Computational study of solution behavior of magainin 2 monomers

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.

Antimicrobial peptides (AMPs) are found widely as part of nonspecific immune defenses. One class of AMPs forms stable pores in membranes, including the two chemically distinct membranes found in the Gram-negative cell envelope. As the Gram-negative cell envelope is a significant barrier to drug development, some have hypothesized that these AMPs could be used clinically, either alone or in combination with other drugs that cannot cross the Gram-negative cell envelope on their own. Here, we use atomic force microscopy (AFM), fluorescence spectroscopy, and fluorescence microscopy to elucidate the biomechanical changes that occur in Escherichia coli treated with various concentrations of the pore-forming AMP magainin 2 (MAG2). We find that near the minimum inhibitory concentration, MAG2 induces a loss of cell stiffness and a decrease in cell height consistent with pore formation and cellular leakage. Surprisingly, treatment with high concentrations of MAG2 leads to cells becoming stiffer and increasing in height. We confirmed that MAG2 forms pores at high concentrations using a standard propidium iodide (PI) uptake assay, in which PI is added to a cell suspension and is detected only after pores form in the cell membrane. However, when PI was added after 30 min of treatment with high concentrations of MAG2, less PI fluorescence was observed than in the standard PI uptake assay, indicating that movement across the cell membrane was restricted at the end of our experiments. We also observed that the modulus of the cell envelope increased with increasing MAG2 concentration, consistent with greater packing of MAG2 into the cell envelope membranes. Finally, our AFM images in air revealed that cells formed blebs when treated with high concentrations of MAG2. These data suggest that MAG2 initially forms pores at high concentrations, but as membrane packing increases, movement across the cell envelope becomes restricted. Understanding the concentration-dependent restriction of movement across the cell envelope could be important if AMPs are to be used clinically.

In mouse Paneth cells, alpha-defensins, termed cryptdins (Crps), are activated by matrix metalloproteinase-7-mediated proteolysis of inactive precursors (pro-Crps) to bactericidal forms. The activating cleavage step at Ser(43) downward arrow Ile(44) in mouse pro-Crp4-(20-92) removes nine acidic amino acids that collectively block the membrane-disruptive behavior of the Crp4 moiety of the proform. This inhibitory mechanism has been investigated further to identify whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) are responsible for blocking bactericidal activity and membrane disruption. To test whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) have specific positional effects that block bactericidal peptide activity and membrane disruption, acidic residues positioned at the distal (Asp(20), Asp(26), Glu(27), and Glu(28)), mid (Glu(32) and Glu(33)), and proximal (Glu(37), Glu(38), and Asp(39)) clusters in pro-Crp4-(20-92) were mutagenized, and variants were assayed for differential effects of mutagenesis on bactericidal peptide activity. Substitution of the mid and proximal Asp and Glu clusters with Gly produced additive effects with respect to the induction of both bactericidal activity and membrane permeabilization of live Escherichia coli ML35 cells. In contrast, substitution of distal Glu and Asp residues with Gly or their deletion resulted in pro-Crp4-(20-92) variants with bactericidal and membrane-disruptive activities equal to or greater than that of fully mature Crp4. These findings support the conclusion that the most distal N-terminal anionic residues of pro-Crp4-(20-92) are primarily responsible for blocking Crp4-mediated membrane disruption in the precursor.

The "pre-assembled state" of magainin 2 lysine-linked dimer determines its enhanced antimicrobial activity.

Synergy between antimicrobial peptides PGLa and Magainin 2 (MAG2) provides an efficient way to enhance their antimicrobial ability. However, the underlying molecular mechanism of such synergy, especially the individual roles of each peptide, remains poorly understood. We combined a giant unilamellar vesicle leakage assay, in situ interfacial photovoltage testing, and molecular dynamics to investigate membrane poration under the action of PGLa, MAG2, or a PGLa/MAG2 mixture. Our results clearly show the different membrane action modes of the three systems and demonstrate the importance of forming PGLa-MAG2 heterodimers in the membrane poration process. PGLa inserted into and extracted from a membrane rapidly and continually with minimal aggregation and produced only transient, small pores. In contrast, MAG2 peptides tended to aggregate together on the membrane surface or only shallowly embed in the membrane. Additionally, the PGLa and MAG2 residues were well integrated into the membrane via the formation of PGLa-MAG2 heterodimers. The membrane defect produced by the rapid insertion of PGLa was stabilized by MAG2, which further recruited other peptides for the formation of PGLa-MAG2 heterodimers and even heterodimer clusters. Growth in pore size then occurred in a step-by-step process involving the formation and assembly of heterodimer clusters within the membrane. Our results provide insight into the complicated synergy that occurs between PGLa and MAG2 during membrane poration and will assist in the design of new antimicrobial peptides.

Antimicrobial peptides (AMPs) are relatively short peptides endowed with broad spectrum antimicrobial, anti-inflammatory immunomodulating and wound healing enhancement activity. They are essential components of the innate immune system of higher eukaryotes providing the first line of defense against microbial invasions. AMPs are extremely heterogeneous in length, structure, aminoacid composition and, more importantly, molecular mechanism of bacterial killing. From this point of view AMPs can be divided in at least four wide classes: (i) cationic antimicrobial peptides (CAMPs); (ii) anionic antimicrobial peptides (AAMPs); (iii) histidine-rich antimicrobial peptides (HAMPs), and (iv) AMPs with intracellular targets (DNA, RNA, ribosomes, chaperons, enzymes). The pharmacological potential of AMPs, and especially of AMP mixtures, is obvious. However the costs of pharmaceutical formulations including two or more peptides separately produced would be so high that even a very interesting pharmacological activity would not compensate for the effort needed to prepare the mixture. Accordingly, the aim of the present project is to develop a strategy to prepare mixtures of two or more AMPs with costs, production times and amount of work comparable to those necessary for the production of a single recombinant AMP. Two alternative strategies were evaluated, one exploiting a previously optimized-carrier protein for the production of recombinant AMPs as fusion proteins, and an alternative strategy aiming to prepare a carrier-less precursor composed only by AMP modules. The carrier developed by our group, ONC-DCless-H6, was able to effectively drive to inclusion bodies both a highly cationic sequence of about 50 residues [(P)PAP-A3(Pro26)] containing two CAMP-like sequences and a modular sequence (AT2H) of more than 100 residues including 2 anionic, 2 cationic and a histidine rich module. In both cases expression yields were high (>200 mg/L and >100 mg/L, respectively). Purification by IMAC provided a protein with good yield and purity. Purified recombinant proteins were hydrolyzed in 0.1 M acetic acid at 60°C to cleave the acid labile Asp-Pro sequences at the junction sites between consecutive AMP modules. The efficiency was very high in the case AT2H, which provided only the desired peptides, thus indicating that all the acid labile Asp-Pro sequences underwent cleavage with an efficiency higher than 95%. Only in the case of (P)PAP-A3(Pro26), the internal Asp-Pro sequence was cleaved with an efficiency of only about 85%. The comparison of the sequences adjacent to the different Asp-Pro cleavage sites in (P)PAP-A3(Pro26) and AT2H suggested that in order to obtain a very high cleavage efficiency a flexible sequence, rich in small and/or coil-preferring amino acids should be present at least on one side of the Asp-Pro site. This information will be useful for the design of further fusion proteins. The carrier-less strategy was tested by preparing two modular precursors, AT5H (containing 5 anionic, 5 cationic and a histidine rich module) and AT4LH (containing 4 anionic, 4 cationic and a histidine rich module). AT5H and AT4LH showed relatively low expression levels (15 and 30 mg/L, respectively) and a different behavior of the inclusion bodies formed upon recombinant expression. In fact, the inclusion bodies, differently from those generated by ONC-DCless-H6, were soluble in 2 M urea thus suggesting a lower compactness/stability. A possible cause of the low expression levels could be the non-optimal codon usage of the coding sequences necessary for avoiding the presence of identical direct repeats which would have made difficult the chemical synthesis of the coding sequences. Low expression levels, in turn, could have contributed to the formation of “unstable” inclusion bodies. Indeed, it is well known that inclusion bodies formation is generally related to high expression levels. In the future, alternative approaches based on ligation of multiple copies of a smaller fragment could allow preparing coding sequences where all the repeats have an optimal codon usage.

Antimicrobial peptides are promising therapeutic agents in view of increasing resistance to conventional antibiotics. Antimicrobial peptides usually fold in α-helical, β-sheet, and extended/random-coil structures. The α-helical antimicrobial peptides are often unstructured in aqueous solution but become structured on bacterial membrane. The α-helical structure allows the partitioning into bacterial membrane. Therefore it is important to understand the mechanism of unfolding and refolding of α-helical structure in antimicrobial peptides. It is not very easy to obverse and study the process of unfolding and refolding of α-helical antimicrobial peptides because of their rapidity. Therefore, molecular simulation provides a way to observe and explain this phenomenon. Plantaricin A is a 26 amino-acid antimicrobial pheromone peptide and can spontaneously unfold and refold under physiological condition. This study demonstrated the unfolding and refolding of plantaricin A by means of molecular simulation, and its mechanism was discussed with its implication to the Levinthal paradox.

Action of Antimicrobial Peptides on Bacterial and Lipid Membranes: A Direct Comparison

Designed histidine-rich amphipathic cationic peptides, such as LAH4, have enhanced membrane disruption and antibiotic properties when the peptide adopts an alignment parallel to the membrane surface. Although this was previously achieved by lowering the pH, here we have designed a new generation of histidine-rich peptides that adopt a surface alignment at neutral pH. In vitro, this new generation of peptides are powerful antibiotics in terms of the concentrations required for antibiotic activity; the spectrum of target bacteria, fungi, and parasites; and the speed with which they kill. Further modifications to the peptides, including the addition of more hydrophobic residues at the N terminus, the inclusion of a helix-breaking proline residue or using D-amino acids as building blocks, modulated the biophysical properties of the peptides and led to substantial changes in toxicity to human and parasite cells but had only a minimal effect on the antibacterial and antifungal activity. Using a range of biophysical methods, in particular solid-state NMR, we show that the peptides are highly efficient at disrupting the anionic lipid component of model membranes. However, we also show that effective pore formation in such model membranes may be related to, but is not essential for, high antimicrobial activity by cationic amphipathic helical peptides. The information in this study comprises a new layer of detail in the understanding of the action of cationic helical antimicrobial peptides and shows that rational design is capable of producing potentially therapeutic membrane active peptides with properties tailored to their function.

The innate immune system in humans consists of both cellular and humoral components that collaborate to eradicate invading bacteria from the body. Here, we discover that the gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, does not grow in human serum. Fractionation of serum by gel filtration chromatography led to the identification of human transferrin as the inhibiting factor. Purified transferrin blocks growth of both the fully virulent encapsulated B. anthracis Ames and the non-encapsulated Sterne strain. Growth inhibition was also observed in serum of wild-type mice but not of hypotransferrinemic mice that only have approximately 1% circulating transferrin levels. We were able to definitely assign the bacteriostatic activity of transferrin to its iron-binding function: neither iron-saturated transferrin nor a recombinant transferrin mutant unable to bind iron could inhibit growth of B. anthracis. Additional iron could restore bacterial growth in human serum. The observation that other important gram-positive pathogens are not inhibited by transferrin suggests they have evolved effective mechanisms to circumvent serum iron deprivation. These findings provide a better understanding of human host defense mechanisms against anthrax and provide a mechanistic basis for the antimicrobial activity of human transferrin.

Magainin-2 (MAG2) is a polycationic antimicrobial peptide isolated from the skin of the African clawed frog Xenopus laevis. It has a wide spectrum of antimicrobial activities against gram-positive and gram-negative bacteria, fungi, and induces osmotic lysis of protozoa. MAG2 also possesses antiviral and antitumoral properties. These activities make this peptide a promising candidate for therapeutic applications. Recombinant expression systems are necessary for the affordable production of large amounts of the biologically active peptide. In this work, MAG2 has been cloned to the N-terminal of a family III carbohydrate-binding module fused to the linker sequence (LK-CBM3) from Clostridium thermocellum; a formic acid recognition site was introduced between the two modules for chemical cleavage of the peptide. The recombinant protein MAG2-LK-CBM3 was expressed in Escherichia coli BL21 (DE3) and MAG2 was successfully cleaved and purified from the fusion partner LK-CBM3. Its functionality was confirmed by testing its activity against gram-negative bacteria.

The emergence and spread of antibiotic‐resistant bacteria pose a significant public health threat, necessitating the exploration of alternative antibacterial strategies. Antibacterial peptide (ABP) is a kind of antimicrobial peptide (AMP) that has the potential ability to fight against bacteria infection, offering a promising avenue for developing novel therapeutic interventions. This study introduces AMPActiPred, a three‐stage computational framework designed to identify ABPs, characterize their activity against diverse bacterial species, and predict their activity levels. AMPActiPred employed multiple effective peptide descriptors to effectively capture the compositional features and physicochemical properties of peptides. AMPActiPred utilized deep forest architecture, a cascading architecture similar to deep neural networks, capable of effectively processing and exploring original features to enhance predictive performance. In the first stage, AMPActiPred focuses on ABP identification, achieving an Accuracy of 87.6% and an MCC of 0.742 on an elaborate dataset, demonstrating state‐of‐the‐art performance. In the second stage, AMPActiPred achieved an average GMean at 82.8% in identifying ABPs targeting 10 bacterial species, indicating AMPActiPred can achieve balanced predictions regarding the functional activity of ABP across this set of species. In the third stage, AMPActiPred demonstrates robust predictive capabilities for ABP activity levels with an average PCC of 0.722. Furthermore, AMPActiPred exhibits excellent interpretability, elucidating crucial features associated with antibacterial activity. AMPActiPred is the first computational framework capable of predicting targets and activity levels of ABPs. Finally, to facilitate the utilization of AMPActiPred, we have established a user‐friendly web interface deployed at https://awi.cuhk.edu.cn/∼AMPActiPred/.

Endophytes associated with medicinal plants are a potential source of valuable natural products. This study aimed to evaluate the antibacterial and antibiofilm activities of endophytic bacteria from Archidendron pauciflorum against multidrug-resistant (MDR) strains. A total of 24 endophytic bacteria were isolated from the leaf, root, and stem of A. pauciflorum. Seven isolates showed antibacterial activity with different spectra against four MDR strains. Extracts derived from four selected isolates (1mg/mL) also displayed antibacterial activity. Among four selected isolates, DJ4 and DJ9 isolates exhibited the strongest antibacterial activity against P. aeruginosa strain M18, as indicated by the lowest minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) (DJ4 and DJ9 MIC: 7.81µg/mL; DJ4 and DJ9 MBC: 31.25µg/mL). 2 × MIC of DJ4 and DJ9 extracts was found to be the most effective concentration to inhibit more than 52% of biofilm formation and eradicate more than 42% of established biofilm against all MDR strains. 16S rRNA-based identification revealed four selected isolates belong to the genus Bacillus. DJ9 isolate possessed nonribosomal peptide synthetase (NRPS) gene, and DJ4 isolate possessed NRPS and polyketide synthase type I (PKS I) gene. Both these genes are commonly responsible for secondary metabolites synthesis. Several antimicrobial compounds, including 1,4-dihydroxy-2-methyl-anthraquinone and paenilamicin A1, were detected in the bacterial extracts. This study highlights endophytic bacteria isolated from A. pauciflorum provide a great source of novel antibacterial compounds.

To investigate the secondary structure, physicochemical properties and antibacterial activity of Brevinin- 2GHk (BR2GK), an antimicrobial peptide from frog skin, and explore its antibacterial mechanism. BR2GK was synthesized, purified with high performance liquid chromatography (HPLC) and identified using mass spectrometry. Circular dichroism was used to determine the secondary structure and physicochemical properties of BR2GK. Two-fold dilution method was used to determine the antibacterial activity of BR2GK, and its antibacterial mechanism was explored using laser scanning confocal microscopy (LSCM) and scanning electron microscopy (SEM). The hemolytic activity of BR2GK was analyzed in red blood cells. Isothermal titration calorimetry (ITC) and surface plasmon resonance imaging (SPRi) were employed to detect the binding of BR2GK to lipopolysaccharide (LPS), and the antioxidant activity of BR2GK was evaluated using biochemical kits. The synthesized BR2GK, with a purity exceeding 95% after purification, had the basic characteristics of cationic antimicrobial peptides. BR2GK consisted mainly of α-helical structure in SDS solution and exhibited a broad-spectrum antibacterial activity. Antibacterial activity assay showed that BR2GK had inhibitory and killing activity against a variety of strains with a minimum inhibitory concentration (MIC) of 2.76 μmol/L against Staphylococcus aureus. Observation with LSCM and SEM showed that BR2GK at an active concentration caused bacterial cell membrane damage, cell swelling, contraction, deformation, and massive exudation of intracellular contents without causing hemolysis. ITC showed that the binding affinity KD of BR2GK to LPS was 18.2±0.8 μmol/L. The antioxidant test showed that BR2GK was capable of effectively scavenging NO, ABTS and DPPH. BR2GK, as a broad-spectrum antibacterial peptide with also a strong antioxidant capacity, does not cause hemolysis and is capable of binding to LPS. BR2GK has an important value for future design and synthesis of antimicrobial peptides with stronger antibacterial activity and lower cytotoxicity.

de Novo-designed antimicrobial peptides with broad-spectrum antimicrobial potency and rapid wound disinfection