Multiple Antimicrobial Effects of Hybrid Peptides Synthesized Based on the Sequence of Ribosomal S1 Protein from Staphylococcus aureus

The need to develop new antimicrobial peptides is due to the high resistance of pathogenic bacteria to traditional antibiotics now and in the future. The creation of synthetic peptide constructs is a common and successful approach to the development of new antimicrobial peptides. In this work, we use a simple, flexible, and scalable technique to create hybrid antimicrobial peptides containing amyloidogenic regions of the ribosomal S1 protein from Staphylococcus aureus. While the cell-penetrating peptide allows the peptide to enter the bacterial cell, the amyloidogenic site provides an antimicrobial effect by coaggregating with functional bacterial proteins. We have demonstrated the antimicrobial effects of the R23F, R23DI, and R23EI hybrid peptides against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa, Escherichia coli, and Bacillus cereus. R23F, R23DI, and R23EI can be used as antimicrobial peptides against Gram-positive and Gram-negative bacteria resistant to traditional antibiotics.     

Antibody–Bactericidal Macrocyclic Peptide Conjugates To Target Gram‐Negative Bacteria

To combat antimicrobial infections, new active molecules are needed. Antimicrobial peptides, ever abundant in nature, are a fertile starting point to develop new antimicrobial agents but suffer from low stability, low specificity, and off‐target toxicity. These drawbacks have limited their development. To overcome some of these limitations, we developed antibody–bactericidal macrocyclic peptide conjugates (ABCs), in which the antibody directs the bioactive macrocyclic peptide to the targeted Gram‐negative bacteria. We used cysteine S_NAr chemistry to synthesize and systematically study a library of large (>30‐mer) macrocyclic antimicrobial peptides (mAMPs) to discover variants with extended proteolytic stability in human serum and low hemolytic activity while maintaining bioactivity. We then conjugated, by using sortase A, these bioactive variants onto an Escherichia coli targeted monoclonal antibody. We found that these ABCs had minimized hemolytic activity and were able to kill E. coli at nanomolar concentrations. Our findings suggest macrocyclic peptides if fused to antibodies may facilitate the discovery of new agents to treat bacterial infections.     

Antimicrobial Activities of LL-37 Fragment Mutant-Poly (Lactic-Co-Glycolic) Acid Conjugate against Staphylococcus aureus,Escherichia coli,and Candida albicans

Various peptides and their derivatives have been reported to exhibit antimicrobial activities. Although these activities have been examined against microorganisms, novel methods have recently emerged for conjugation of the biomaterials to improve their activities. Here, we prepared CKR12-PLGA, in which CKR12 (a mutated fragment of human cathelicidin peptide, LL-37) was conjugated with poly (lactic-co-glycolic) acid (PLGA), and compared the antimicrobial and antifungal activities of the conjugated peptide with those of FK13 (a small fragment of LL-37) and CKR12 alone. The prepared CKR12-PLGA was characterized by dynamic light scattering and measurement of the zeta potential, critical micellar concentration, and antimicrobial activities of the fragments and conjugate. Although CKR12 showed higher antibacterial activities than FK13 against Staphylococcus aureus and Escherichia coli, the antifungal activity of CKR12 was lower than that of FK13. CKR12-PLGA showed higher antibacterial activities against S. aureus and E. coli and higher antifungal activity against Candida albicans compared to those of FK13. Additionally, CKR12-PLGA showed no hemolytic activity in erythrocytes, and scanning and transmission electron microscopy suggested that CKR12-PLGA killed and disrupted the surface structure of microbial cells. Conjugation of antimicrobial peptide fragment analogues was a successful approach for obtaining increased microbial activity with minimized cytotoxicity.     

Discovery of the Class I Antimicrobial Lasso Peptide Arcumycin

Lasso peptides are a structurally diverse superfamily of conformationally constrained peptide natural products, of which a subset exhibits broad antimicrobial activity. Although advances in bioinformatics have increased our knowledge of strains harboring the biosynthetic machinery for lasso peptide production, relating peptide sequence to bioactivity remains a continuous challenge. To this end, genome mining investigation of Actinobacteria-produced antimicrobial lasso peptides was performed to correlate predicted structure with antibiotic activity. Bioinformatic evaluation revealed eight putative novel class I lasso peptide sequences. Fermentation of one of these hits, Streptomyces NRRL F-5639, resulted in the production of a novel class I lasso peptide, arcumycin. Arcumycin exhibited antibiotic activity against Gram-positive bacteria including Bacillus subtilis (4 μg/mL), Staphylococcus aureus (8 μg/mL), and Micrococcus luteus (8 μg/mL). Arcumycin treatment of B. subtilis liaI-β-gal promoter fusion reporter strain resulted in upregulation of the liaRS system by the promoter liaI, indicating arcumycin interferes with lipid II biosynthesis. Cumulatively, the results illustrate the relationship between phylogenetically related lasso peptides and their bioactivity as validated through the isolation, structural determination, and evaluation of bioactivity of the novel class I antimicrobial lasso peptide arcumycin.     

New Milk Protein-Derived Peptides with Potential Antimicrobial Activity: An Approach Based on Bioinformatic Studies

New peptides with potential antimicrobial activity, encrypted in milk protein sequences, were searched for with the use of bioinformatic tools. The major milk proteins were hydrolyzed in silico by 28 enzymes. The obtained peptides were characterized by the following parameters: molecular weight, isoelectric point, composition and number of amino acid residues, net charge at pH 7.0, aliphatic index, instability index, Boman index, and GRAVY index, and compared with those calculated for known 416 antimicrobial peptides including 59 antimicrobial peptides (AMPs) from milk proteins listed in the BIOPEP database. A simple analysis of physico-chemical properties and the values of biological activity indicators were insufficient to select potentially antimicrobial peptides released in silico from milk proteins by proteolytic enzymes. The final selection was made based on the results of multidimensional statistical analysis such as support vector machines (SVM), random forest (RF), artificial neural networks (ANN) and discriminant analysis (DA) available in the Collection of Anti-Microbial Peptides (CAMP database). Eleven new peptides with potential antimicrobial activity were selected from all peptides released during in silico proteolysis of milk proteins.     

The impact of Nb2O5 on in-vitro bioactivity and antibacterial activity of CaF2–CaO–B2O3–P2O5–SrO glass system

This work consists of in vitro bioactivity (in SBF) and antibacterial studies (against S. aureus and E. coli bacteria) of Nb₂O₅ doped bioactive glasses. X-ray diffraction and scanning electron microscopy investigations indicated deposition of Nb-HAp (hydroxyapatite) crystalline layer on the samples after exposing to SBF. The spectroscopy investigations also indicated the deposition of HAp layer on these samples. The magnitude of HAp deposited on the glasses found to be relying on concentration of Nb₂O₅ dopant; this conclusion was drawn by determining weight loss of the glasses due to exposure to SBF and also by assessing the variation of pH of the remnant fluid as functions of Nb₂O₅content. The studies further indicated the maximal content of hydroxyapatite was deposited on the surface the glasses doped with 4.0 mol% of Nb₂O₅. The antibacterial studies (against E. coli and S. aureus bacteria) of these glasses indicated the maximal killing effect of bacteria of the samples admixed with 4.0 mol% of Nb₂O₅. This result is attributed to the occupancy of maximal fraction of Nb ions in NbO₆ structural units (confirmed by IR and Raman spectroscopic results) in this sample that paved the way for easy disintegration of the glass and to act on the bacteria. Overall, the results of bioactivity studies of Nb₂O₅ doped bioglasses indicated that the Nb₂O₅ not only enhanced bioactivity potential but also exhibited antimicrobial activity.     

Adhesive Antimicrobial Peptides Containing 3,4-Dihydroxy-L-Phenylalanine Residues for Direct One-Step Surface Coating

Bacterial colonization and transmission via surfaces increase the risk of infection. In this study, we design and employ novel adhesive antimicrobial peptides to prevent bacterial contamination of surfaces. Repeats of 3,4-dihydroxy-L-phenylalanine (DOPA) were added to the C-terminus of NKC, a potent synthetic antimicrobial peptide, and the adhesiveness and antibacterial properties of the resulting peptides are evaluated. The peptide is successfully immobilized on polystyrene, titanium, and polydimethylsiloxane surfaces within 10 min in a one-step coating process with no prior surface functionalization. The antibacterial effectiveness of the NKC-DOPA₅-coated polystyrene, titanium, and polydimethylsiloxane surfaces is confirmed by complete inhibition of the growth of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus within 2 h. The stability of the peptide coated on the substrate surface is maintained for 84 days, as confirmed by its bactericidal activity. Additionally, the NKC-DOPA₅-coated polystyrene, titanium, and polydimethylsiloxane surfaces show no cytotoxicity toward the human keratinocyte cell line HaCaT. The antimicrobial properties of the peptide-coated surfaces are confirmed in a subcutaneous implantation animal model. The adhesive antimicrobial peptide developed in this study exhibits potential as an antimicrobial surface-coating agent for efficiently killing a broad spectrum of bacteria on contact.     

The study of antimicrobial activity and preservative effects of nanosilver ingredient

In this study, we investigated the antimicrobial activity of silver nanoparticles (Ag-NPs) and platinum nanoparticles (Pt-NPs) aqueous solution, which were prepared using different stabilizer, such as sodium dodecylsulfate (SDS) and poly-(N-vinyl-2-pyrrolidone) (PVP), for Staphylococcus aureus (S. aureus) and Escherichia coli (E.coli) by measuring the minimum inhibitory concentration (MIC). Antimicrobial effect of Ag-NPs for S. aureus and E. coli was investigated using cup diffusion method. The growth of Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria were inhibited by Ag-NPs. The MIC of Ag-NPs for S. aureus and E. coli were 5 and 10 ppm, respectively. But the Au-NPs stabilized with SDS did not show antimicrobial activity. Also, the Pt-NPs stabilized with PVP (or SDS) did not show antimicrobial activity for the test organisms.     

Antibacterial Activity and Amphidinol Profiling of the Marine Dinoflagellate Amphidinium carterae (Subclade III)

Microalgae have received growing interest for their capacity to produce bioactive metabolites. This study aimed at characterising the antimicrobial potential of the marine dinoflagellate Amphidinium carterae strain LACW11, isolated from the west of Ireland. Amphidinolides have been identified as cytotoxic polyoxygenated polyketides produced by several Amphidinium species. Phylogenetic inference assigned our strain to Amphidinium carterae subclade III, along with isolates interspersed in different geographic regions. A two-stage extraction and fractionation process of the biomass was carried out. Extracts obtained after stage-1 were tested for bioactivity against bacterial ATCC strains of Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa. The stage-2 solid phase extraction provided 16 fractions, which were tested against S. aureus and E. faecalis. Fractions I, J and K yielded minimum inhibitory concentrations between 16 μg/mL and 256 μg/mL for both Gram-positive. A targeted metabolomic approach using UHPLC-HRMS/MS analysis applied on fractions G to J evidenced the presence of amphidinol type compounds AM-A, AM-B, AM-22 and a new derivative dehydroAM-A, with characteristic masses of m/z 1361, 1463, 1667 and 1343, respectively. Combining the results of the biological assays with the targeted metabolomic approach, we could conclude that AM-A and the new derivative dehydroAM-A are responsible for the detected antimicrobial bioactivity.     

Conformational Changes of Anoplin,W-MreB1–9, and (KFF)3K Peptides near the Membranes

Many peptides interact with biological membranes, but elucidating these interactions is challenging because cellular membranes are complex and peptides are structurally flexible. To contribute to understanding how the membrane-active peptides behave near the membranes, we investigated peptide structural changes in different lipid surroundings. We focused on two antimicrobial peptides, anoplin and W-MreB_1–9, and one cell-penetrating peptide, (KFF)₃K. Firstly, by using circular dichroism spectroscopy, we determined the secondary structures of these peptides when interacting with micelles, liposomes, E. coli lipopolysaccharides, and live E. coli bacteria. The peptides were disordered in the buffer, but anoplin and W-MreB_1–9 displayed lipid-induced helicity. Yet, structural changes of the peptide depended on the composition and concentration of the membranes. Secondly, we quantified the destructive activity of peptides against liposomes by monitoring the release of a fluorescent dye (calcein) from the liposomes treated with peptides. We observed that only for anoplin and W-MreB_1–9 calcein leakage from liposomes depended on the peptide concentration. Thirdly, bacterial growth inhibition assays showed that peptide conformational changes, evoked by the lipid environments, do not directly correlate with the antimicrobial activity of the peptides. However, understanding the relation between peptide structural properties, mechanisms of membrane disruption, and their biological activities can guide the design of membrane-active peptides.     

Antimicrobial Catanionic Vesicular Self-Assembly with Improved Spectrum of Action

A catanionic mixture based on three components, N-dodecyldiethanolamine, decanoic acid and azelaic acid, was prepared and investigated from a physico-chemical point of view and for its antimicrobial activity. The spontaneously formed vesicles were characterized by dynamic light scattering, zeta potential and electron microscopy. These experiments showed spherical vesicles with an average diameter of 200 nm and a zeta potential of +22 mV. The antimicrobial activity of the vesicle dispersion against different strains (Staphylococcus aureus, Escherichia coli, Staphylococcus epidermidis, Propionibacterium acnes and Candida albicans), was evaluated and compared with the activity of the components separately. The synergistic effect of these bioactive self-assembled catanionic vesicles on the spectrum of activity and on the cytoplasmic membrane is discussed.     

Comparative Antimicrobial Activity of Hp404 Peptide and Its Analogs against Acinetobacter baumannii

An amphipathic α-helical peptide, Hp1404, was isolated from the venomous gland of the scorpion Heterometrus petersii. Hp1404 exhibits antimicrobial activity against methicillin-resistant Staphylococcus aureus but is cytotoxic. In this study, we designed antimicrobial peptides by substituting amino acids at the 14 C-terminal residues of Hp1404 to reduce toxicity and improve antibacterial activity. The analog peptides, which had an amphipathic α-helical structure, were active against gram-positive and gram-negative bacteria, particularly multidrug-resistant Acinetobacter baumannii, and showed lower cytotoxicity than Hp1404. N-phenyl-1-naphthylamine uptake and DisC₃-5 assays demonstrated that the peptides kill bacteria by effectively permeating the outer and cytoplasmic membranes. Additionally, the analog peptides inhibited biofilm formation largely than Hp1404 at low concentrations. These results suggest that the analog peptides of Hp1404 can be used as therapeutic agents against A. baumannii infection.     

Antimicrobial and Antioxidative Activity of Newly Synthesized Peptides Absorbed into Bacterial Cellulose Carrier against Acne vulgaris

The ongoing search for effective treatment of Acne vulgaris is concentrated, i.a., on natural peptides with antimicrobial properties. The aim of this work was the development of new amino acid derivatives with potential activity on dermal infections against selected microorganisms, including the facultative anaerobe C. acne. The peptides P1–P6 were synthesized via Fmoc solid phase peptide synthesis using Rink amide AM resin, analyzed by RP-HPLC-MS, FTIR, DPPH radical scavenging activity, and evaluated against C. acne and S. aureus, both deposited and non-deposited in BC. Peptides P1–P6 presented a lack of cytotoxicity, antimicrobial activity, or antioxidative properties correlated with selected structural properties. P2 and P4–P6 sorption in BC resulted in variable data, i.a., confirming the prospective topical application of these peptides in a BC carrier.     

Synthesis and in Silico Modelling of the Potential Dual Mechanistic Activity of Small Cationic Peptides Potentiating the Antibiotic Novobiocin against Susceptible and Multi-Drug Resistant Escherichia coli

Cationic antimicrobial peptides have attracted interest, both as antimicrobial agents and for their ability to increase cell permeability to potentiate other antibiotics. However, toxicity to mammalian cells and complexity have hindered development for clinical use. We present the design and synthesis of very short cationic peptides (3–9 residues) with potential dual bacterial membrane permeation and efflux pump inhibition functionality. Peptides were designed based upon in silico similarity to known active peptides and efflux pump inhibitors. A number of these peptides potentiate the activity of the antibiotic novobiocin against susceptible Escherichia coli and restore antibiotic activity against a multi-drug resistant E. coli strain, despite having minimal or no intrinsic antimicrobial activity. Molecular modelling studies, via docking studies and short molecular dynamics simulations, indicate two potential mechanisms of potentiating activity; increasing antibiotic cell permeation via complexation with novobiocin to enable self-promoted uptake, and binding the E. coli RND efflux pump. These peptides demonstrate potential for restoring the activity of hydrophobic drugs.     

Porous chitosan/ZnO-doped bioglass composites as carriers of bioactive peptides

In this study, we aimed to assess whether the composite of chitosan/ZnO-doped bioglass can be applied as a suitable scaffold for the incorporation of bioactive peptides. Material of a porous composite with 1:1 ratio of bioglass:polymer was produced and used as a matrix for delivery of peptide. A peptide with the PEPTIDES sequence (Pro-Glu-Pro-Thr-Ile-Asp-Glu-Ser) was chosen as a model peptide. Microstructure and pore sizes of chitosan/ZnO-doped bioglass were assessed. Open porosity and pore sizes of the composite were suitable for enabling the migration of cells and ensuring the easy delivery of nutrients within the implant. In addition, composite showed bioactivity and bactericidal activity against Staphylococcus aureus and Pseudomonas aeruginosa strains. Peptide alone did not have any cytotoxic activity on human fibroblasts and keratinocytes. Also it did not show any antibacterial properties and did not cause hemolysis of red blood cells. The peptide incorporated in composite showed a rapid release in the kinetics profile. The obtained results indicate that there is the technological possibility to incorporate peptides in chitosan/ZnO-doped bioglass scaffolds. Such biomaterials have potential application in bone tissue engineering.     

Cyclization of the Antimicrobial Peptide Gomesin with Native Chemical Ligation: Influences on Stability and Bioactivity

Gomesin is an 18‐residue peptide originally isolated from the hemocytes of the Brazilian spider Acanthoscurria gomesiana. A broad spectrum of bioactivities have been attributed to gomesin, including in vivo and in vitro cytotoxicity against tumour cells, antimicrobial, antifungal, anti‐Leishmania and antimalarial effects. Given the potential therapeutic applications of gomesin, it was of interest to determine if an engineered version with a cyclic backbone has improved stability and bioactivity. Cyclization has been shown to confer enhanced stability and activity to a range of bioactive peptides and, in the case of a cone snail venom peptide, confer oral activity in a pain model. The current study demonstrates that cyclization improves the in vitro stability of gomesin over a 24 hour time period and enhances cytotoxicity against a cancer cell line without being toxic to a noncancerous cell line. In addition, antimalarial activity is enhanced upon cyclization. These findings provide additional insight into the influences of backbone cyclization on the therapeutic potential of peptides.     

Phenol-Soluble-Modulin-Inspired Amphipathic Peptides Have Bactericidal Activity against Multidrug-Resistant Bacteria

Phenol-soluble modulins (PSMs) are a large family of cytolytic peptide toxins produced by Staphylococcus aureus. Based on their amino acid sequences, we have constructed a small library of cationic isoleucine-rich peptides for antimicrobial evaluation. Relative to the parent PSMs, peptide zp3 (GIIAGIIIKIKK-NH₂) was found to possess greatly improved physicochemical properties (soluble in water) and antibacterial activity (MIC=8 μm for E. coli, B. subtilis, and C. freundii) while maintaining low hemolytic activity (<5 % at 256 μm ) and cytotoxicity (HEK293 cells IC₅₀>80 μm ). We reasoned that the selective activity of zp3 toward bacterial cells is due to its amphiphilic nature and positive net charge. Moreover, it is difficult for bacteria to develop resistance against zp3 . Through microscopic studies of E. coli, we demonstrated that zp3 can penetrate the bacterial membrane, thereby causing leakage of the bacterial cytoplasm. Our findings present a promising antimicrobial peptide lead, which has great potential for further chemical modification.     

Monitoring Human Milk β-Casein Phosphorylation and O-Glycosylation Over Lactation Reveals Distinct Differences between the Proteome and Endogenous Peptidome

Human milk is a vital biofluid containing a myriad of molecular components to ensure an infant’s best start at a healthy life. One key component of human milk is β-casein, a protein which is not only a structural constituent of casein micelles but also a source of bioactive, often antimicrobial, peptides contributing to milk’s endogenous peptidome. Importantly, post-translational modifications (PTMs) like phosphorylation and glycosylation typically affect the function of proteins and peptides; however, here our understanding of β-casein is critically limited. To uncover the scope of proteoforms and endogenous peptidoforms we utilized mass spectrometry (LC-MS/MS) to achieve in-depth longitudinal profiling of β-casein from human milk, studying two donors across 16 weeks of lactation. We not only observed changes in β-casein’s known protein and endogenous peptide phosphorylation, but also in previously unexplored O-glycosylation. This newly discovered PTM of β-casein may be important as it resides on known β-casein-derived antimicrobial peptide sequences.     

Peptide-Mimicking Poly(2-oxazoline)s Displaying Potent Antimicrobial Properties

Poly(2-oxazoline)s have excellent biocompatibility and have been used as FDA-approved indirect food additives. The inert property of the hydrophilic poly(2-oxazoline)s suggests them as promising substitutes for poly(ethylene glycol) (PEG) in various applications such as anti-biofouling agents. It was recently reported that poly(2-oxazoline)s themselves have antimicrobial properties as synthetic mimics of host defense peptides. These studies revealed the bioactive properties of poly(2-oxazoline)s as a new class of functional peptide mimics, by mimicking host defense peptides to display potent and selective antimicrobial activities against methicillin-resistant Staphylococcus aureus both in vitro and in vivo, without concerns about antimicrobial resistance. The high structural diversity, facile synthesis, and potent and tunable antimicrobial properties underscore the great potential of poly(2-oxazoline)s as a class of novel antimicrobial agents in dealing with drug-resistant microbial infections and antimicrobial resistance.     

EcDBS1R4, an Antimicrobial Peptide Effective against Escherichia coli with In Vitro Fusogenic Ability

Discovering antibiotic molecules able to hold the growing spread of antimicrobial resistance is one of the most urgent endeavors that public health must tackle. The case of Gram-negative bacterial pathogens is of special concern, as they are intrinsically resistant to many antibiotics, due to an outer membrane that constitutes an effective permeability barrier. Antimicrobial peptides (AMPs) have been pointed out as potential alternatives to conventional antibiotics, as their main mechanism of action is membrane disruption, arguably less prone to elicit resistance in pathogens. Here, we investigate the in vitro activity and selectivity of EcDBS1R4, a bioinspired AMP. To this purpose, we have used bacterial cells and model membrane systems mimicking both the inner and the outer membranes of Escherichia coli, and a variety of optical spectroscopic methodologies. EcDBS1R4 is effective against the Gram-negative E. coli, ineffective against the Gram-positive Staphylococcus aureus and noncytotoxic for human cells. EcDBS1R4 does not form stable pores in E. coli, as the peptide does not dissipate its membrane potential, suggesting an unusual mechanism of action. Interestingly, EcDBS1R4 promotes a hemi-fusion of vesicles mimicking the inner membrane of E. coli. This fusogenic ability of EcDBS1R4 requires the presence of phospholipids with a negative curvature and a negative charge. This finding suggests that EcDBS1R4 promotes a large lipid spatial reorganization able to reshape membrane curvature, with interesting biological implications herein discussed.