BamA-targeted antimicrobial peptide design for enhanced efficacy and reduced toxicity

The emergence of drug-resistant superbugs has necessitated a pressing need for innovative antibiotics. Antimicrobial peptides (AMPs) have demonstrated broad-spectrum antibacterial activity, reduced susceptibility to resistance, and immunomodulatory effects, rendering them promising for combating drug-resistant microorganisms. This study employed computational simulation methods to screen and design AMPs specifically targeting ESKAPE pathogens. Particularly, AMPs were rationally designed to target the BamA and obtain novel antimicrobial peptide sequences. The designed AMPs were assessed for their antibacterial activities, mechanisms, and stability. Molecular docking and dynamics simulations demonstrated the interaction of both designed AMPs, 11pep and D-11pep, with the β1, β9, β15, and β16 chains of BamA, resulting in misfolding of outer membrane proteins and antibacterial effects. Subsequent antibacterial investigations confirmed the broad-spectrum activity of both 11pep and D-11pep, with D-11pep demonstrating higher potency against resistant Gram-negative bacteria. D-11pep exhibited MICs of 16, 8, and 32 μg/mL against carbapenem-resistant Escherichia coli, carbapenem-resistant Pseudomonas aeruginosa, and multi-drug-resistant Acinetobacter baumannii, respectively, with a concomitant lower resistance induction. Mechanism of action studies confirmed that peptides could disrupt the bacterial outer membrane, aligning with the findings of molecular dynamics simulations. Additionally, D-11pep demonstrated superior stability and reduced toxicity in comparison to 11pep. The findings of this study underscore the efficacy of rational AMP design that targets BamA, along with the utilization of D-amino acid replacements as a strategy for developing AMPs against drug-resistant bacteria.


Introduction
Drug-resistant bacterial infections have become a critical global public health concern, significantly contributing to the worldwide mortality rate.The prevalence of drug resistance has escalated, leading to a significant number of annual fatalities.In 2019, drug-resistant infections caused an estimated 4.95 million deaths.Among these fatalities, the six superbugs known as ESKAPE pathogens [Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp (Antimicrobial Resistance 2022)] accounted Handling editor: J. Gonzalez-Lopez.
for approximately 929,000 deaths.The primary approach currently used to tackle drug-resistant bacterial infections involves modifying existing drug structures (Walesch et al. 2023;Lewis 2020).However, discovering new antibiotics through this strategy is becoming increasingly challenging.Exploring alternative methods to address this problem is of utmost importance (Furniss et al. 2022).
Antimicrobial peptides (AMPs) are small host defense proteins with the potential to combat drug-resistant microorganisms (Mukhopadhyay et al. 2020;Boparai and Sharma 2020;Mwangi et al. 2019).However, the potential of AMPs as a promising alternative to conventional antibiotics has been hindered by obstacles, such as cytotoxicity, instability, and high cost (Kim et al. 2014;Wei and Bobek 2005;Bowdish et al. 2005).Several strategies have been developed to overcome these challenges (Kim et al. 2017).For example, novel AMPs can be rationally designed using computational methods to address antibiotic resistance (Upert et al. 2021).Additionally, non-natural amino acids like D-amino acids (Vlieghe et al. 2010), cyclization, and chemical modifications have been introduced to enhance peptide stability (Hunter et al. 2005;Nguyen et al. 2005;Hancock and Sahl 2006).Studies have shown that synthetic antimicrobial peptides in D-isomeric form can enhance stability, particularly against enzymatic degradation (Lu et al. 2020;Maxian et al. 2021;Zhao et al. 2016).
This study utilized BamA (Ishikawa et al. 2004), a recognized bacterial vulnerability (Furniss et al. 2022), as the target for screening peptides from the Antimicrobial Peptide Database (APD3) (Wang et al. 2016).Subsequently, the peptides underwent rational design (Basu et al. 2022) modifications to create a positively charged helix composed of alternating hydrophobic and basic amino acids, resulting in a non-natural peptide sequence called 11pep.To enhance the antimicrobial activity of 11pep against drugresistant bacteria and minimize resistance, a strategy was implemented to replace all its amino acids with D-amino acids and reverse the sequence, thereby preserving the alpha helix of the peptides (Tan et al. 2021).Subsequently, the novel peptide D-11pep was obtained.This study presents the design, synthesis, activity, and stability of BamA-targeted antimicrobial peptides and their mechanisms of action.

Docking-based virtual screening
The BamA protein (PDB ID: 7NRE (Kaur et al. 2021)) was acquired from the Protein Data Bank (https:// www.rcsb.org/) (Burley et al. 2021).The organism specified is Escherichia coli O157:H7, and it is a synthetic construct.A total of 385 AMPs were obtained from the APD3 (https:// aps.unmc.edu) (Wang et al. 2016) antimicrobial library, consisting of antimicrobial peptides isolated from bacteria, predicted antimicrobial peptides, and synthetic antimicrobial peptides.The active binding sites of the protein correspond to the original ligand-binding sites.Standard precision docking was performed using SYBYL under default settings during the docking operation.Subsequently, the ten antimicrobial peptides with favorable docking results underwent fine molecular docking using the CDOCKER module of Discover studio (2021).The ESMFold (https:// esmat las.com/) (Lin et al. 2022) was utilized to predict the secondary structures of the intended antimicrobial peptides.

Molecular dynamics simulation
Molecular dynamics simulations were performed using Amber20.The peptides (11pep, D-11pep, and Darobactin) were parameterized using the GAFF force field (He et al. 2020;Jakalian et al. 2002), and the BamA were parameterized using the AMBER ff19SB force field (Tian et al. 2020).Phospholipid bilayers were generated using CHARMM-GUI (https:// charmm-gui.org/) (Lee et al. 2020) to create protein-peptide-membrane complexes.The TIP3P water model {98, 95, 124} was used to set the full cubic water box as the com box for each complex.The pmemd.MPI and pmemd.cuda modules in the AMBER20 package were utilized to conduct 100 ns molecular dynamics simulations of protein-peptide-membrane complexes.The CPPTRAJ module was employed to calculate RMSD and RMSF for the entire molecular simulation process, serving as an assessment of equilibration and stabilization.The MM/PBSA program (Lwin et al. 2006;Homeyer and Gohlke 2015;Miller et al. 2012) was utilized to calculate the binding free energies of the complexes.
The minimum inhibitory concentration (MIC) of the designed peptides against the tested microbes was determined using a standard method described in reference (Pal et al. 2006).The colonies on the plates were initially diluted with Mueller-Hinton broth (MHB) medium to a concentration of 10 6 CFU/mL.Next, 100 μL of a bacterial solution containing different concentrations of antimicrobial peptides was added to the 96-well plates.After 24 h of incubation at 37 °C, the absorbance of each well at 600 nm was measured using an enzyme marker.Sterile water was used as a substitute for medication in the positive control, while a sterile medium replaced the bacterial solution in the negative control.
After determining the MIC, a sample was taken from the well that exhibited no signs of bacterial growth.The sample was streaked onto a solid culture medium-containing Luria-Bertani broth (LB).The culture was incubated overnight at 37 °C.The minimum bactericidal concentration (MBC) was defined as the lowest concentration at which no bacterial growth was observed.

Time-kill curve
The CRE colonies, inoculated on LB solid medium overnight, were initially diluted to a concentration of 10 8 CFU/ mL using saline, followed by a subsequent dilution to 10 5 CFU/mL using LB medium.The bacterial solution was then combined with antimicrobial peptides to attain combination concentrations of 1/2 × MIC, 1 × MIC, and 2 × MIC.The optical density at 600 nm (OD600) was monitored at 2-h intervals over a 12-h period.

Prolonged induction of antimicrobial peptide resistance
The long-term effects of antimicrobial peptides on CRE were assessed by evaluating the changes in MIC values over a 10-day period.CRE at sub-inhibitory concentrations was cultured overnight in MHB, and then, the MIC of the antimicrobial peptide was measured.The bacteria with the newly established sub-inhibitory concentration were cultured overnight in MHB, and the MIC of the antimicrobial peptide was measured once more.This process was repeated for 10 consecutive days, with polymyxin B used as a control.
Protease, salt ion, temperature, pH, and serum stability CRE was selected as the representative bacterial strain to assess the stability of antimicrobial peptides.After combining with the antimicrobial peptide solution at different concentrations, the final enzyme concentration of trypsin, papain, pepsin, and proteinase K was adjusted to 1 mg/mL.
The mixture was then incubated for one hour in a water bath at 37 °C.The MIC of the protease-treated antimicrobial peptides was measured and compared to the untreated control.
To further examine the impact of environmental conditions on the antimicrobial activity of the peptides, the MIC was determined by incubating them with various salt ion solutions (150 mM NaCl, 6 mM NH 4 Cl, 2.5 mM CaCl 2 , 1 mM MgCl 2 , 4 mM FeCl 3 , and 2.5 mM BaCl 2 ) for 30 min at 37 °C.Finally, the antimicrobial peptides were incubated at varying temperatures (0 °C, 37 °C, and 100 °C) and pH levels (4, 6, 8, and 10) for 30 min, followed by measuring the MIC values.
Various concentrations of Fetal Bovine Serum (FBS) were added to the medium-bacterial solution mixture.The final concentrations of FBS in the mixture were 0%, 5%, and 10%, respectively.The mixture was then incubated for 30 min to determine the MIC.

Cell viability assay
A cell (human normal lung epithelial cells, BEAS-2B cells ATCC CRL-9609) suspension with a concentration of 10 4 CFU/mL was added to each well of a 96-well plate (100 μL per well).The plate was then incubated overnight with different concentrations of antimicrobial peptide solutions and subjected to a 6 h incubation in a 5% CO 2 environment.The cytotoxicity was assessed using MTT kits.

Hemolytic toxicity assay
Sterile suspensions of sheep blood cells containing varying peptide concentrations were transferred to 96-well plates.Hemoglobin release was assessed by measuring the absorbance at 414 nm.Positive controls consisted of 10% Triton X-100 and sterile sheep blood cell suspension (OD T ), while negative controls comprised PBS and sterile sheep blood cell suspension (OD PBS ).The extent of hemolytic toxicity was assessed using the equation provided below

Cell membrane permeability assay
Enterococcus faecalis, CRE, CRPA, and MDRAB strains were diluted to a concentration of 10 7 CFU/mL in a fresh medium.Subsequently, they were exposed to an antibiotic peptide at a concentration of 1 × MIC and incubated at 37 °C.The OD 260 was measured after incubating the samples for 0, 0.5, 1.5, 3, 6, and 12 h, followed by filtration through a 0.22 μm aqueous membrane.PBS served as the negative control, while Triton X-100 was used as the positive control.

Biofilm assay
The overnight cultures of CRE were initially diluted to a concentration of 10 8 CFU/mL using LB medium.The bacterial solutions containing different concentrations of antimicrobial peptides were added to 96-well plates and incubated at 37 °C for 1 h to allow bacterial immobilization.The bacterial samples were stained with crystalline violet, and the OD 450 was measured to evaluate the impact of the antimicrobial peptide on initial bacterial adherence.
The bacterial solution was diluted to a concentration of 10 5 CFU/mL.The diluted bacterial solutions, with varying doses of antimicrobial peptides, were added to the 96-well plate, which was then incubated at 37 °C for 24 h.Crystalline violet staining was performed to assess the inhibitory effect of antimicrobial peptides on biofilm development.
The diluted bacterial solution (10 4 CFU/mL) was added to the 96-well plate and incubated at 37 °C for 24 h to facilitate biofilm formation.The liquid was aspirated from the 96-well plate, and media with varying concentrations of antimicrobial peptides were added.The plate was subsequently incubated at 37 °C for 24 h.Crystalline violet staining was used to assess the disruptive impact of the antimicrobial peptide on the established biofilm.

Scanning electron microscope
The CRE was incubated overnight using LB medium, followed by low-speed centrifugation and resuspension in PBS.Subsequently, the bacteria were combined with an antimicrobial peptide solution at a concentration of 2 × MIC, and incubated on a shaker at 37 °C for 2 h.After another round of low-speed centrifugation, the cells were fixed in 5% glutaraldehyde for 4 h.Following fixation, the cells were dehydrated with sequential ethanol treatments in preparation for scanning electron microscopy (JSM-IT700HR, Japan).The bacteria treated with PBS served as the negative control.

Virtual screening results based on molecular docking
We utilized the native ligand-binding site of the protein as the active binding site, which is located at the membranefacing side gate of BamA.This gate is formed by the β1 and β16 chains, with the β16 chain adopting a random coiling conformation at residue Gly384 (Fig. 1).The results are presented in Table 1.

Rational design of antimicrobial peptides
Based on the amphiphilic properties of antimicrobial peptides and the impact of secondary structure on their antimicrobial activity, we designed a peptide sequence by combining three antimicrobial peptide fragments: RWV, RRW, and RKW.This sequence was constructed using alternating hydrophobic and basic amino acids, following the length criterion specified in AP02776.
We used EMSFold to predict the secondary structures and proceeded with sequences that exhibited α-helical secondary structures for precise molecular docking.Detailed information about the peptides can be found in Table 2, where we assigned the molecule 05 with the highest docking score as 11pep.
Previous studies have demonstrated the potential of replacing L-amino acids with D-amino acids as a promising strategy for enhancing both the antimicrobial activity and stability of peptides.To preserve the directional orientation of the helix, we developed a novel peptide, D-11pep, by substituting the amino acids in 11pep with their D-amino acid counterparts and reversing the sequence.

Molecular dynamics simulations
Molecular dynamics simulations were performed to investigate the interactions between BamA-peptide complexes (11pep and D-11pep) and the membrane.The results demonstrate a decrease in the longitudinal diameter of the entire BamA complex following treatment with 11pep, reducing it from 29.9 Å to 27.1 Å.Similarly, the complex treated with D-11pep exhibited a diameter decrease from 28.4 Å to 25.9 Å (Fig. 2).
RMSD analysis was performed to monitor the thermodynamic robustness and conformational stability of the peptide-protein (BamA) complexes.Furthermore, it provided insights into the variations in average atomic separation of the protein throughout the simulation.Figure 4 displays the RMSD plots of the peptide-protein complexes (11pep, D-11pep) and the control drug Darobactin-BamA complex.The current study demonstrates that all three peptide-complex systems achieved stability during the final 20 ns of the molecular simulation, suggesting an adequate simulation duration.Subsequent analysis of the RMSD plots indicated average RMSD ranges of approximately 4.25 Å, 3.52 Å, and 1.72 Å for the protein BamA in the complexes with 11pep, D-11pep, and Darobactin, respectively.In the initial phase (0-10 ns), the maximum deviations were approximately − 0.87 Å, − 0.70 Å, and 0.59 Å for 11pep, D-11pep, and Darobactin, respectively.However, D-11pep was the only peptide     3), suggesting that D-type amino acids exhibit stronger interactions with BamA.These findings provide valuable insights into the peptide-BamA interactions and offer guidance for future investigations in this field.

Antibacterial and bactericidal activity
The sequences, molecular weights, and purity (over 98%) of the synthesized antimicrobial peptides, namely 11pep and D-11pep, are provided in Table 4.
Using the microdilution method, we determined the MIC of the antimicrobial peptides against 11 bacterial strains, as summarized in Table 5.Our results unequivocally demonstrate the broad-spectrum antibacterial activity of both 11pep and D-11pep, including efficacy against Gram-negative bacteria that exhibit antibiotic resistance.Notably, 11pep exhibited higher bioactivity against Enterococcus faecalis, Pseudomonas aeruginosa, and carbapenem-resistant Pseudomonas aeruginosa, with MIC values of 8, 16, and 16 μg/mL, respectively.Conversely, D-11pep displayed MIC values of 4 μg/mL  Furthermore, we made an intriguing observation that the MBC of the antimicrobial peptides was nearly double the MIC.This suggests that a higher concentration of the peptides was required to achieve bactericidal activity.

Time-kill curve analysis
The time-kill curves evaluating the activity of 11pep and D-11pep against CRE were examined in this study.The results demonstrated that both peptides, at a concentration of 2 × MIC, significantly reduced the colony counts of CRE by nearly 99.9% (3 log 10 of CFU/mL) after 12 h of incubation, which was significantly lower than that of the control (P < 0.0001).However, the inhibitory effects of D-11pep at 1/2 × MIC against CRE were only significant within 2 h (P < 0.001) compared to the blank control, and this phenomenon disappeared after 2 h of incubation.Additionally, the bactericidal activity of 11pep and D-11pep varied significantly with different concentrations (P < 0.001) (Fig. 6).Notably, both peptides exhibited bactericidal activity at a concentration of 2 × MIC, while the inhibitory activity at a concentration of 1 × MIC depended mainly on the drug concentration.These findings underscore the efficacy of 11pep and D-11pep in treating CRE infections and highlight their potential as therapeutic agents against drug-resistant bacteria.

Antimicrobial peptide resistance analysis
Compared to conventional antibiotics, antimicrobial peptides offer a notable advantage in terms of reduced susceptibility to drug resistance.Figure 7 illustrates this advantage, showing that the MIC of D-11pep against CRE remained relatively stable even after 10 days of continuous exposure.In contrast, the MIC of 11pep increased threefold, and the MIC of polymyxin B increased sevenfold.These findings highlight the high efficacy of D-11pep against drug-resistant bacteria and demonstrate the advantage of incorporating D-amino acids in reducing drug resistance.

Protease stability analysis
One of the main challenges in the in vivo application of peptide-based therapeutics is their vulnerability to proteases.To assess the resistance of 11pep and D-11pep to proteases, their MIC against CRE were determined after incubating the antimicrobial peptides with trypsin, papain, pepsin, and proteinase K for 1 h, using various concentrations.Table 6 presents the results, indicating that D-11pep exhibited remarkable stability in the presence of proteases, maintaining its MIC value, whereas 11pep displayed negligible antimicrobial activity.These findings underscore the susceptibility of 11pep, composed of L-type natural amino acids, to enzymatic degradation, while D-type amino acids confer significantly higher resistance to enzymatic digestion.The enhanced protease stability of D-11pep suggests its potential as a promising candidate for in vivo applications as an antimicrobial peptide.

Salt ion stability analysis
The antibacterial activity of 11pep and D-11pep against CRE was evaluated under different physiological salt ion concentrations to investigate the influence of salt ions on the antimicrobial efficacy of these peptides.The results, presented in Table 7, demonstrate that the MIC of both 11pep and D-11pep against CRE remained constant across various physiological salt conditions.

Temperature and pH stability analysis
The results of the temperature and pH stability experiments conducted on antimicrobial peptides, 11pep and D-11pep, suggest that D-amino acids have limited impact on enhancing stability in this context.However, D-11pep exhibits superior antibacterial activity compared to 11pep, except when exposed to pH 10 conditions.Specifically, under neutral conditions at a temperature of 37 °C (control), D-11pep demonstrates optimal antibacterial activity (1 × MIC).Furthermore, it maintains a consistent MIC of 2 × MIC against CRE under temperatures of 0 °C, 100 °C, and acidic conditions.Notably, under alkaline conditions (pH 8 and pH 10), D-11pep exhibits a broader range of MIC variations, reaching 4-8 × MIC.In contrast, 11pep only reaches a 2 × MIC under alkaline conditions, with the MIC remaining unchanged across other conditions.The detailed variations in MIC are provided in Table 8.

Serum stability analysis
The stability of a potential drug in serum is a critical factor to consider when evaluating its suitability for drug development.Table 9 demonstrates that elevating serum levels in the medium result in a decline in the antibacterial efficacy of the antimicrobial peptide against CRE, compared

Cell viability of antimicrobial peptides
The cytotoxicity of 11pep and D-11pep was assessed on human normal lung epithelial cells in an in vitro setting.

Hemolytic toxicity of antimicrobial peptides
The hemolytic toxicity of 11pep and D-11pep on sterile sheep blood cells was investigated to assess the safety of antimicrobial peptides on mammalian cells.The results demonstrated that the 11pep and D-11pep exhibited lower hemolytic toxicity.Figure 9 illustrates that even at the maximum concentration (256 μg/mL), the hemolytic activity of 11pep and D-11pep on sheep erythrocytes was only 18.67% and 20.11%, respectively.

Cell membrane permeability of antimicrobial peptides
The ability to penetrate cell membranes is a crucial factor in determining the efficacy of antimicrobial peptides.In this study, Fig. 10 illustrates that the cell membrane permeability of 11pep, D-11pep, and the positive control imipenem (IPM) was assessed in four bacterial strains: CRE, MDRAB, CRPA, and Enterococcus faecalis, using a concentration of 1 × MIC.After 12 h of treatment, all three agents induced significant alterations in cell membrane permeability in the tested bacteria (P < 0.0001).Among the tested peptides, 11pep displayed maximum cell membrane permeability values of 52.65, 56.15, 59.91, and 64.01%against the four strains, whereas D-11pep exhibited significantly higher values of 86.87, 74.05, 87.28, and 85.51%, respectively.Importantly, D-11pep exhibited significantly different results compared to IPM (P < 0.0001), indicating that D-11pep may function by disrupting the cell membrane's permeability.

Biofilm analysis
The objective of this study was to investigate the effects of antimicrobial peptides on CRE bacterial biofilms, with a specific focus on their capacity to suppress bacterial adhe-    Overall, the results indicate that D-11pep exerts the most significant influence on the three processes of biofilm development and performs optimally at lower doses.The differences in biofilm inhibition and disruption between 11pep and D-11pep were substantial at lower concentrations but diminished as the concentrations increased.These findings underscore the potential of antimicrobial peptides.

Observation of the effects of antimicrobial peptides on membranes using scanning electron microscopy
The antibacterial mechanisms of 11pep and D-11pep on CRE were evaluated using SEM.The results (Fig. 12) demonstrated the ability of the peptides to damage the surface of CRE.Treatment with 11pep and D-11pep caused significant wrinkling, damage, and rupture of the CRE surfaces compared to PBS.Treatment with 11pep led to noticeable crumpling of the CRE cell surface, whereas D-11pep treatment resulted in cell rupture and the release of cell contents.These findings suggest that both 11pep and D-11pep exert antibacterial effects by disrupting the cell membrane, with D-11pep demonstrating greater potency than 11pep.

Discussion and conclusions
Antimicrobial resistance is a complex and multifaceted issue that has emerged as a leading cause of death worldwide (Antimicrobial Resistance 2022).To combat this problem, novel antimicrobial agents are urgent needed to be developed (Walesch et al. 2023;Wang et al. 2020;Brown and Wright 2016).Rational design could create diverse peptide sequences and structures to enrich the precursor chemical library of antimicrobial drugs.Moreover, computer software could assist in targeted design, reducing redundancy and improving the overall efficiency of the process.
BamA is a critical core component of the outer membrane protein (OMP) family (Kuszak et al. 2015), playing a crucial role in facilitating the insertion of OMPs into the outer membrane (Noinaj et al. 2015).This region, known as the Achilles' heel of bacteria, is an effective target for designing antimicrobial peptides (AMPs) against ESKAPE pathogens.Studies have shown that AMPs targeting BamA, such as Darobactin (Imai et al. 2019), exert antibacterial activity by binding to its skeletal structure and closing the BamA lateral gate (Kaur et al. 2021) through simulated β signaling.This mode of BamA inhibition by Darobactin can significantly overcome the resistance effects caused by specific amino acid mutations.
To overcome the drawbacks of antimicrobial peptides, such as toxicity, instability, and high cost (Mahlapuu et al. 2016), D-amino acids were substituted for L-amino acids, and the sequence of 11pep was reversed.This approach preserved the critical α-helix structure of the antimicrobial peptide and enhanced stability by generating peptide bonds resistant to enzymatic degradation (Chen et al. 2016), thereby improving the in vivo half-life of the antimicrobial peptides (Zhong et al. 2020).
Therefore, in this study, the active binding site of Darobactin in the BamA protein was utilized to screen AMPs from the APD3 library.Peptide sequences with high affinity for binding to the lateral gate and cavity regions of BamA were identified through molecular docking and molecular dynamics simulations.These sequences were combined to create the novel antimicrobial peptides, 11pep and D-11pep.The results demonstrated that the designed antimicrobial peptides, 11pep and D-11pep, could inhibit the insertion of BamA into the outer membrane by occupying the cavity and lateral gate regions, thus exhibiting antimicrobial activity.Through the analysis of simulation trajectories such as RMSD and RMSF, it was found that D-11pep exhibited greater stability in its interaction with the BamA protein compared to 11pep and Darobactin.
The results of antibacterial activity testing demonstrated that both 11pep and D-11pep, synthesized using Subsequent investigations aimed to study the antibacterial mechanisms of 11pep and D-11pep.Scanning electron microscopy results revealed that exposure to both peptides resulted in notable surface wrinkling, damage, and rupture of CRE.Additionally, D-11pep was able to eliminate 69.31% of bacterial biofilm at a concentration of 1 × MIC, which was consistent with the results of molecular dynamics simulations.These findings provide evidence of D-11pep's superior antibacterial activity compared to 11pep, as it targets the cell membrane, thereby supporting its alignment with the targeted direction of peptide design.
In conclusion, the designed BamA-targeted antimicrobial peptides have good antibacterial activities, and the replacement of natural amino acids with D-amino acids could enhance the antibacterial efficacy, stability, and toxicity of linear peptides, while decreasing their resistance.This study gives fresh insights into the rational design of novel BamA-targeted antimicrobial peptides against drugresistant bacteria via computational techniques and the D-amino acids' replacement on antimicrobial peptides.

Data Availability Statement
The data supporting the findings of this study are available from the corresponding author upon reasonable request.Additionally, a portion of the data supporting this study is publicly available through the following sources: APD3 (https:// aps.unmc.edu), PDB (https:// www.rcsb.org/), and EMSFold (https:// esmat las.com/).

Declarations
Conflict of interest The authors declare no competing financial interest.
Ethical approval This research project does not involve any human or animal subjects and therefore does not require ethical approval.The study will be conducted using only publicly available data or secondary data analysis.As such, it does not pose any risks or harm to individuals, and the privacy and confidentiality of any data analyzed will be protected.We are committed to conducting this study in accordance with the highest ethical standards and will adhere to all relevant laws and regulations.

Fig. 1
Fig. 1 Docking interactions between BamA and the polypeptides with the highest scores.A AP00141; B AP02776; C AP02856 that maintained stability throughout the subsequent 80 ns period, with a fluctuation mean of 0.01 Å and an absolute mean of 0.08 Å.In contrast, 11pep exhibited a fluctuation mean of 0.01 Å and an absolute mean of 0.2 Å, and Darobactin displayed an absolute mean fluctuation of 0.10 Å and an absolute mean of 0.13 Å.In conclusion, the complexes of D-11pep and Darobactin exhibited greater stability compared to 11pep, with D-11pep demonstrating the highest stability in terms of overall fluctuation in the MD trajectory.Root-mean-square fluctuation (RMSF) is a critical metric for characterizing the regions of flexibility and rigidity in a receptor structure.Furthermore, it offers insights into the mobility of amino acid residues during the simulation and their deviation from the initial positions.Higher RMSF values suggest reduced stability or increased mobility of

Fig. 2 Fig. 3
Fig. 2 Changes in the longitudinal diameter of BamA before and after molecular simulations: A before 11pep molecular dynamics simulation; B after 11pep molecular dynamics simulation; C before D-11pep molecular dynamics simulation; D after D-11pep molecular dynamics simulation

Figure 8
illustrates that neither 11pep nor D-11pep demonstrated significant cytotoxicity at the MIC level.Nevertheless, at a concentration of 256 μg/mL for both 11pep and D-11pep, there was a significant reduction in cell viability (P < 0.0001), and D-11pep still exhibited a cell viability of over 60%.These findings suggest that both 11pep and D-11pep exhibit low cytotoxicity, with D-11pep showing a notable decrease in comparison to 11pep.

Fig. 11
Fig. 11 The effect of antimicrobial peptides on biofilm.A Biofilm adherence.B Biofilm formation inhibition.C The detrimental impact on the biofilm.Compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 Author contribution YW and LY collaborated on the conception and design of the study, with LY conducting the experiments, analyzing the data, and writing the manuscript.ML and SG offered insights into data interpretation and critically revised the manuscript.ZL, YL and ZL provided valuable technical guidance throughout the study.All authors reviewed the manuscript.Funding This study was supported by the Natural Science Foundation of Chongqing (CSTB2022NSCQ-MSX1327), the Innovation Project of Chongqing Stay and Create Program (cx2020012), the Graduate Education Research Fund of Chongqing Municipal Education Commission, and the Graduate Innovation Project of Chongqing University of Technology (gzlcx20223347).

Table 1
Molecular docking scores of the screened antimicrobial peptides

Table 2
The molecular docking scores and sequence of the designed antibacterial peptides

Table 3
Calculated binding free energy of 11pep, D-11pep, and Darobactin

Table 4
The confirmed sequences and molecular weights of 11pep and D-11pep against Enterococcus faecalis, Escherichia coli, carbapenem-resistant Enterobacteriaceae, and 16 μg/mL against carbapenem-resistant Pseudomonas aeruginosa.These findings unambiguously indicate that D-11pep surpasses 11pep in effectiveness against drug-resistant bacteria.

Table 6 The
MIC values of 11pep and D-11pep in the presence of various proteasesPeptide MIC (μg/mL)