence of CatBF (Figure S11). Area per lipid calculations are also useful to monitor the interaction of peptides with model membranes. In the presence of pronounced interactions, the membrane surface tends to invaginate, resulting in an increase and decrease in the surface area per lipid of the internal and external leaflets of the bilayer, respectively ⁷⁸. This parameter remains constant for the two leaflets in the case of POPC bilayers, even when the peptide concentration is increased by introducing 6 peptides in the simulations (Figure S25).

The activity of AMPs can be impaired by several factors, such as temperature, pH, and buffer. As most of the media used to determine their activity contain $Na^+$, $Mg^{2+}$, and $Ca^{2+}$, we have performed simulations with each

of these ions separately. Magnesium is an essential factor for the action of many antibiotics, such as tetracycline ⁷⁹⁻⁸¹, but can impair the action of peptides targeting the cell membrane ⁸². For some peptides, the equilibrium between self-aggregation and the interaction with a membrane may be modulated by calcium ⁸³⁻⁸⁹. $Ca^{2+}$ binds to the phosphate groups of the bilayer, thus shielding its negative charge and reducing the affinity for positively charged residues of the peptide. However, calcium can form salt bridges connecting charged glutamate or aspartate residues of the peptide to the phosphate groups of the membrane, enhancing the peptide interaction ⁸⁸,⁹⁰.

The substitution of $Na^+$ ion with $Ca^{2+}$ or $Mg^{2+}$ did not change significantly what was previously observed. CatBF establishes more polar contacts with PE/PG than POPC (Figures S27, S28). Interestingly, we noticed an increased internalization of the peptide in the presence of $Ca^{2+}$ as testified by the high number of van der Waals contacts (Figure S29) as compared to $Na^+$ (Figure S24) or $Mg^{2+}$ (Figure S30) and a more significant effect on the order parameter of lipid acyl chains (Figures S31–S34).

### Effect on CatBF on phosphatidylserine membranes

CatBF has been reported to exhibit further biological activities, acting as an immunomodulator, anticancer, and antifungal (against *Candida* species) agent ²⁵⁻²⁷,³⁵,³⁶. In all these functions, phosphatidylserine (PS) is an essential lipid because it is found exposed on eukaryotic cell membranes ⁹,¹⁰,⁹¹⁻⁹⁴. To get deeper insight into these activities, we explored the interaction of CatBF with POPS membranes by MD. Polar (Figure S19) and apolar (Figure S23) contacts are very similar to those obtained for POPG, probably because of the negative charge of both phospholipids. We also observe an important membrane invagination (Figure S25), as previously observed for other PS-targeting anticancer peptides ⁷⁰,⁷³,⁷⁴ and membrane rigidification reflected by higher values of the order parameter (Figure S13), especially in models containing multiple peptides as in the case of POPG (Figure S14). Finally, some degree of CatBF oligomerization is observed in the presence of POPS membranes, as in the case of other charged lipids (Figure S10).

These preliminary data will constitute the basis for further future investigations.

## Conclusion

In this work, we have evaluated the activity of CatBF towards clinically relevant multidrug-resistant bacterial isolates giving rise to nosocomial infections. We found that CatBF displays MIC values as low as sub-micromolar against most isolates, independently of the bacterial species and of their mechanisms of resistance (expression of carbapenemases, porin loss, expression of efflux pumps).

To get insight into the mechanism of action of cationic AMPs at the level of the inner and outer membrane of Gram-negative and Gram-positive bacteria,
respectively, we investigated the interaction of CatBF with
phospholipid bilayers, exposing head groups typically
found on their surface: PG, PE, and CL. We found that
CatBF is unstructured in solution, but its N-terminus
(residues 1–20) assumes an α-helical structure in the
presence of bacterial membrane models, triggered by its
affinity for PG and CL. NMR indicates that the relatively
hydrophobic C-terminus is unstructured and points outside
DPC micelles or oriented bilayers. According to ssNMR
and MD, the α-helix formed by CatBF orients with its axis
almost parallel to the surface and partially penetrates the
bilayers by its N-terminus. NMR, MD, and CD data
suggest that the peptide oligomerizes and recruits PG
phospholipids, contributing to a very significant
deformation of the membrane surface, possibly a prelude
to its irreversible destabilization.

**Experimental section**

E. coli lipid extract and the phospholipids (POPE, POPG) were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification.

**Peptide synthesis**

The peptide CatBF (KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF) was synthesized following the standard Fmoc-synthesis protocol on a Millipore 9050 automatic peptide synthesizer. The peptides were purified by reverse phase HPLC (Gilson, Villiers-le-bel, France) using a preparative C18 column (Luna, C-18-100 Å-5 mm, Phenomenex, Le Pecq, France) and an acetonitrile/water gradient (Figure S35). Their identity and purity (>90%) were checked by MALDI mass spectrometry (MALDI-TOF Autoflex, Bruker Daltonics, Bremen, Germany) (Figure S36).

**Susceptibility testing**

To test antimicrobial susceptibilities to the CatBF peptide on various bacteria isolates, broth microdilution using Muller-Hinton broth (MHB) as recommended by EUCAST was used. Roughly an inoculate of McFarland 0.5 was obtained with fresh colonies grown on a Mueller-Hinton agar plate. Within 15 minutes, the adjusted bacterial suspension was diluted in MHB to reach approximately 10x10⁵ CFU/mL in a final volume of 50 µL. To this, 50 µL of the diluted CatBF were added to reach a final concentration of 5x10⁵ CFU/mL of bacteria in each well.

A stock solution of CatBF was prepared at 9.8 mg/mL and sequentially diluted in water. 50 µl of bacterial suspension was added to each dilution of CatBF to test a range of final concentrations between 128 µg/mL and 0.125 µg/mL. One row of each well plate served as a growth control without antibiotics. Sterility control was also performed. Microdilution plates were incubated at 310 K for 20 h and subsequently analyzed by the naked eye. Each MIC determination was done in triplicate.

The bacterial isolates used were pre-
characterized clinical multidrug-resistant isolates from the
strain collection of the German National Reference Centre
for Multidrug-resistant Gram-negative Bacteria (Bochum,
Germany), from the French national reference center for
antibiotic resistance, and from the bacteriology ward of the
Bicêtre Hospital, France. Pre-characterization was

performed as previously described ⁹⁵. Testing was
performed according to ISO 20776-1.

Liquid-state NMR spectroscopy and sample preparation

Titration of CatBF with DPC micelles

50 mM phosphate buffer with 10% of D₂O and 0.1 mM deuterated sodium 3-(trimethylsilyl)propionate:d4 (TSP:d4) (used as an internal reference) was used to solubilize CatBF in a final volume of 500 µl. The final concentration was 0.8 mM. Small amounts of 1 M stock solution of dodecylphosphocholine-d38 (DPC:d38) were added gradually up to a final concentration of 60 mM. All the experiments were run at a temperature of 310 K.

**Paramagnetic NMR experiments**

The paramagnetic probe 16-doxyl stearic acid (16-DSA) was dissolved in deuterated methanol and was gradually added to the samples of CatBF with DPC micelles until the paramagnetic effect was observed as an increase of the linewidth of at least one peak (usually for a ratio of 16-DSA/DPC of 1:100). The percentage reduction of intensity of H/C cross peaks in ¹H,¹³C-HMQC spectra was used to assess paramagnetic contributions. Errors were evaluated using the propagation formula in ratios A = $\frac{B}{C} : \Delta A=A(\frac{AB}{B} + \frac{AC}{C})$, where B is the cross-peak intensity without 16-DSA, C is the cross-peak intensity with 16-DSA, and ΔB and ΔC are the spectral noise. Tests were carried out at 310 K.

Titration of CatBF with isotropic bicelles

CatBF was solubilized in the same buffer as the
one used for titration with DPC micelles (see Section
4.3.1). The final concentration was 0.8 mM. Small amounts
(in the order of microliters) of a stock solution of bicelles
were gradually added until reaching a final concentration
of 60 mM. Isotropic bicelles were obtained by mixing in
chloroform 1,2-dihexanoyl-sn-glycero-3-phosphocholine
(DHPC), 1,2-dimyristoyl-sn-glycero-3- phosphocholine
(DMPC), and 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-
rac-glycerol) (DMPG) at molar ratios 66.7/25.0/8.3. After
the mixing, chloroform was evaporated under nitrogen
steam and lyophilized overnight. Experiments were run at
310 K.

**NMR acquisition and processing**

A 500 MHz Bruker spectrometer equipped with a
5 mm Broadband Inverse (BBI) probe was used to perform
all the liquid-state NMR experiments. ¹H, ¹³C-HMQC,
¹H, ¹H-TOCSY (mixing time of 60 ms), and ¹H, ¹H-NOESY
(mixing time of 200 ms) were used to assign backbone
resonances. Spectra were analyzed by TopSpin 4 (Bruker
BioSpin) and NMRFAM-SPARKY ⁹⁶. Secondary structure
prediction by examining the chemical shift deviations ⁹⁷⁻⁹⁹
was done by comparing them with reference random coil
values obtained by the POTENCI web server (https://st-
protein02.chem.au.dk/potenci/) ⁵⁵ at 310K.
## ssNMR spectroscopy and sample preparation

Samples for ssNMR spectroscopy were prepared by co-dissolving POPE:POPG 3:1 (molar ratio) or *E. coli* total lipid extract with an appropriate amount of CatBF peptide in methanol:chloroform (1:1 by volume). Solution was carefully applied onto 25 ultrathin cover glasses (8 x 18 mm; Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany), dried under vacuum overnight, and hydrated at 93% r.h. at 37 °C for 2 days. Thereafter, glass plates were stacked on top of each other, sealed, and subjected to NMR measurements.

The preparation of non-oriented samples started by mixing POPE/POPG-d31 (3:1, 4 mg/1.5 mg) or POPE/POPE-d31/POPG (2:1:1, 2.9 mg/1.5 mg/1.5 mg) and the appropriate amount of peptide to reach a 2% mole ratio in methanol/chloroform 1/1 (v/v). The sample solution was vortexed and sonicated using a bath sonicator for 5 min, and the solvent was evaporated using exposure to a stream of nitrogen and to a high vacuum overnight in such a manner to form a film along the walls of a glass tube. The sample was resuspended in 23.5 µL (water content h = 0.81) of 100 mM Tris buffer (pH 7.4) and involved vortexing and bath sonication, as well as 3 freeze/heat cycles at -20 °C and 40 °C. The glass tube (6 mm outer diameter) with the sample was inserted into the solenoidal coil of the ssNMR probe to measure ²H and ³¹P spectra. In the case of ¹³C MAS spectra, the sample was carefully transferred into a 3.2 mm rotor.

Proton-decoupled ³¹P ssNMR spectra were acquired at 121.575 MHz using a Bruker Avance wide-bore 300 ssNMR spectrometer equipped with a commercial double-resonance flat-coil probe (Bruker, Rheinstetten, Germany)¹⁰⁰. A Hahn-echo pulse sequence¹⁰¹ was used with a π/2 pulse of 5 µs, a spectral width of 100 kHz, an echo delay of 100 µs, an acquisition time of 10.2 ms, and a recycle delay of 3 s. An exponential line broadening of 50 Hz was applied before the Fourier transformation. External 85% H₃PO₄ at 0 ppm was used for calibration. The temperature was set to 310 K.

The proton-decoupled ¹⁵N cross-polarization spectra of statically aligned samples were acquired at 30.43 MHz using a Bruker Avance wide bore 300 NMR spectrometer. An adiabatic CP pulse sequence was used with a spectral width, acquisition time, CP contact time, and recycle delay times of 25 kHz, 10.2 ms, 0.4 ms, and 3 s, respectively ¹⁰²,¹⁰³. The ¹H π/2 pulse and spinal-64 heteronuclear decoupling field strengths B₁ corresponded to a nutation frequency of 31 kHz. Then, 40 k scans were accumulated, and the spectra were zero-filled to 4 k points. An exponential line broadening of 100 Hz was applied before the Fourier transformation. The spectra were externally referenced to ¹⁵NH₄Cl at 39.3 ppm ¹⁰³. Samples were cooled with a stream of air at a temperature of 310 K.

Next, ²H ssNMR spectra were acquired using a quadrupole pulse-echo sequence ¹⁰⁴ with a recycle delay of 0.3 s, an echo time of 100 µs, a dwell time of 0.5 µs, and a π/2 pulse of 5 µs. Before the Fourier transformation of free induction decay, exponential apodization with a line broadening of 300 Hz was applied in the case of an oriented sample and only 100 Hz for non-oriented membrane samples. Spectra were externally referenced to ²H₂O (0 Hz). The temperature was set to 310 K.

At last, 13C ssNMR spectra were measured with a MAS 3.2 mm triple probe at 125MHz using a Bruker 500MHz spectrometer. The ¹H π/2 pulse and spinal-64 heteronuclear decoupling field strengths B₁ corresponded to a nutation frequency of 50 kHz. Then, 40 k scans were accumulated, and the spectra were zero-filled to 16 k points. An exponential line broadening of 20 Hz was applied before the Fourier transformation. The spectra were externally referenced to 13C-adamantine at 0 ppm. Samples were cooled with a stream of air at a temperature of 310 K with 5 kHz spinning speed.

The deuterium order parameters ($S_{CD}$) for each CD2 and CD3 group were determined according to $S_{CD}=4/3(h/e^2qQ)\Delta^2u$, where $\Delta^2u$ is the quadrupolar splitting of segment i and (e²qQ/h) is the static quadrupole coupling constant (167 kHz) observed for deuterons within C-D bonds [68].

Orientational restraints from oriented solid-state NMR spectra were obtained as before ¹⁰⁵.

## Circular dichroism

The peptides were dissolved at 50 µM in a 10 mM Tris buffer at pH 7. CD spectra recorded at 37°C using a J-810 spectropolarimeter (Jasco, Tokyo, Japan) using 2 scans, a step resolution of 1 nm, and an adaptive integration time between 1 and 8 s. The path length of the quartz cuvette was 1 mm. The spectra were recorded between $\lambda = 190$ and 250 nm. POPE/POPG (3/1 mol/mol) vesicles were prepared by extrusion of a multilaminar suspension through a polycarbonate film with a 100 nm pore size. The vesicles contained 1% of DSPE-PEG2000 (Avanti 880120) to prevent vesicle agglutination.

## Molecular dynamics simulations

Lipid bilayer systems were generated using the CHARMM-GUI web server ¹⁰⁶, and GROMACS software ¹⁰⁶ was employed to run the simulations under the CHARMM36m forcefield ¹⁰⁷. Bilayers contained a total of 128 lipids, and the initial structure of the peptide was obtained by I-Tasser ⁵⁴. Peptide molecules were placed over the upper leaflet at a distance of >10 Å. Lysine and arginine side chains were protonated. Water molecules were described by the TIP3P model ⁷⁷, and a 50-Å thick water layer was added above and below the membrane. Ions were added to obtain a concentration of 150 mM of NaCl, CaCl₂, or MgCl₂, where appropriate. Bacterial membranes were simulated using molar ratios close to those found in many Gram-negative bacteria ⁹, namely 70/30 for POPE/POPG and 67/27/6 for POPE/POPG/CL.

The particle mesh Ewald (PME) method was used for long-range electrostatic interactions ¹⁰⁸,¹⁰⁹ and periodic boundary conditions were applied for all the simulations. All bonds were constrained by the LINear Constraint
Solver (LINCS) algorithm<sup>110</sup>, allowing a 2 fs integration step. Every system was energy-minimized with a 5000-step steepest descent method. Berendsen barostat <sup>111</sup> was used during the equilibration, while Parrinello-Rahman barostat <sup>112,113</sup> was employed to maintain pressure (1 bar) semi-isotropically with a time constant of 5 ps and a compressibility of $4.5 \times 10^{-5} \text{ bar}^{-1}$. The temperature was maintained at 310.15 K using the Nose-Hoover thermostat <sup>114,115</sup> with a time constant of 1 ps. Simulations were run for 500 ns (except when a different duration is specified in the manuscript) after the standard CHARMM-GUI minimization and equilibration steps <sup>116</sup>. Minimization, equilibration, and production runs were repeated to have two repetitions of membranes in the absence of peptide and three in their presence. Convergence was assessed by the analysis of polar contacts and RMSD.

MD trajectories were analyzed by GROMACS tools and Fatslim <sup>117</sup>. Graphs and images were created with GNUplot <sup>118</sup> and PyMol <sup>119</sup>. VMD <sup>120</sup> was used for visualization.

## Supporting Information

The authors have cited additional references within the Supporting Information. Supplementary_CatBF.pdf

**Keywords:** antimicrobial peptide, drug resistance, paramagnetic NMR, molecular dynamics, biological membranes

## Acknowledgements

We would like to thank the Matrics platform at the University Picardie Jules Verne for providing computing resources. We would also like to thank Anja Kaminski for excellent technical assistance and Dominique Cailleu for his help on setting up NMR experiments.

This work and the PhD fellowship of Morgane Adélaïde were funded by the ANR-BMBF NATURAL-ARSENAL project (ANR-19-AMRB-0004). Additional financial contributions of the Agence Nationale de la Recherche (AmphiPep 20-CE18-0021, SAFEST 21-CE18-0043, in particular the Interdisciplinary Thematic Institute SysChem, via the IdEx Unistra (ANR-10-IDEX-0002) within the French Investments for the Future Program), the University of Strasbourg, the CNRS, and the Région Grand-Est are gratefully acknowledged.

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CatBF's potent antimicrobial activity stems from its high charge, able to destabilize bacterial membranes by recruiting charged lipids. This peculiar mechanism of action bypasses conventional resistance pathways, making it effective against a wide range of drug-resistant bacteria.