Transmembrane Pore Research Articles

Antimicrobial peptides: An alternative strategy to combat antimicrobial resistance.

Antimicrobial peptides (AMPs) are a diverse group of naturally occurring molecules produced by eukaryotes and prokaryotes. They have an important role in innate immunity via their direct microbicidal properties or immunomodulatory activities against pathogens. With the widespread occurrence of antimicrobial resistance (AMR), AMPs are considered as viable alternatives for the treatment of multidrug-resistant microbes, inflammation, and, wound healing. The broad-spectrum antibacterial activity of AMPs is predominantly attributed to membrane disruption, leading to the formation of transmembrane pores and, eventually, cell lysis. However, mechanisms related to inhibition of cell wall synthesis, nucleic acid synthesis, protein synthesis, or enzymatic activity are also associated with these peptides. In this review, we discuss our current understanding, therapeutic uses and challenges associated with the clinical applications of AMPs.

Interleaflet Translocation of Second-Harmonic-Generation-Active Dye Molecules in Phospholipid Bilayers with Transmembrane Pores.

Second harmonic generation (SHG) measurements using SHG-active dye molecules have recently attracted attention as a method to detect the formation of pores in phospholipid bilayers. The bilayers, in which the dye molecules are embedded in the outer leaflet, exhibit a noncentrosymmetric structure, generating SHG signals. However, when pores form, these dye molecules translocate through the pores into the inner leaflet, leading to a more centrosymmetric structure and the subsequent loss of the SHG signals. A decrease in the SHG signals has been experimentally observed in membranes subjected to electrical stimuli. However, the characteristics of the interleaflet translocation of SHG-active dye molecules through pores remain unclear, hindering quantitative estimation of the membrane conditions, such as the pore size and density, based on the SHG signal reduction. In this study, we investigated the interleaflet translocation characteristics of Ap3, an SHG-active dye molecule, using molecular dynamics (MD) simulations and two-dimensional random-walk (RW) simulations. The MD simulations revealed that Ap3 molecules only translocate between the leaflets along the pore sidewalls. We determined the lateral diffusion coefficient of Ap3 within the membrane plane and its propensity for interleaflet movement at the pore wall. Based on these movement characteristics, the RW model successfully reproduced the characteristic time scale of the interleaflet translocation observed in the MD simulations. By varying the pore size and density in the RW simulations, we estimated that the characteristic time scale of interleaflet translocation depends on the -0.31 power of the pore radius and the -1.13 power of the pore density. Using these findings, we estimated the number of pores that probably formed in membranes during previous electroporation experiments. These results indicate the potential of optical measurement of the dye molecule movement for the indirect quantitative estimation of the pore size and number, which are challenging to measure optically.

Mechanisms of RCD-1 pore formation and membrane bending

Regulator of cell death-1 (RCD-1) governs the heteroallelic expression of RCD-1-1 and RCD-1-2, a pair of fungal gasdermin (GSDM)-like proteins, which prevent cytoplasmic mixing during allorecognition and safeguard against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (e.g., senescence plasmids) by effecting a form of cytolytic cell death. However, the underlying mechanisms by which RCD-1 acts on the cell membrane remain elusive. Here, we demonstrate that RCD-1 binds acidic lipid membranes, forms pores, and induces membrane bending. Using atomic force microscopy (AFM) and AlphaFold, we show that RCD-1-1 and RCD-1-2 form heterodimers that further self-assemble into ~14.5 nm-wide transmembrane pores (~10 heterodimers). Moreover, through AFM force spectroscopy and micropipette aspiration, we reveal that RCD-1 proteins bend membranes with low bending moduli. This combined action of pore formation and membrane deformation may constitute a conserved mechanism within the broader GSDM family.

Fatty acid desaturase 3-mediated α-linolenic acid biosynthesis in plants.

Omega (ω)-3 fatty acids (FAs) are essential components of cell membranes that also serve as precursors of numerous regulatory molecules. α-linolenic acid (ALA), one of the most important ω3 FAs in plants, is synthesized in both the plastid and extraplastidial compartments. FA Desaturase (FAD) 3 is an extraplastidial enzyme that converts linoleic acid (LA) to ALA. Phylogenetic analysis suggested that FAD3 proteins are distinct from FAD7 and FAD8 desaturases, which convert LA to ALA in plastids. Structural analysis of FAD3 proteins indicated a positive relationship between enzymatic activity and transmembrane pore length and width. An inverse relationship between temperature and ALA biosynthesis was also evident, with ALA accumulation decreasing with increasing temperature. These findings suggest that certain FAD3 enzymes are more effective at converting LA to ALA than others. This information could potentially be used to engineer crop plants with higher levels of ALA.

Acidic pH can attenuate immune killing through inactivation of perforin.

Cytotoxic lymphocytes are crucial to our immune system, primarily eliminating virus-infected or cancerous cells via perforin/granzyme killing. Perforin forms transmembrane pores in the plasma membrane, allowing granzymes to enter the target cell cytosol and trigger apoptosis. The prowess of cytotoxic lymphocytes to efficiently eradicate target cells has been widely harnessed in immunotherapies against haematological cancers. Despite efforts to achieve a similar outcome against solid tumours, the immunosuppressive and acidic tumour microenvironment poses a persistent obstacle. Using different types of effector cells, including therapeutically relevant anti-CD19 CAR T cells, we demonstrate that the acidic pH typically found in solid tumours hinders the efficacy of immune therapies by impeding perforin pore formation within the immunological synapse. A nanometre-scale study of purified recombinant perforin undergoing oligomerization reveals that pore formation is inhibited specifically by preventing the formation of a transmembrane β-barrel. The absence of perforin pore formation directly prevents target cell death. This finding uncovers a novel layer of immune effector inhibition that must be considered in the development of effective immunotherapies for solid tumours.

Cryo-EM structure of the human Pannexin-3 channel.

Pannexin-3 (PANX3) is a member of the pannexin family of large-pore, ATP-permeable channels conserved across vertebrates. PANX3 contributes to various developmental and pathophysiological processes by permeating ATP and Ca2+ ions; however, the structural basis of PANX3 channel function remains unclear. Here, we present the cryo-EM structure of human PANX3 at 2.9-3.2Å. The PANX3 channel is heptameric and forms a transmembrane pore along the central symmetric axis. The narrowest constriction of the pore is composed of an isoleucine ring located in the extracellular region, and its size is comparable to that of other pannexins. A structural variability analysis revealed prominent structural dynamics in intracellular regions. Our structural studies provide a foundation for understanding the detailed properties of pannexin channels.

Cholesterol-driven modulation of membrane-membrane interactions by an antimicrobial peptide, NK-2, in phospholipid vesicles

Antimicrobial peptides (AMPs) are essential components of the innate immune system, demonstrating their antimicrobial effects primarily through the creation of transmembrane pores that result in membrane disruption. Cholesterol within the membrane can significantly affect the interaction between AMPs and the membrane, as it is known to alter both the permeability and elastic properties of the membrane. In this study, we have investigated the influence of cholesterol on the interaction of the AMP, NK-2 with phospholipid vesicles. We prepared giant unilamellar vesicles (GUVs) composed of DOPC-DOPG and Egg PC, varying the cholesterol concentrations, and analyzed them using phase contrast microscopy. The aggregation of vesicles is evident in the phase contrast microscopy observations of GUVs. The aggregation of GUVs with cholesterol ultimately leads to a collapse state, a condition not typically seen in GUVs lacking cholesterol. Furthermore, the aggregation kinetics were determined from the analysis of phase contrast micrographs. This biophysical investigation offers valuable insights into how cholesterol affects the interactions between membranes induced by antimicrobial peptides.

Dissecting the Membrane Association Mechanism of Aerolysin Pores at Femtomolar Concentrations Using Water as a Probe.

Aerolysin is a bacterial toxin that forms transmembrane pores at the host plasma membrane and has a narrow internal diameter and great stability. These assets make it a highly promising nanopore for detecting biopolymers such as nucleic acids and peptides. Although much is known about aerolysin from a microbiological and structural perspective, its membrane association and pore-formation mechanism are not yet fully understood. Here, we used angle-resolved second harmonic scattering (AR-SHS) and single-channel current measurements to investigate how wild-type (wt) aerolysin and its mutants interact with liposomes in aqueous solutions at femtomolar concentrations. Our AR-SHS experiments were sensitive enough to detect changes in the electrostatic properties of membrane-bound aerolysin, which were induced by variations in pH levels. We reported for the first time the membrane binding affinity of aerolysin at different stages of the pore formation mechanism: while wt aerolysin has a binding affinity as high as 20 fM, the quasi-pore and the prepore states show gradually decreasing membrane affinities, incomplete insertion, and a pore opening signature. Moreover, we quantitatively characterized the membrane affinity of mutants relevant for applications to nanopore sensing. Our study provides a label-free method for efficiently screening biological pores suitable for conducting molecular sensing and sequencing measurements as well as for probing pore-forming processes.

Nanoelectrospray based synthesis of large, transportable membranes with integrated membrane proteins

Membrane proteins tend to be difficult to study since they need to be integrated into a lipid bilayer membrane to function properly. This study presents a method to synthesize a macroscopically large and freely transportable membrane with integrated membrane proteins which is useful for studying membrane proteins and protein complexes in isolation. The method could serve as a blueprint for the production of larger quantities of functionalised membranes for integration into technical devices similar to the MinION DNA sequencer. It is possible to self-assemble larger biological membranes on solid surfaces. However, they cannot be removed from their solid support without destroying them. In transportable form, self-assembled membranes are limited to sizes of about 17 nm in nanodiscs. Here we electrospray a series of molecular layers onto the liquid surface of a buffer solution which creates a flat, liquid environment on the surface that directs the self-assembly of the membrane. This method enables us to experimentally control the membrane composition and to succeed in producing large membranes with integrated OmpG, a transmembrane pore protein. The technique is compatible with the assembly of membrane based protein complexes. Listeriolysin O and pneumolysin efficiently assemble into non-covalent membrane pore complexes of approximately 30 units or more within the surface layer.

Simulation-Guided Molecular Modeling of Nisin and Lipid II Assembly and Membrane Pore Formation.

The lantibiotic pore-forming peptide nisin is a promising candidate in the fight against multidrug-resistant bacteria due to its unique structure, which allows it to disrupt bacteria in two distinct ways─Lipid II trafficking and transmembrane pore formation. However, exactly how nisin and Lipid II assemble into oligomeric pore structures in the bacterial membrane is not known. Spontaneous peptide assembly into pores is difficult to observe in even the very long-time scale molecular dynamics (MD) simulations. In this study, we adopted an MD-guided modeling approach to investigate the nisin-nisin and nisin-Lipid II associations in the membrane environment. Through extensive microsecond-time scale all-atom MD simulations, we established that nisin monomers dimerize by forming β-sheets in a POPE:POPG lipid bilayer and oligomerize further to form stable transmembrane channels. We determined that these nisin dimers use Lipid II as a dimer interface to incur enhanced stability. Our results provide a clearer understanding of the self-assembly of nisin monomers within the membrane and insights into the role of Lipid II in the structural integrity of oligomeric structures.

Entangling roles of cholesterol-dependent interaction and cholesterol-mediated lipid phase heterogeneity in regulating listeriolysin O pore-formation.

Cholesterol-dependent cytolysins (CDCs) are the distinct class of β-barrel pore-forming toxins (β-PFTs) that attack eukaryotic cell membranes, and form large, oligomeric, transmembrane β-barrel pores. Listeriolysin O (LLO) is a prominent member in the CDC family. As documented for the other CDCs, membrane cholesterol is essential for the pore-forming functionality of LLO. However, it remains obscure how exactly cholesterol facilitates its pore formation. Here, we show that cholesterol promotes both membrane-binding and oligomerization of LLO. We demonstrate cholesterol not only facilitates membrane-binding, it also enhances the saturation threshold of LLO-membrane association, and alteration of the cholesterol-recognition motif in the LLO mutant (LLOT515G-L516G) compromises its pore-forming efficacy. Interestingly, such defect of LLOT515G-L516G could be rescued in the presence of higher membrane cholesterol levels, suggesting cholesterol can augment the pore-forming efficacy of LLO even in the absence of a direct toxin-cholesterol interaction. Furthermore, we find the membrane-binding and pore-forming abilities of LLOT515G-L516G, but not those of LLO, correlate with the cholesterol-dependent rigidity/ordering of the membrane lipid bilayer. Our data further suggest that the line tension derived from the lipid phase heterogeneity of the cholesterol-containing membranes could play a pivotal role in LLO function, particularly in the absence of cholesterol binding. Therefore, in addition to its receptor-like role, we conclude cholesterol can further facilitate the pore-forming, membrane-damaging functionality of LLO by asserting the optimal physicochemical environment in membranes. To the best of our knowledge, this aspect of the cholesterol-mediated regulation of the CDC mode of action has not been appreciated thus far.

Pneumolysin-responsive liposomal platform for selective treatment of Streptococcus pneumoniae.

The bacterium Streptococcus pneumoniae has become a leading cause of meningitis, sepsis, and bacterial pneumonia worldwide, with increased prevalence of antibiotic-resistant serotypes serving to exacerbate the issue. The main factor responsible for colonization and immune response escape in pneumococcal infections is the secreted molecule pneumolysin, which is a subset within a family of related toxins that form transmembrane pores in biological membranes through cholesterol recognition and binding. The conserved activity and structure of pneumolysin between all observed S. pneumoniae serotypes, along with its requirement for pathogenicity, has made this molecule an attractive target for vaccination, diagnostic, and sequestration platforms, but not yet as a facilitative agent for therapeutic treatment. Consequently, the present work aimed to examine the impact of liposomal cholesterol content for pneumolysin-induced release of the encapsulated antimicrobial peptide nisin. It was determined that a cholesterol content above 45mol% was necessary to facilitate interactions with both purified pneumolysin toxin and S. pneumoniae culture, demonstrated through enhanced nisin release and a reduction in hemolytic rates upon exposure of the toxin with cholesterol-rich vesicles. Antibacterial testing highlighted the ability of the developed platform to elicit a potent and specific bactericidal response in vitro against cultured S. pneumoniae when compared to a control strain, Staphylococcus epidermidis. It further improved viability of a fibroblast cell line upon S. pneumoniae challenge, outperforming free nisin via the synergistic impact of simultaneous bacterial clearance and pneumolysin neutralization. These findings collectively indicate that cholesterol-rich liposomes hold promise as a selective treatment platform against pneumococcal infections.

Derivatives of Pyrimidine Nucleosides Affect Artificial Membranes Enriched with Mycobacterial Lipids.

The mechanisms of action of pyrimidine nucleoside derivatives on model lipid membranes of various compositions were studied. A systematic analysis of the tested agents' effects on the membrane physicochemical properties was performed. Differential scanning microcalorimetry data indicated that the ability of nucleoside derivatives to disorder membrane lipids depended on the types of nucleoside bases and membrane-forming lipids. The 5'-norcarbocyclic uracil derivatives were found to be ineffective, while N4-alkylcytidines demonstrated the most pronounced effects, significantly decreasing the dipalmitoylphosphocholine melting temperature and cooperativity of phase transition. The elongation of hydrophobic acyl radicals potentiated the disordering action of N4-alkylcytidines, while an increase in hydrophilicity due to replacing deoxyribose with ribose inhibited this effect. The ability of compounds to form transmembrane pores was also tested. It was found that 5-alkyluridines produced single, ion-permeable pores in phosphatidylglycerol membranes, and that methoxy-mycolic acid and trehalose monooleate potentiated the pore-forming activity of alkyloxymethyldeoxyuridines. The results obtained open up perspectives for the development of innovative highly selective anti-tuberculosis agents, which may be characterized by a low risk of developing drug resistance due to the direct action on the membranes of the pathogen.

Computational Design of Pore-Forming Peptides with Potent Antimicrobial and Anticancer Activities.

Peptides that form transmembrane barrel-stave pores are potential alternative therapeutics for bacterial infections and cancer. However, their optimization for clinical translation is hampered by a lack of sequence-function understanding. Recently, we have de novo designed the first synthetic barrel-stave pore-forming antimicrobial peptide with an identified function of all residues. Here, we systematically mutate the peptide to improve pore-forming ability in anticipation of enhanced activity. Using computer simulations, supported by liposome leakage and atomic force microscopy experiments, we find that pore-forming ability, while critical, is not the limiting factor for improving activity in the submicromolar range. Affinity for bacterial and cancer cell membranes needs to be optimized simultaneously. Optimized peptides more effectively killed antibiotic-resistant ESKAPEE bacteria at submicromolar concentrations, showing low cytotoxicity to human cells and skin model. Peptides showed systemic anti-infective activity in a preclinical mouse model of Acinetobacter baumannii infection. We also demonstrate peptide optimization for pH-dependent antimicrobial and anticancer activity.

The conformational properties of alamethicin in ethanol studied by NMR.

Alamethicin, a peptide consisted of 20 amino acid residues, has been known to function as an antibiotic. The peptides self-associate in biological membranes, form an ion channel, and then induce cell death by leaking intracellular contents through a transmembrane pore of an ion channel. We investigated conformation and its thermal stability of alamethicin-A6 and -U6 in ethanol using proton nuclear magnetic resonance (NMR) spectroscopy; alamethicin-A6 and -U6 have the amino acid sequences of UPUAUAQUVUGLUPVUUQQO and UPUAUUQUVUGLUPVUUQQO, respectively, where U and O represent α-aminoisobutyric acid and phenylalaninol, respectively. As indicated by the under bars in the sequences, only the residue 6 differs between the alamethicins. We show that the alamethicins in ethanol form helix conformation in the region of the residues 2-11 and a non-regular conformation in the regions of the N- and C-termini, and that the helices are maintained up to 66°C at least. Conformations in the region of the residues 12-18 of the alamethicins, however, are not well identified due to the lack of NMR data. In addition, we demonstrate that the amide proton chemical shift temperature coefficients' method, which is known as an indicator for intramolecular hydrogen bonds in peptides and proteins in aqueous solutions, can be also applied to the alamethicins in ethanol. Further, we show that the conformation around the C-terminus of alamethicin-A6 is restrained by intramolecular hydrogen bonds, whereas that of alamethicin-U6 is either restrained or unrestrained by intramolecular hydrogen bonds; the alamethicin-U6 molecules having the restrained and unrestrained conformations coexist in ethanol. We discuss the two types of conformations using a model chain consisting of particles linked by rigid bonds called as the free jointed chain.

Nanoscale Perturbations of Lipid Bilayers Induced by Magainin 2: Insights from AFM Imaging and Force Spectroscopy

This study explores the impact of the antimicrobial peptide magainin 2 (Mag2) on lipid bilayers with varying compositions. We employed high-resolution atomic force microscopy (AFM) to reveal a dynamic spectrum of structural changes induced by Mag2. Our AFM imaging unveiled distinct structural alterations in zwitterionic POPC bilayers upon Mag2 exposure, notably the formation of nanoscale depressions within the bilayer surface, which we term as "surface pores" to differentiate them from transmembrane pores. These surface pores are characterized by a limited depth that does not appear to fully traverse the bilayer and reach the opposing leaflet. Additionally, our AFM-based force spectroscopy investigation on POPC bilayers revealed a reduction in bilayer puncture force (FP) and Young's modulus (E) upon Mag2 interaction, indicating a weakening of bilayer stability and increased flexibility, which may facilitate peptide insertion. The inclusion of anionic POPG into POPC bilayers elucidated its modulatory effects on Mag2 activity, highlighting the role of lipid composition in peptide-bilayer interactions. In contrast to surface pores, Mag2 treatment of E. coli total lipid extract bilayers resulted in increased surface roughness, which we describe as a fluctuation-like morphology. We speculate that the weaker cohesive interactions between heterogeneous lipids in E. coli bilayers may render them more susceptible to Mag2-induced perturbations. This could lead to widespread disruptions manifested as surface fluctuations throughout the bilayer, rather than the formation of well-defined pores. Together, our findings of nanoscale bilayer perturbations provide useful insights into the molecular mechanisms governing Mag2-membrane interactions.

Poptosis or Peptide-Induced Transmembrane Pore Formation: A Novel Way to Kill Cancer Cells without Affecting Normal Cells.

Recent advances in cancer treatment like personalized chemotherapy and immunotherapy are aimed at tumors that meet certain specifications. In this review, we describe a new approach to general cancer treatment, termed peptide-induced poptosis, in which specific peptides, e.g., PNC-27 and its shorter analogue, PNC-28, that contain the segment of the p53 transactivating 12-26 domain that bind to HDM-2 in its 1-109 domain, bind to HDM-2 in the membranes of cancer cells, resulting in transmembrane pore formation and the rapid extrusion of cancer cell contents, i.e., tumor cell necrosis. These peptides cause tumor cell necrosis of a wide variety of solid tissue and hematopoietic tumors but have no effect on the viability and growth of normal cells since they express at most low levels of membrane-bound HDM-2. They have been found to successfully treat a highly metastatic pancreatic tumor as well as stem-cell-enriched human acute myelogenous leukemias in nude mice, with no evidence of off-target effects. These peptides also are cytotoxic to chemotherapy-resistant cancers and to primary tumors. We performed high-resolution scanning immuno-electron microscopy and visualized the pores in cancer cells induced by PNC-27. This peptide forms 1:1 complexes with HDM-2 in a temperature-independent step, followed by dimerization of these complexes to form transmembrane channels in a highly temperature-dependent step parallel to the mode of action of other membranolytic but less specific agents like streptolysin. These peptides therefore may be effective as general anti-cancer agents.

Novel insights into the inhibitory effect of pterostilbene targeting Aeromonas hydrophila virulence

The overuse of antibiotics in aquaculture brings a range of problems. Therefore, novel approaches alternating antibiotics are urgently needed. Strategies targeting bacterial virulence have become one of the routes developing drugs against bacterial infections. Here, we found that pterostilbene (PSE) could inhibit the activity of aerolysin (AerA) and disrupt quorum sensing (QS) at sub-inhibitory concentrations. Molecular docking and dynamics simulation assays were performed to predict the binding mode and binding sites of AerA-PSE complex. Then we confirmed that PSE bound to the sites of Val189 and Ile302 and prevented AerA forming heptamer with transmembrane pores. In addition, PSE could protect to A549 cells and the channel catfish (Ictalurus punctatus) against AerA mediated cell injury and mortality infected with A. hydrophila. The findings demonstrated that targeting bacterial virulence could be a promising measure identifying agents against A. hydrophila infections.

The interaction of plant flavones with amphotericin B: Consequences for its pore-forming ability

The growth of antibiotic resistance to antifungal drugs contributes to the search for new ways to enhance their effectiveness and reduce toxicity. The undeniable advantage of polyene macrolide antibiotic amphotericin B (AmB) which ensures low pathogen resistance is its mechanism of action related to the formation of transmembrane pores in target lipid membranes. Here, we investigated the effects of plant flavones, chrysin, wogonin, baicalein, apigenin, scutellarein, luteolin, morin and fisetin on the pore-forming activity of AmB in the sterol-enriched membranes by electrophysiological assays. Сhrysin, wogonin, baicalein, apigenin, scutellarein, and luteolin were shown to decrease the AmB pore-forming activity in the bilayers composed of palmitoyloleylphosphocholine independently of their sterol composition. Morin and fisetin led to the increase and decrease in the AmB pore-forming activity in the ergosterol- and cholesterol-containing bilayers respectively. Differential scanning microcalorimetry of the gel-to-liquid crystalline phase transition of membrane forming lipids, molecular dynamics simulations, and absorbance spectroscopy revealed the possibility of direct interactions between AmB and some flavones in the water and/or in the lipid bilayer. The influence of these interactions on the antibiotic partitioning between aqueous solution and membrane and/or its transition between different states in the bilayer was discussed.

PAM-1: an antimicrobial peptide with promise against ceftazidime-avibactam resistant Escherichia coli infection.

Antibiotic misuse and overuse have led to the emergence of carbapenem-resistant bacteria. The global spread of resistance to the novel antibiotic combination ceftazidime-avibactam (CZA) is becoming a severe problem. Antimicrobial peptide PAM-1 offers a novel approach for treating infections caused by antibiotic-resistant bacteria. This study explores its antibacterial and anti-biofilm activities and mechanisms against CZA-resistant Escherichia. Coli (E. coli), evaluating its stability and biosafety as well. The broth microdilution method, growth curve analysis, crystal violet staining, scanning electron microscopy, and propidium iodide staining/N-phenyl-1-naphthylamine uptake experiments were performed to explore the antibacterial action and potential mechanism of PAM-1 against CZA-resistant E. coli. The biosafety in diverse environments of PAM-1 was evaluated by red blood cell hemolysis, and cytotoxicity tests. Its stability was further assessed under different temperatures, serum concentrations, and ionic conditions using the broth microdilution method to determine its minimum inhibitory concentration (MIC). Galleria mellonella infection model and RT-qPCR were used to investigate the in vivo antibacterial and anti-inflammatory effects. In vitro antibacterial experiments demonstrated that the MICs of PAM-1 ranged from 2 to 8 μg/mL, with its effectiveness sustained for a duration of 24 h. PAM-1 exhibited significant antibiofilm activities against CZA-resistant E. coli (p < 0.05). Furthermore, Membrane permeability test revealed that PAM-1 may exert its antibacterial effect by disrupting membrane integrity by forming transmembrane pores (p < 0.05). Red blood cell hemolysis and cytotoxicity tests revealed that PAM-1 exerts no adverse effects at experimental concentrations (p < 0.05). Moreover, stability tests revealed its effectiveness in serum and at room temperature. The Galleria mellonella infection model revealed that PAM-1 can significantly improve the survival rate of Galleria mellonella (>50%)for in vivo treatment. Lastly, RT-qPCR revealed that PAM-1 downregulates the expression of inflammatory cytokines (p < 0.05). Overall, our study findings highlight the potential of PAM-1 as a therapeutic agent for CZA-resistant E. coli infections, offering new avenues for research and alternative antimicrobial therapy strategies.