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.     

Effect of Acylation on the Antimicrobial Activity of Temporin B Analogues

New peptides derived from the natural antimicrobial temporin B were obtained. The design, antimicrobial activity, and characterization of the secondary structure of peptides in the presence of bacterial cells is described herein. TB_KKG6K (KKLLPIVKNLLKSLL) has been identified as the most active analogue against Gram‐positive and ‐negative bacteria, compared with natural temporin B (LLPIVGNLLKSLL) and TB_KKG6A (KKLLPIVANLLKSLL). Acylation with hydrophobic moieties generally led to reduced activity; however, acylation at the 6‐position of TB_KKG6K led to retained sub‐micromolar activity against Staphylococcus epidermidis.     

Biosynthetic Gene Cluster of a d‐Tryptophan‐Containing Lasso Peptide,MS‐271

MS‐271, produced by Streptomyces sp. M‐271, is a lasso peptide natural product comprising 21 amino acid residues with a d ‐tryptophan at its C terminus. Because lasso peptides are ribosomal peptides, the biosynthesis of MS‐271, especially the mechanism of d ‐Trp introduction, is of great interest. The MS‐271 biosynthetic gene cluster was identified by draft genome sequencing of the MS‐271 producer, and it was revealed that the precursor peptide contains all 21 amino acid residues including the C‐terminal tryptophan. This suggested that the d ‐Trp residue is introduced by epimerization. Genes for modification enzymes such as a macrolactam synthetase (mslC), precursor peptide recognition element (mslB1), cysteine protease (mslB2), disulfide oxidoreductases (mslE, mslF), and a protein of unknown function (mslH) were found in the flanking region of the precursor peptide gene. Although obvious epimerase genes were absent in the cluster, heterologous expression of the putative MS‐271 cluster in Streptomyces lividans showed that it contains all the necessary genes for MS‐271 production including a gene for a new peptide epimerase. Furthermore, a gene‐deletion experiment indicated that MslB1, ‐B2, ‐C and ‐H were indispensable for MS‐271 production and that some interactions of the biosynthetic enzymes were essential for the biosynthesis of MS‐271.     

Biochemical,Structural, and Genetic Characterization of Tridecaptin A1, an Antagonist of Campylobacter jejuni

Bacillus circulans NRRL B‐30644 (now Paenibacillus terrae) was previously reported to produce SRCAM 1580, a bacteriocin active against the food pathogen Campylobacter jejuni. We have been unable to isolate SRCAM 1580, and did not find any genetic determinants in the genome of this strain. We now report the reassignment of this activity to the lipopeptide tridecaptin A₁. Structural characterization of tridecaptin A₁ was achieved through NMR, MS/MS and GC‐MS studies. The structure was confirmed through the first chemical synthesis of tridecaptin A₁, which also revealed the stereochemistry of the lipid chain. The impact of this stereochemistry on antimicrobial activity was examined. The biosynthetic machinery responsible for tridecaptin production was identified through bioinformatic analyses. P. terrae NRRL B‐30644 also produces paenicidin B, a novel lantibiotic active against Gram‐positive bacteria. MS/MS analyses indicate that this lantibiotic is structurally similar to paenicidin A.     

Search for Shorter Portions of the Proline-Rich Antimicrobial Peptide Fragment Bac5(1–25) That Retain Antimicrobial Activity by Blocking Protein Synthesis

The spread of antibiotic-resistant pathogens has boosted the search for new antimicrobial drugs. Proline-rich antimicrobial peptides are promising lead compounds for the development of next-generation antibiotics, given their very low cytotoxicity and their good antimicrobial activity targeting the bacterial ribosome. Bac5(1–25) is an N-terminal fragment of the bovine proline-rich antimicrobial peptide Bac5, whose mode of action has been recently described. In this work we tested a number of Bac5(1–25) fragments, and we characterized their antimicrobial activity against Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella enterica, and Pseudomonas aeruginosa. We evaluated their cytotoxicity toward human cells and their efficacy in inhibiting bacterial protein synthesis. This allowed us to identify some shorter fragments of Bac5(1–25) with a good balance between antibacterial efficacy, protein synthesis inhibition, and ease/cost-effectiveness of synthesis, suitable as lead compounds to develop new antibacterials.     

Sequence determinants governing the topology and biological activity of a lasso peptide, microcin J25

Microcin J25 is a potent antibacterial peptide produced by Escherichia coli AY25. It displays a lasso structure, which consists of a knot involving an N-terminal macrolactam ring through which the C-terminal tail is threaded and sterically trapped. In this study, we rationally designed and performed site-specific mutations in order to pinpoint the sequence determinants of the lasso topology. Structures of the resulting variants were analysed by a combination of methods (mass spectrometry, NMR spectroscopy, enzymatic digestion), and correlated to the antibacterial activity. The selected mutations resulted in the production of branched-cyclic or lasso variants. The C-terminal residues below the ring (Tyr20, Gly21) and the size of the macrolactam ring were revealed to be critical for both the lasso scaffold and bioactivity, while shortening the loop region (Tyr9-Ser18) or extending the C-terminal tail below the ring did not alter the lasso structure, but differentially affected the antibacterial activity. These results provide new insights for the bioengineering of antibacterial agents using a lasso peptide as template.     

Antimicrobial and Anticancer Properties of Synthetic Peptides Derived from the Wasp Parachartergus fraternus

Agelaia-MPI and protonectin are antimicrobial peptides isolated from the wasp Parachartergus fraternus that show antimicrobial and neuroactive activities. Previously, two analogues of these peptides, neuroVAL and protonectin-F, were designed to reduce nonspecific toxicity and improve potency. Here, the three-dimensional structures of neuroVAL, protonectin and protonectin-F were determined by using circular dichroism and NMR spectroscopy. Antibacterial, antifungal, cytotoxic and hemolytic activities were tested for the parent peptides and analogues. All peptides showed moderate antimicrobial activity against Gram-positive bacteria, with agelaia-MPI being the most active. Protonectin and protonectin-F were found to be toxic to cancerous and noncancerous cell lines. Internalization experiments revealed that these peptides accumulate inside both cell types. By contrast, neuroVAL was nontoxic to all tested cells and was able to enter cells without accumulating. In summary, neuroVAL has potential as a nontoxic cell-penetrating peptide, while protonectin-F needs further modification to realize its potential as an antitumor peptide.     

Antimicrobial Peptide Mimetics Based on a Diphenylacetylene Scaffold: Synthesis,Conformational Analysis,and Activity

Mimics of natural antimicrobial peptides are promising compounds to fight the rising threat of multi-drug resistant bacteria. Here we report the design, synthesis and conformational analysis of a new class of antimicrobial peptide mimetics incorporating a diphenylacetylene scaffold. Within a small set of compounds, we observe a correlation between amphiphilicity, the efficiency of partitioning into negatively charged membranes and antibacterial activity. The most amphiphilic compound, which contains four isoleucine residues and four lysine residues, displays species-selective antibacterial activity (most active against Bacillus subtills) and low haemolytic activity. Solution-phase conformational analysis of this compound indicates that a defined structure is adopted in the presence of negatively charged phospholipid membranes and aqueous 2,2,2-trifluoroethanol but not in water. A conformation model indicates that the cationic and hydrophobic functional groups are segregated. These results may inform the development of highly selective antimicrobial peptide mimetics for therapeutic applications.     

Rational Design of Tryptophan‐Rich Antimicrobial Peptides with Enhanced Antimicrobial Activities and Specificities

Trp‐rich antimicrobial peptides play important roles in the host innate defense mechanism of many plants and animals. A series of short Trp‐rich peptides derived from the C‐terminal region of Bothrops asper myothoxin II, a Lys49 phospholipase A₂ (PLA₂), were found to reproduce the antimicrobial activities of their parent molecule. Of these peptides, KKWRWWLKALAKK—designated PEM‐2—was found to display improved activity against both Gram‐positive and Gram‐negative bacteria. To improve the antimicrobial activity of PEM‐2 for potential clinical applications further, we determined the solution structure of PEM‐2 bound to membrane‐mimetic dodecylphosphocholine (DPC) micelles by two‐dimensional NMR methods. The DPC micelle‐bound structure of PEM‐2 adopts an α‐helical conformation and the positively charged residues are clustered together to form a hydrophilic patch. The surface electrostatic potential map indicates that two of the three tryptophan residues are packed against the peptide backbone and form a hydrophobic face with Leu7, Ala9, and Leu10. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that PEM‐2 interacted with negatively charged phospholipid vesicles and efficiently induced dye release from these vesicles, suggesting that the antimicrobial activity of PEM‐2 could be due to interactions with bacterial membranes. Potent analogues of PEM‐2 with enhanced antimicrobial and less pronounced hemolytic activities were designed with the aid of these structural studies.     

New Antimicrobial Hexapeptides: Synthesis,Antimicrobial Activities,Cytotoxicity, and Mechanistic Studies

Synthetic antimicrobial peptides have recently emerged as promising candidates against drug‐resistant pathogens. We identified a novel hexapeptide, Orn‐D ‐Trp‐D ‐Phe‐Ile‐D ‐Phe‐His(1‐Bzl)‐NH₂, which exhibits broad‐spectrum antifungal and antibacterial activity. A lead optimization was undertaken by conducting a full amino acid scan with various proteinogenic and non‐proteinogenic amino acids depending on the hydrophobic or positive‐charge character of residues at various positions along the sequence. The hexapeptide was also cyclized to study the correlation between the linear and cyclic structures and their respective antimicrobial activities. The synthesized peptides were found to be active against the fungus Candida albicans and Gram‐positive bacteria such as methicillin‐resistant Staphylococcus aureus and methicillin‐resistant Staphylococcus epidermidis, as well as the Gram‐negative bacterium Escherichia coli; MIC values for the most potent structures were in the range of 1–5 μg mL^?1 (IC₅₀ values in the range of 0.02–2 μg mL^?1). Most of the synthesized peptides showed no cytotoxic effects in an MTT assay up to the highest test concentration of 200 μg mL^?1. A tryptophan fluorescence quenching study was performed in the presence of negatively charged and zwitterionic model membranes, mimicking bacterial and mammalian membranes, respectively. The results of the fluorescence study demonstrate that the tested peptides are selective toward bacterial over mammalian cells; this is associated with a preferential interaction between the peptides and the negatively charged phospholipids of bacterial cells.     

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.     

Structural and Functional Analysis of Human Liver‐Expressed Antimicrobial Peptide 2

Human liver‐expressed antimicrobial peptide 2 (LEAP‐2) is a cationic antimicrobial peptide (CAMP) believed to have a protective role against bacterial infection. Little is known about the structure–activity relationships of LEAP‐2 or its mechanism of action. In this study we describe the structure of LEAP‐2, analyze its interaction with model membranes, and relate them to the antimicrobial activity of the peptide. The structure of LEAP‐2, determined by NMR spectroscopy, reveals a compact central core with disorder at the N and C termini. The core comprises a β‐hairpin and a 3₁₀‐ helix that are braced by disulfide bonds between Cys17–28 and Cys23–33 and further stabilized by a network of hydrogen bonds. Membrane‐affinity studies show that LEAP‐2 membrane binding is governed by electrostatic attractions, which are sensitive to ionic strength. Truncation studies show that the C‐terminal region of LEAP‐2 is irrelevant for membrane binding, whereas the N‐terminal (hydrophobic domain) and core regions (cationic domain) are essential. Bacterial‐growth‐inhibition assays reveal that the antimicrobial activity of LEAP‐2 correlates with membrane affinity. Interestingly, the native and reduced forms of LEAP‐2 have similar membrane affinity and antimicrobial activities; this suggests that disulfide bonds are not essential for the bactericidal activity. This study reveals that LEAP‐2 has a novel fold for a CAMP and suggests that although LEAP‐2 exhibits antimicrobial activity under low‐salt conditions, there is likely to be another physiological role for the peptide.     

Discovery of Natural Products Possessing Selective Eukaryotic Readthrough Activity: 3‐epi‐Deoxynegamycin and Its Leucine Adduct

Herein we report the first discovery of natural readthrough products that do not display antimicrobial activity. Two natural negamycins, 3‐epi‐deoxynegamycin and its leucine adduct, isolated 37 years ago, were found to be potent readthrough agents against nonsense mutations of eukaryotes, but not prokaryotes, without displaying antimicrobial activity. These results suggest that the compounds are valuable leads for the development of readthrough drugs against nonsense‐mediated genetic diseases without the potential for contributing to the emergence of drug‐resistant bacteria.     

Gramicidin A Mutants with Antibiotic Activity against Both Gram‐Positive and Gram‐Negative Bacteria

Antimicrobial peptides (AMPs) have shown potential as alternatives to traditional antibiotics for fighting infections caused by antibiotic‐resistant bacteria. One promising example of this is gramicidin A (gA). In its wild‐type sequence, gA is active by permeating the plasma membrane of Gram‐positive bacteria. However, gA is toxic to human red blood cells at similar concentrations to those required for it to exert its antimicrobial effects. Installing cationic side chains into gA has been shown to lower its hemolytic activity while maintaining the antimicrobial potency. In this study, we present the synthesis and the antibiotic activity of a new series of gA mutants that display cationic side chains. Specifically, by synthesizing alkylated lysine derivatives through reductive amination, we were able to create a broad selection of structures with varied activities towards Staphylococcus aureus and methicillin‐resistant S. aureus (MRSA). Importantly, some of the new mutants were observed to have an unprecedented activity towards important Gram‐negative pathogens, including Escherichia coli, Klebsiella pneumoniae and Psuedomonas aeruginosa.     

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.     

Lasioglossins: Three Novel Antimicrobial Peptides from the Venom of the Eusocial Bee Lasioglossum laticeps (Hymenoptera: Halictidae)

Three novel structurally related pentadecapeptides, named lasioglossins, were isolated from the venom of the eusocial bee Lasioglossum laticeps. Their primary sequences were established as H‐Val‐Asn‐Trp‐Lys‐Lys‐Val‐Leu‐Gly‐Lys‐Ile‐Ile‐Lys‐Val‐Ala‐Lys‐NH₂ (LL‐I), H‐Val‐Asn‐Trp‐Lys‐Lys‐Ile‐Leu‐Gly‐Lys‐Ile‐Ile‐Lys‐Val‐Ala‐Lys‐NH₂ (LL‐II) and H‐Val‐Asn‐Trp‐Lys‐Lys‐Ile‐Leu‐Gly‐Lys‐Ile‐Ile‐Lys‐Val‐Val‐Lys‐NH₂ (LL‐III). These lasioglossins exhibited potent antimicrobial activity against both Gram‐positive and Gram‐negative bacteria, low haemolytic and mast cell degranulation activity, and a potency to kill various cancer cells in vitro. The lasioglossin CD spectra were measured in the presence of trifluoroethanol and sodium dodecyl sulfate solution and indicated a high degree of α‐helical conformation. NMR spectroscopy, which was carried out in trifluoroethanol/water confirmed a curved α‐helical conformation with a concave hydrophobic and convex hydrophilic side. To understand the role of this bend on biological activity, we studied lasioglossin analogues in which the Gly in the centre of the molecule was replaced by other amino acid residues (Ala, Lys, Pro). The importance of the N‐terminal part of the molecule to the antimicrobial activity was revealed through truncation of five residues from both the N and C termini of the LL‐III peptide. C‐terminal deamidation of LL‐III resulted in a drop in antimicrobial activity, but esterification of the C terminus had no effect. Molecular modelling of LL‐III and the observed NOE contacts indicated the possible formation of a bifurcated H‐bond between hydrogen from the Lys15 CONH peptide bond and one H of the C‐terminal CONH₂ to the Ile11 oxygen atom. Such interactions cannot form with C‐terminal esterification.     

Factors Governing the Thermal Stability of Lasso Peptides

Lasso peptides belong to the natural product superfamily of ribosomally synthesized and post-translationally modified peptides (RiPPs). They are defined by an N-terminal macrolactam ring that is threaded by the C-terminal tail. In class II lasso peptides, this fold is maintained only through steric hindrance. Nonetheless, this fold can often withstand prolonged incubation at highly elevated temperatures. However, some lasso peptides will irreversibly unthread into their branched-cyclic counterparts upon heating. In recent years, an increasing number of research studies have focused on studying the factors that govern the thermal stability (or the lack thereof) of lasso peptides by using in vitro stability assays, mutational analysis, and molecular dynamics simulations. In this review, the current state of understanding the physicochemical parameters deciding the fate of a lasso peptide at elevated temperatures is discussed, and an overview is given of the techniques developed to streamline the separation and discrimination of lasso peptides from their branched-cyclic topoisomers.     

Site‐Directed and Global Incorporation of Orthogonal and Isostructural Noncanonical Amino Acids into the Ribosomal Lasso Peptide Capistruin

Expansion of the structural diversity of peptide antibiotics was performed through two different methods. Supplementation‐based incorporation (SPI) and stop‐codon suppression (SCS) approaches were used for co‐translational incorporation of isostructural and orthogonal noncanonical amino acids (ncAAs) into the lasso peptide capistruin. Two ncAAs were employed for the SPI method and five for the SCS method; each of them probing the incorporation of ncAAs in strategic positions of the molecule. Evaluation of the assembly by HR‐ESI‐MS proved more successful for the SCS method. Bio‐orthogonal chemistry was used for post‐biosynthetic modification of capistruin congener Cap_Alk10 containing the ncAA Alk (Nε‐Alloc‐L ‐lysine) instead of Ala. A second‐generation Hoveyda–Grubbs catalyst was used for an in vitro metathesis reaction with Cap_Alk10 and an allyl alcohol, which offers options for post‐biosynthetic modifications. The use of synthetic biology allows for the in vivo production of new peptide‐based antibiotics from an expanded amino acid repertoire.     

Lipidation of Temporin-1CEb Derivatives as a Tool for Activity Improvement,Pros and Cons of the Approach

The alarming raise of multi-drug resistance among human microbial pathogens makes the development of novel therapeutics a priority task. In contrast to conventional antibiotics, antimicrobial peptides (AMPs), besides evoking a broad spectrum of activity against microorganisms, could offer additional benefits, such as the ability to neutralize toxins, modulate inflammatory response, eradicate bacterial and fungal biofilms or prevent their development. The latter properties are of special interest, as most antibiotics available on the market have limited ability to diffuse through rigid structures of biofilms. Lipidation of AMPs is considered as an effective approach for enhancement of their antimicrobial potential and in vivo stability; however, it could also have undesired impact on selectivity, solubility or the aggregation state of the modified peptides. In the present work, we describe the results of structural modifications of compounds designed based on cationic antimicrobial peptides DK5 and CAR-PEG-DK5, derivatized at their N-terminal part with fatty acids with different lengths of carbon chain. The proposed modifications substantially improved antimicrobial properties of the final compounds and their effectiveness in inhibition of biofilm development as well as eradication of pre-formed 24 h old biofilms of Candida albicans and Staphylococcus aureus. The most active compounds (C5-DK5, C12-DK5 and C12-CAR-PEG-DK5) were also potent against multi-drug resistant Staphylococcus aureus USA300 strain and clinical isolates of Pseudomonas aeruginosa. Both experimental and in silico methods revealed strong correlation between the length of fatty acid attached to the peptides and their final membranolytic properties, tendency to self-assemble and cytotoxicity.     

Peptides from the Intestinal Tract of Breast Milk-Fed Infants Have Antimicrobial and Bifidogenic Activity

For bioactive milk peptides to be relevant to infant health, they must be released by gastrointestinal proteolysis and resist further proteolysis until they reach their site of activity. The intestinal tract is the likeliest site for most bioactivities, but it is currently unknown whether bioactive milk peptides are present therein. The purpose of the present study was to identify antimicrobial and bifidogenic peptides in the infant intestinal tract. Milk peptides were extracted from infant intestinal samples, and the activities of the bulk peptide extracts were determined by measuring growth of Escherichia coli, Staphylococcus aureus, and Bifidobacterium longum spp. infantis after incubation with serial dilutions. The peptide profiles of active and inactive samples were determined by peptidomics analysis and compared to identify candidate peptides for bioactivity testing. We extracted peptides from 29 intestinal samples collected from 16 infants. Five samples had antimicrobial activity against S. aureus and six samples had bifidogenic activity for B. infantis. We narrowed down a list of 6645 milk peptides to 11 candidate peptides for synthesis, of which 6 fully inhibited E. coli and S. aureus growth at concentrations of 2500 and 3000 µg/mL. This study provides evidence for the potential bioactivity of milk peptides in the infant intestinal tract.