Leaflet by Leaflet Synergistic Effects of Antimicrobial Peptides on Bacterial and Mammalian Membrane Models.

Antimicrobial peptides (AMPs) offer significant hope in the fight against antibiotic resistance. Operating via a mechanism different from that of antibiotics, they target the microbial membrane and ideally should damage it without impacting mammalian cells. Here, the interactions of two AMPs, magainin 2 and PGLa, and their synergistic effects on bacterial and mammalian membrane models were studied using electrochemical impedance spectroscopy, atomic force microscopy (AFM), and fluorescence correlation spectroscopy. Toroidal pore formation was observed by AFM when the two AMPs were combined, while individually AMP effects were confined to the exterior leaflet of the bacterial membrane analogue. Using microcavity-supported lipid bilayers, the diffusivity of each bilayer leaflet could be studied independently, and we observed that combined, the AMPs penetrate both leaflets of the bacterial model but individually each peptide had a limited impact on the proximal leaflet of the bacterial model. The impact of AMPs on a ternary, mammalian mimetic membrane was much weaker.

Beta cell regeneration upon magainin and growth hormone treatment as a possible alternative to insulin therapy.

Insulin therapy, pancreas transplantation and ß cell regeneration are among the suggested treatment strategies for type 1 diabetes. It has been shown that some antimicrobial peptides have the potential to increase insulin release and to improve glucose tolerance, although the mechanism by which they promote the regeneration of damaged pancreatic cells to functional ß-like cells remains unknown. To answer this question, we evaluated the in vivo effects of magainin-AM2 and growth hormone (GH) on the regeneration of streptozotocin (STZ)-damaged mouse pancreas. Treatment with magainin-AM2 and GH ameliorated the effects of STZ on fasting blood glucose and glucose tolerance test values, and also resulted in a significant increase in total cell counts (α and ß) and the number of insulin+ and glucagon+ cells per islet and a decrease in the number of T and B cells. In addition, we observed a 1.43- and 2.21-fold increase in expression of paired box 4, one of the main factors for α to ß-like cell conversion, in normal- and diabetes-treated mice, respectively. Similarly, expression of P-S6 and extracellular signal-regulated kinases 1 and 2, required for cell proliferation/differentiation, increased by 3.27- and 2.19-fold among the diabetes-treated and control diabetic mice, respectively. Furthermore, in all experiments, amelioration of the effects of STZ were greatest upon Mag treatment followed by GH administration. The present in vivo data provide evidence in support of the possibility of pharmaceutical induction of α cell production and their trans-differentiation to functional ß-like cells.

Magainin 2 and PGLa in bacterial membrane mimics IV: Membrane curvature and partitioning.

We previously reported that the synergistically enhanced antimicrobial activity of magainin 2 (MG2a) and PGLa is related to membrane adhesion and fusion. Here, we demonstrate that equimolar mixtures of MG2a and L18W-PGLa induce positive monolayer curvature stress and sense, at the same time, positive mean and Gaussian bilayer curvatures already at low amounts of bound peptide. The combination of both abilities-membrane curvature sensing and inducing-is most likely the base for the synergistically enhanced peptide activity. In addition, our coarse-grained simulations suggest that fusion stalks are promoted by decreasing the free-energy barrier for their formation rather than by stabilizing their shape. We also interrogated peptide partitioning as a function of lipid and peptide concentration using tryptophan fluorescence spectroscopy and peptide-induced leakage of dyes from lipid vesicles. In agreement with a previous report, we find increased membrane partitioning of L18W-PGLa in the presence of MG2a. However, this effect does not prevail to lipid concentrations higher than 1 mM, above which all peptides associate with the lipid bilayers. This implies that synergistic effects of MG2a and L18W-PGLa in previously reported experiments with lipid concentrations >1 mM are due to peptide-induced membrane remodeling and not their specific membrane partitioning.

Interaction between Antimicrobial Peptide Magainin 2 and Nonlipid Components in the Bacterial Outer Envelope.

Antimicrobial peptides (AMPs) offer advantages over conventional antibiotics; for example, bacteria develop more resistance to small-molecule antibiotics than to AMPs. The interaction of the AMPs with the lipopolysaccharide (LPS) layer of the Gram-negative bacteria cell envelope is not well understood. A MARTINI model was constructed of a Gram-negative bacterial outer membrane interacting with the AMP Magainin 2. In a 20 µs molecular dynamics (MD) simulation, the AMP diffused to the LPS layer of the cell envelope and remained there, suggesting interactions between the Magainin 2 and the LPS layer, causing the AMP to concentrate at that position. The free energy profile for the insertion of the Magainin 2 into the membrane was also calculated using umbrella sampling, which showed that the AMP positioned such that the cationic side chains of the AMP coordinated to the negatively charged phosphate groups of the LPS layer. These simulations indicate that the AMP Magainin 2 partition into the LPS layer of a bacterial membrane.

Single-Cell Analysis of the Antimicrobial and Bactericidal Activities of the Antimicrobial Peptide Magainin 2.

Antimicrobial peptides (AMPs) inhibit the proliferation of or kill bacterial cells. To measure these activities, several methods have been used, which provide only the average value of many cells. Here, we report the development of a method to examine the antimicrobial and bactericidal activities of AMPs at the single-cell level (i.e., single-cell analysis) and apply this strategy to examine the interaction of an AMP, magainin 2 (Mag), with Escherichia coli cells. Using this method, we monitored the proliferation of single cells on agar in a microchamber and measured the distribution of the number of cells in each microcolony using optical microscopy. For method A, we incubated cells in the presence of various concentrations of AMPs for 3 h. The fraction of microcolonies containing only a single cell, Psingle, increased with the Mag concentration and reached 1 at a specific concentration, which corresponded to the MIC. For method B, after the interaction of a cell suspension with an AMP for a specific time, an aliquot was diluted to stop the interaction, and the proliferation of single cells then was monitored after a 3-h incubation; this method permits the definition of Psingle(t), the fraction of dead cells after the interaction. For the interaction of Mag with E. coli cells, Psingle(t) increased with the interaction time, reaching ~1 at 10 and 20 min for 25 and 13 µM Mag, respectively. Thus, these results indicate that a short interaction time between Mag and E. coli cells is sufficient to induce bacterial cell death. IMPORTANCE To elucidate the activity of antimicrobial peptides (AMPs) against bacterial cells, it is important to estimate the interaction time that is sufficient to induce cell death. We have developed a method to examine the antimicrobial and bactericidal activities of AMPs at the single-cell level (i.e., single-cell analysis). Using this method, we monitored the proliferation of single cells on agar in a microchamber and measured the distribution of the number of cells in each microcolony using optical microscopy. We found that during the interaction of magainin 2 (Mag) with E. coli cells, the fraction of dead cells, Psingle(t), increased with the interaction time, rapidly reaching 1 (e.g., 10 min for 25 µM Mag). This result indicates that Mag induces cell death after a short time of interaction.

Anurans against SARS-CoV-2: A review of the potential antiviral action of anurans cutaneous peptides.

At the end of 2019, in China, clinical signs and symptoms of unknown etiology have been reported in several patients whose sample sequencing revealed pneumonia caused by the SARS-CoV-2 virus. COVID-19 is a disease triggered by this virus, and in 2020, the World Health Organization declared it a pandemic. Since then, efforts have been made to find effective therapeutic agents against this disease. Identifying novel natural antiviral drugs can be an alternative to treatment. For this reason, antimicrobial peptides secreted by anurans' skin have gained attention for showing a promissory antiviral effect. Hence, this review aimed to elucidate how and which peptides secreted by anurans' skin can be considered therapeutic agents to treat or prevent human viral infectious diseases. Through a literature review, we attempted to identify potential antiviral frogs' peptides to combat COVID-19. As a result, the Magainin-1 and -2 peptides, from the Magainin family, the Dermaseptin-S9, from the Dermaseptin family, and Caerin 1.6 and 1.10, from the Caerin family, are molecules that already showed antiviral effects against SARS-CoV-2 in silico. In addition to these peptides, this review suggests that future studies should use other families that already have antiviral action against other viruses, such as Brevinins, Maculatins, Esculentins, Temporins, and Urumins. To apply these peptides as therapeutic agents, experimental studies with peptides already tested in silico and new studies with other families not tested yet should be considered.

Magainin 2 and PGLa in bacterial membrane mimics III: Membrane fusion and disruption.

We previously speculated that the synergistically enhanced antimicrobial activity of Magainin 2 and PGLa is related to membrane adhesion, fusion, and further membrane remodeling. Here we combined computer simulations with time-resolved in vitro fluorescence microscopy, cryoelectron microscopy, and small-angle X-ray scattering to interrogate such morphological and topological changes of vesicles at nanoscopic and microscopic length scales in real time. Coarse-grained simulations revealed formation of an elongated and bent fusion zone between vesicles in the presence of equimolar peptide mixtures. Vesicle adhesion and fusion were observed to occur within a few seconds by cryoelectron microscopy and corroborated by small-angle X-ray scattering measurements. The latter experiments indicated continued and time-extended structural remodeling for individual peptides or chemically linked peptide heterodimers but with different kinetics. Fluorescence microscopy further captured peptide-dependent adhesion, fusion, and occasional bursting of giant unilamellar vesicles a few seconds after peptide addition. The synergistic interactions between the peptides shorten the time response of vesicles and enhance membrane fusogenic and disruption properties of the equimolar mixture compared with the individual peptides.

The effects of magainin 2-derived and rationally designed antimicrobial peptides on Mycoplasma pneumoniae.

Combating the spread of antimicrobial resistance (AMR) among bacteria requires a new class of antimicrobials, which desirably have a narrow spectrum because of their low propensity for the spread of AMR. Antimicrobial peptides (AMPs), which target the bacterial cell membrane, are promising seeds for novel antimicrobials because the cell membrane is essential for all cells. Previously, we reported the antimicrobial and haemolytic effects of a natural AMP, magainin 2 (Mag2), isolated from the skin of Xenopus laevis (the African clawed frog), four types of synthesised Mag2 derivatives, and three types of rationally designed AMPs on gram-positive and gram-negative bacteria. To identify novel antimicrobial seeds, we evaluated the effect of AMPs on Mycoplasma pneumoniae, which also exhibits AMR. We also evaluated the antimicrobial effects of an AMP, NK2A, which has been reported to have antimicrobial effects on Mycoplasma bovis, in addition to Mag2 and previously synthesised seven AMPs, on four strains of M. pneumoniae using colorimetric, biofilm, and killing assays. We found that three synthesised AMPs, namely 17base-Ac6c, 17base-Hybrid, and Block, had anti-M. pneumoniae (anti-Mp) effect at 8-30 µM, whereas others, including NK2A, did not have any such effect. For the further analysis, the membrane disruption activities of AMPs were measured by propidium iodide (PI) uptake assays, which suggested the direct interaction of AMPs to the cell membrane basically following the colorimetric, biofilm, and killing assay results. PI uptake assay, however, also showed the NK2A strong interaction to cell membrane, indicating unknown anti-Mp determinant factors related to the peptide sequences. Finally, we conclude that anti-Mp effect was not simply determined by the membrane disruption activities of AMPs, but also that the sequence of AMPs were important for killing of M. pneumoniae. These findings would be helpful for the development of AMPs for M. pneumoniae.

In vitro activity of the antimicrobial peptides h-Lf1-11, MSI-78, LL-37, fengycin 2B, and magainin-2 against clinically important bacteria.

We investigated the antibacterial activity of the antimicrobial peptides h-Lf1-11, MSI-78, LL-37, fengycin 2B, and magainin-2. The minimum inhibitory concentration (MIC) was determined by microdilution technique according to CLSI (M07-A9, 2012) against Escherichia coli, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, carbapenem-resistant Klebsiella pneumoniae, and Acinetobacter baumannii. The MSI-78 showed potent bactericidal activity with MIC range of 1.25-40 mg/L against all bacterial strains. The h-Lf1-11, magainin-2, and LL-37 exhibited moderate activity (MIC range of 40-160, 80-160, and 40-160 mg/L, respectively) while the fengycin 2B did not show significant activity against all bacterial strains tested. These results revealed that MSI-78, h-Lf1-11, magainin-2, and LL-37 have great potential as antibacterial agents and their activity deserves to be more explored in further studies for the treatment of antibiotic-resistant bacteria.

A magainin-2 like bacteriocin BpSl14 with anticancer action from fish gut Bacillus safensis SDG14.

Bacteriocins are gaining utmost importance in antimicrobial and chemotherapy due to their diverse structure and activity. This study centres on magainin-2 like bacteriocin with anticancer action, produced by Bacillus safensis strain SDG14 isolated from gut of marine fish Sardinella longiceps. The purified bacteriocin designated as BpSl14 was thermostable and pH tolerant. The molecular weight of BpS114 was estimated to be 6061.2 Da using MALDI-ToF MS. The partial primary sequence was elucidated by peptide mass fingerprinting using MALDI MS/MS. The tertiary structure of the partial sequence was similar to that of two magainin-2 α-helices joined together by extended indolicidin. The BpSl14 protein inhibited the cells of lung carcinoma, one of the deadliest cancers. Docking studies conducted with DR5 and TGF-ß, two of the most prominent apoptotic receptors in adenocarcinoma, also proved the anti-apoptotic action of BpSl14.

Development of Antimicrobial Stapled Peptides Based on Magainin 2 Sequence.

Magainin 2 (Mag2), which was isolated from the skin of the African clawed frog, is a representative antimicrobial peptide (AMP) that exerts antimicrobial activity via microbial membrane disruption. It has been reported that the helicity and amphipathicity of Mag2 play important roles in its antimicrobial activity. We investigated and recently reported that 17 amino acid residues of Mag2 are required for its antimicrobial activity, and accordingly developed antimicrobial foldamers containing α,α-disubstituted amino acid residues. In this study, we further designed and synthesized a set of Mag2 derivatives bearing the hydrocarbon stapling side chain for helix stabilization. The preferred secondary structures, antimicrobial activities, and cell-membrane disruption activities of the synthesized peptides were evaluated. Our analyses revealed that hydrocarbon stapling strongly stabilized the helical structure of the peptides and enhanced their antimicrobial activity. Moreover, peptide 2 stapling between the first and fifth position from the N-terminus showed higher antimicrobial activity than that of Mag2 against both gram-positive and gram-negative bacteria without exerting significant hemolytic activity. To investigate the modes of action of tested peptides 2 and 8 in antimicrobial and hemolytic activity, electrophysiological measurements were performed.

Palmitoylation of Membrane-Penetrating Magainin Derivatives Reinforces Necroptosis in A549 Cells Dependent on Peptide Conformational Propensities.

Anticancer lipopeptides (ACLPs) are considered promising alternatives to combat resistant cancer cells, but the influence of peptide conformational propensity alone on their selectivity and mechanism remains obscure. In this study, we developed N-palmitoylated MK5E (P1MK5E) and MEK5 (P1MEK5) that have the same composition of 23 residues undergoing the pH-dependent structural alterations but differ in the conformational tendency of their amino acid composites. Nonlipidated peptides were readily accumulated in the A549 cell nucleus by the direct membrane translocation and the heparan sulfate-mediated endocytosis than the lipid-raft-dependent pathway. The increased hydrophobicity favored the amino acid-position-dependent folding of P1MK5E and P1MEK5, respectively, toward the α-helical coiled-coil nanofibrils and amyloidlike ß-protofibrils. At the close concentrations (∼7.5 µM) to the toxic effects of doxorubicin (DOX), P1MK5E exhibited (i) an increased anticancer toxicity through a time-dependent S-phase arrest, (ii) enhanced plasma membrane permeability, and (iii) dose-dependent changes in the cell death characteristic features in the A549 cells relative to P1MEK5 that was almost inactive at ∼75 µM. These observations were in accordance with the TNF-α-mediated necroptotic signaling in the c-MYC/PARP1-overexpressed A549 cells exposed to P1MK5E and accompanied by the ultrastructure of plasma membrane protrusions, extensive endoplasmic reticulum (ER) membrane expansion, mitochondrial swelling, and the formation of distinct cytoplasmic vacuolation. The structural results and the bioactivity behaviors, herein, declared the significance of α-helical propensity in the peptide sequence and the nanostructure morphologies of self-assembling ACLPs upon the selectivity and enhanced anticancer effectiveness, which notably holds promise in the design and development of efficient therapeutics for cancer.

Review: Examining the Natural Role of Amphibian Antimicrobial Peptide Magainin.

Host defense peptides (HDPs) are a group of antimicrobial peptides (AMPs) that are crucial components of the innate immune system of many different organisms. These small peptides actively kill microbes and prevent infection. Despite the presence of AMPs in the amphibian immune system, populations of these organisms are in decline globally. Magainin is an AMP derived from the African clawed frog (Xenopus laevis) and has displayed potent antimicrobial effects against a wide variety of microbes. Included in this group of microbes are known pathogens of the African clawed frog and other amphibian species. Arguably, the most deleterious amphibious pathogen is Batrachochytrium dendrobatidis, a chytrid fungus. Investigating the mechanism of action of magainin can help understand how to effectively fight off infection. By understanding amphibian AMPs' role in the frog, a potential conservation strategy can be developed for other species of amphibians that are susceptible to infections, such as the North American green frog (Rana clamitans). Considering that population declines of these organisms are occurring globally, this effort is crucial to protect not only these organisms but the ecosystems they inhabit as well.

Highly synergistic antimicrobial activity of magainin 2 and PGLa peptides is rooted in the formation of supramolecular complexes with lipids.

Magainin 2 and PGLa are cationic, amphipathic antimicrobial peptides which when added as equimolar mixture exhibit a pronounced synergism in both their antibacterial and pore-forming activities. Here we show for the first time that the peptides assemble into defined supramolecular structures along the membrane interface. The resulting mesophases are quantitatively described by state-of-the art fluorescence self-quenching and correlation spectroscopies. Notably, the synergistic behavior of magainin 2 and PGLa correlates with the formation of hetero-domains and an order-of-magnitude increased membrane affinity of both peptides. Enhanced membrane association of the peptide mixture is only observed in the presence of phophatidylethanolamines but not of phosphatidylcholines, lipids that dominate bacterial and eukaryotic membranes, respectively. Thereby the increased membrane-affinity of the peptide mixtures not only explains their synergistic antimicrobial activity, but at the same time provides a new concept to increase the therapeutic window of combinatorial drugs.

Effect of membrane potential on pore formation by the antimicrobial peptide magainin 2 in lipid bilayers.

The effect of membrane potential on plasma membrane damage generated by antimicrobial peptides (AMPs) is an important, yet poorly characterized, process. Here, we studied the effect of membrane potential (φm) on pore formation by magainin 2 (Mag) in single giant unilamellar vesicles (GUVs) composed of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes. Various membrane potentials in GUVs containing gramicidin A were generated as a result of K+ concentration gradients. First, we examined Mag-generated membrane permeation of the water-soluble fluorescent probe calcein in single DOPG/DOPC-GUVs in the presence of membrane potential. The results indicate that the rate constant (kp) of Mag-induced pore formation increased with increasing negative membrane potentials. Analysis of the rim intensity of single GUVs interacting with low concentrations of a fluorescent probe, carboxyfluorescein-labeled Mag (CF-Mag), using confocal laser scanning microscopy (CLSM) shows that the concentration of CF-Mag in the membrane greatly increased with negative membrane potentials. This indicates that the binding constant of CF-Mag to the membrane increased with more negative membrane potentials. To elucidate the location of Mag in a GUV with φm during Mag-induced pore formation, we examined the interaction of Mag and a low concentration of a CF-Mag mixture with single GUVs containing the water-soluble fluorescent probe AF647 using CLSM. The data indicate that CF-Mag locates in the external leaflet of single GUVs until just before pore formation. Based on these data, we conclude that the increase in the surface concentration of Mag is one of the primary causes of the increase in kp with negative membrane potential.

Potent antibacterial activity of MSI-1 derived from the magainin 2 peptide against drug-resistant bacteria.

The structural modification of existing AMPs is an effective strategy to develop antimicrobial agents with high-efficiency, low-cost and low-toxicity antimicrobial agents. Methods: Here, we truncated 14-amino-acids at the N-terminus of MSI-78 to obtain MSI and further modified MSI to obtain four peptide analogs: MSI-1, MSI-2, MSI-3 and MSI-4. These peptide mutants were evaluated regarding their antibacterial activity against various sensitive or resistant bacteria; toxicity against mammalian cells or mice; and stability against violent pH, temperature variations and high NaCl concentrations. Finally, we also elucidated the possible mechanisms underlying its mode of action. Results: The results showed that MSI-1 and MSI-3 displayed activity that was superior to that of MSI-78 with MICs of 4-16 µg/ml and MBCs of 8-64 µg/ml, respectively, especially against drug-resistant bacteria, due to the increase in percent helicity and amphiphilicity. However, MSI-3, with higher hydrophobicity and antibacterial activity, had a relatively higher hemolysis rate and toxicity than MSI-1. MSI-1 exerted rapid bactericidal activity and effectively improved the survival rate and wound closure in penicillin-resistant E. coli-infected mice by eliminating bacterial counts in mouse organs or subeschar, further inhibiting the systemic dissemination of bacteria. Additionally, MSI-1 displayed perfect stability against violent pH, temperature variations and high NaCl concentrations and has the ability to circumvent the development of drug resistance. In terms of the mode of action, we found that at the super-MIC level, MSI-1 exhibited direct antimicrobial activity by disrupting the integrity of the bacterial cell membrane, while at the sub-MIC level, it bound to bacterial DNA to inhibit DNA replication and protein expression and ultimately disrupted bacterial biological function. Conclusions: This novel peptide MSI-1 could be a potential candidate for drug development against infection induced by drug-resistant bacteria.

Study of molecular transport through a single nanopore in the membrane of a giant unilamellar vesicle using COMSOL simulation.

The antimicrobial peptide (AMP) magainin 2 induces nanopores in the lipid membranes of giant unilamellar vesicles (GUVs), as observed by the leakage of water-soluble fluorescent probes from the inside to the outside of GUVs through the pores. However, molecular transport through a single nanopore has not been investigated in detail yet and is studied in the present work by simulation. A single pore was designed in the membrane of a GUV using computer-aided design software. Molecular transport, from the outside to the inside of GUV through the nanopore, of various fluorescent probes such as calcein, Texas-Red Dextran 3000 (TRD-3k), TRD-10k and TRD-40k was then simulated. The effect of variation in GUV size (diameter) was also investigated. A single exponential growth function was fitted to the time course of the fluorescence intensity inside the GUV and the corresponding rate constant of molecular transport was calculated, which decreases with an increase in the size of fluorescent probe and also with an increase in the size of GUV. The rate constant found by simulation agrees reasonably well with reported experimental results for inside-to-outside probe leakage. Based on Fick's law of diffusion an analytical treatment is developed for the rate constant of molecular transport that supports the simulation results. These investigations contribute to a better understanding of the mechanism of pore formation using various membrane-active agents in the lipid membranes of vesicles and the biomembranes of cells.

Qualitative and Quantitative Changes to Escherichia coli during Treatment with Magainin 2 Observed in Native Conditions by Atomic Force Microscopy.

The bacterial membrane has been suggested as a good target for future antibiotics, so it is important to understand how naturally occurring antibiotics like antimicrobial peptides (AMPs) disrupt those membranes. The interaction of the AMP magainin 2 (MAG2) with the bacterial cell membrane has been well characterized using supported lipid substrates, unilamellar vesicles, and spheroplasts created from bacterial cells. However, to fully understand how MAG2 kills bacteria, we must consider its effect on the outer membrane found in Gram-negative bacteria. Here, we use atomic force microscopy (AFM) to directly investigate MAG2 interaction with the outer membrane of Escherichia coli and characterize the biophysical consequences of MAG2 treatment under native conditions. While propidium iodide penetration indicates that MAG2 permeabilizes cells within seconds, a corresponding decrease in cellular turgor pressure is not observed until minutes after MAG2 application, suggesting that cellular homeostasis machinery may be responsible for helping the cell maintain turgor pressure despite a loss of membrane integrity. AFM imaging and force measurement modes applied in tandem reveal that the outer membrane becomes pitted, more flexible, and more adhesive after MAG2 treatment. MAG2 appears to have a highly disruptive effect on the outer membrane, extending the known mechanism of MAG2 to the Gram-negative outer membrane.

Design, recombinant expression, and antibacterial activity of a novel hybrid magainin-thanatin antimicrobial peptide.

Antimicrobial peptides are small molecule polypeptides with biological activity, which can avoid the drug resistance. Magainin and thanatin are antimicrobial peptides with a broad spectrum of inhibitory microbes, and the core sequence of magainin is linked to a core sequence of thanatin. Here, the hybrid magainin-thanatin (MT) antimicrobial peptide was designed through bioinformatics analysis. The recombinant MT antimicrobial peptide was successfully expressed and purified in Escherichia coli BL21 (DE3). The molecular weight of the hybrid MT antimicrobial peptide was about 3.35 kDa. Moreover, the target protein indeed has an inhibitory effect on Staphylococcus aureus, E. coli DH5α, and Bacillus subtilis, with the minimum inhibitory concentrations 16.5, 20, and 9 µM, respectively. The rational designed hybrid MT antimicrobial peptide will hopefully provide large-scale fermentable antimicrobial peptides in the industrial production in the future.

Grafted Polymer Coatings Enhance Fouling Inhibition by an Antimicrobial Peptide on Reverse Osmosis Membranes.

Bacterial biofilms that are formed on surfaces are highly detrimental to many areas of industry and medicine. Seawater desalination by reverse osmosis (RO) suffers from biofilm growth on the membranes (biofouling), which limits its widespread use because biofouling decreases water permeance and necessitates module cleaning and replacement, leading to increased economic and environmental costs. Antimicrobial peptides (AMPs) bound covalently to RO membranes inhibit biofilm growth and might delay membrane biofouling. Here we examined how various hydrophilic membrane coatings composed of zwitterionic, neutral, positively charged, and poly(ethylene glycol) (PEG)-grafted polymers affected the biocidal activity and the biofilm inhibition of a covalently bonded AMP on RO membranes. AMP magainin-2 was linked by the copper-catalyzed azide-alkyne cycloaddition reaction to a series of RO membranes that were grafted with different methacrylate polymers. Surface characterization by infrared spectroscopy, X-ray photoelectron spectroscopy, and water drop contact angle gave evidence of successful RO modifications, and zeta potential analysis reflected the increase in surface charge due to the linked, positively charged peptide. All AMP-modified membranes inhibited Pseudomonas aeruginosa growth compared to unmodified membranes, and the grafted methacrylic polymers did not significantly interfere with the peptide activity. On the other hand, membranes coated with zwitterionic and other acrylate polymers including AMP attachment inhibited biofilm growth more than either the AMP or the polymer coating alone. This enhancement led to ∼20% less biofilm biovolume on the membrane surfaces. The combination of antimicrobial coatings with polymer coatings known to resist fouling might aid future designs of surface coatings susceptible to biofilm growth.