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Antimicrobial Peptides: Structure and Mechanism of Biological Activity: 3rd Edition

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Microbiology".

Deadline for manuscript submissions: closed (30 November 2024) | Viewed by 8640

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Dr. Juan Borrero

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Departamento de Nutrición y Ciencia de los Alimentos (NUTRYCIAL), Sección Departamental de Nutrición y Ciencia de los Alimentos (SD-NUTRYCIAL), Facultad de Veterinaria, Universidad Complutense de Madrid (UCM), Avenida Puerta de Hierro, s/n, 28040 Madrid, Spain
Interests: microbiology; antimicrobial peptides; synthetic biology; biotechnology; bacteriocins

Special Issue Information

Dear Colleagues,

Since the early 1960s, the resistance of microbes against antibiotics has been recognized as a potential global health issue. This issue is now critical due to the reemergence of several infectious diseases of microbial origin and prevalence of multidrug-resistant microbes. Antimicrobial peptides (AMPs) have offered a good potential for novel drugs against drug-resistant microbial organisms, and extensive research has been dedicated to the discovery, characterization, de novo design, and assessment of the antimicrobial activity of these peptides since the late 1980s. So far, more than 3000 AMPs have been characterized and documented. These peptides have diverse natural origins and are found in unicellular organisms (bacteria, archaea, protists, and fungi), plants, and animals. Close to 75% of AMPs are found in animals, ~11% in plants, and about the same number in bacteria. Based on the sequences of these naturally found peptides, new chemically modified synthetic peptides have been designed to enhance or modify the biological activity of the original peptides. AMPs are also diverse in their biological activities and can be multifunctional. In addition to their antimicrobial activity, AMPs can have other biological functions, such as antioxidant, anticancer, antimalarial, chemotactic (modulation of immune systems), and wound healing. The diversity of AMPs expands to their physicochemical properties, structure, and mechanism of biological activity, which are the foci of this Special Issue. Most, but not all, AMPs are positively charged, and negatively and neutrally charged peptides can also be found. AMPs have different structures (α-helix, β-sheet, turn, or nonspecific interconvertible dynamic structures), overall hydrophobicity and amphipathicity, and can be linear, cyclic, or a combination of both. Many AMPs interact with the lipid membranes of the microbial/nonmicrobial cells and destroy these cells by disrupting the osmotic balance across the membrane. Some AMPs can pass across cell membranes and interact with intracellular targets such as organelle membranes, receptor proteins, or DNA. In the late 1980s and during the 1990s, several models were proposed for the mechanism of interaction of AMPs with model cell membranes, which generally include self-association of peptides and/or peptide–lipid association from specific well-defined pores or to induce nonspecific leakage. The mechanisms of translocation of AMPs through cell membranes and their successive interaction with intracellular molecules are less investigated. Understanding and visualizing the structural dynamics (subtle and fast conformational changes prior to and after interaction with cell membranes) and the entirety of the complex biophysical nature of the mechanism of the biological activity of AMPs are essential steps toward the discovery and design of new antimicrobial peptide drugs.

Dr. Juan Borrero
Guest Editor

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Keywords

antimicrobial peptides
mechanism of biological activity
functional diversity of peptides
structural analysis of peptides
dynamic conformation of peptides
lipid composition of the cell membrane
peptide–lipid interactions
peptide self-association
peptide-lipid complex formation
peptide–intracellular receptor interaction
peptide translocation through membrane
cell morphology
peptide interaction with infectious agents
surface properties of the cell
peptide interaction with the cell in vivo

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Related Special Issues

Antimicrobial Peptides: Structure and Mechanism of Biological Activity in International Journal of Molecular Sciences (12 articles)
Antimicrobial Peptides: Structure and Mechanism of Biological Activity 2.0 in International Journal of Molecular Sciences (11 articles)

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Research

19 pages, 2724 KiB

Open AccessArticle

1,3,5-Triazine as Branching Connector for the Construction of Novel Antimicrobial Peptide Dendrimers: Synthesis and Biological Characterization

by Rotimi Sheyi, Jessica T. Mhlongo, Marta Jorba, Ester Fusté, Anamika Sharma, Miguel Viñas, Fernando Albericio, Paula Espinal and Beatriz G. de la Torre

Int. J. Mol. Sci. 2024, 25(11), 5883; https://doi.org/10.3390/ijms25115883 - 28 May 2024

Viewed by 992

Abstract

Peptides displaying antimicrobial properties are being regarded as useful tools to evade and combat antimicrobial resistance, a major public health challenge. Here we have addressed dendrimers, attractive molecules in pharmaceutical innovation and development displaying broad biological activity. Triazine-based dendrimers were fully synthesized in the solid phase, and their antimicrobial activity and some insights into their mechanisms of action were explored. Triazine is present in a large number of compounds with highly diverse biological targets with broad biological activities and could be an excellent branching unit to accommodate peptides. Our results show that the novel peptide dendrimers synthesized have remarkable antimicrobial activity against Gram-negative bacteria (E. coli and P. aeruginosa) and suggest that they may be useful in neutralizing the effect of efflux machinery on resistance. Full article

(This article belongs to the Special Issue Antimicrobial Peptides: Structure and Mechanism of Biological Activity: 3rd Edition)

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Figure 1

Figure 1
Generation 1 and 2 (G1, G2) of triazine-based dendrimeric peptides. Full article ">Figure 2
Growth curves of (A). E. coli ATCC 25922; (B). P. aeruginosa ATCC 27853 in the presence of dendrimers 1, 2 and 3 at MIC (red line), ½ MIC (purple line) and ¼ MIC (blue line). Control without peptide (green line). Full article ">Figure 3
Time-kill curves of (A). E. coli ATCC 25922; (B). P. aeruginosa ATCC 27853 in the presence of dendrimers 1, 2 and 3 at MIC (blue line), ½ MIC (orange line) and ¼ MIC (red line). Control without peptide (green line). Full article ">Figure 4
AFM images obtained at a scan size of 100 μm2. (A,C,E,G) Topography images and (B,D,F,H) amplitude images. (A,B) Untreated E. coli; (C,D) E. coli after treatment with dendrimer 1 at MIC (8 µg/mL); (E,F) E. coli after treatment with dendrimer 2 at MIC (2 µg/mL); (G,H) E. coli after treatment with dendrimer 3 at MIC (8 µg/mL). Full article ">Figure 5
Percentage of Acridine Orange (AO) accumulation with dendrimers 1, 2 and 3. The control represents the assay with bacteria and AO. Assays were performed in E. coli 208691, P. aeruginosa 666 SJD and P. aeruginosa ATCC 27853. Full article ">Scheme 1
General protocol used in the synthesis of dendrimeric constructs: (a) Solid-phase peptide synthesis (SPPS) at rt: (i) Fmoc removal: 20% v/v piperidine in DMF, (1 × 1 min + 1 × 7 min), resin washing: DMF (3×), (ii) Fmoc-AA-OH (3 eq), DIC (3 eq), OxymaPure (3 eq) (60 min); resin washing: DMF (3×) (ii); (b) (i) p-hydroxybenzoic acid (3 eq), DIC (3 eq), OxymaPure (3 eq), double coupling; (ii) 20% v/v piperidine in DMF, (1 × 1 min + 1 × 7 min), resin washing: DMF (3×); (c) (i) 2,4,6-trichloro-1,3,5-triazine (3 eq), DIEA (3 eq) in DCM at −20 °C, 1 h; (d) EDA (50 eq), DIEA (50 eq) at rt overnight; (e) TFA-TIS-H2O (95:2.5:2.5), at r.t, 1 h, and RP-HPLC purification. Full article ">Scheme 2
Protocol used in the synthesis of dendrimer 2 and 3. (a) Solid-phase peptide synthesis (SPPS) at rt: (i) Fmoc removal: 20% v/v piperidine in DMF, (1 × 1 min + 1 × 7 min), resin washing: DMF (3×), (ii) Fmoc-AA-OH (3 eq), DIC (3 eq), OxymaPure (3 eq) (60 min); resin washing: DMF (3×) (b) (i) 2,4,6-trichloro-1,3,5-triazine (3 eq), DIEA (3 eq) in DCM at −20 °C, 1 h; (c) EDA (50 eq), DIEA (50 eq) at 45 °C for 3 h; (d) TFA-TIS-H2O (95:2.5:2.5), at r.t, 1 h, and RP-HPLC purification. Full article ">

20 pages, 2433 KiB

Open AccessArticle

Production of Pumilarin and a Novel Circular Bacteriocin, Altitudin A, by Bacillus altitudinis ECC22, a Soil-Derived Bacteriocin Producer

by Irene Lafuente, Ester Sevillano, Nuria Peña, Alicia Cuartero, Pablo E. Hernández, Luis M. Cintas, Estefanía Muñoz-Atienza and Juan Borrero

Int. J. Mol. Sci. 2024, 25(4), 2020; https://doi.org/10.3390/ijms25042020 - 7 Feb 2024

Cited by 5 | Viewed by 2246

Abstract

The rise of antimicrobial resistance poses a significant global health threat, necessitating urgent efforts to identify novel antimicrobial agents. In this study, we undertook a thorough screening of soil-derived bacterial isolates to identify candidates showing antimicrobial activity against Gram-positive bacteria. A highly active antagonistic isolate was initially identified as Bacillus altitudinis ECC22, being further subjected to whole genome sequencing. A bioinformatic analysis of the B. altitudinis ECC22 genome revealed the presence of two gene clusters responsible for synthesizing two circular bacteriocins: pumilarin and a novel circular bacteriocin named altitudin A, alongside a closticin 574-like bacteriocin (CLB) structural gene. The synthesis and antimicrobial activity of the bacteriocins, pumilarin and altitudin A, were evaluated and validated using an in vitro cell-free protein synthesis (IV-CFPS) protocol coupled to a split-intein-mediated ligation procedure, as well as through their in vivo production by recombinant E. coli cells. However, the IV-CFPS of CLB showed no antimicrobial activity against the bacterial indicators tested. The purification of the bacteriocins produced by B. altitudinis ECC22, and their evaluation by MALDI-TOF MS analysis and LC-MS/MS-derived targeted proteomics identification combined with massive peptide analysis, confirmed the production and circular conformation of pumilarin and altitudin A. Both bacteriocins exhibited a spectrum of activity primarily directed against other Bacillus spp. strains. Structural three-dimensional predictions revealed that pumilarin and altitudin A may adopt a circular conformation with five- and four-α-helices, respectively. Full article

(This article belongs to the Special Issue Antimicrobial Peptides: Structure and Mechanism of Biological Activity: 3rd Edition)

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Figure 1

Figure 1
Bacillus altitudinis ECC22 genome map generated using the CGView server. The red squares represent the coding sequences (CDS). The black plot shows the GC content, the green plot shows the CG skew +, and the purple plot shows the CG skew −. The position of the gene clusters for pumilarin, altitudin A, and the closticin 574-like bacteriocin (CLB) are highlighted with a blue rectangle. Full article ">Figure 2
Genetic organization of the pumilarin and altitudin A gene clusters in the B. altitudinis ECC22 genome. Full article ">Figure 3
(A) Amino acid sequences of the pumilarin and altitudin A prepeptides, with the leader sequences underlined and separated from the mature peptides. (B) Multiple-sequence alignment of altitudin A with the circular bacteriocins amylocyclicin and enterocin NKR-5-α. The dark grey highlighting shows the allocation of experimentally confirmed α-helices. The cationic amino acid residues are in red. (C) Amino acid sequence alignment of the bacteriocin closticin 574 with the predicted closticin 574-like peptide (CLB). The leader sequence is underlined, and the mature peptide is in bold. An asterisk (*) indicates a single fully conserved residue, a colon (:) indicates conservation within groups of residues with strongly similar properties, and a period (.) indicates conservation within groups of residues with weakly similar properties. Full article ">Figure 4
Bacteriocin circularization by the IV-CFPS protocol coupled to the SIML procedure. (A) Amino acid sequence of mature pumilarin and altitudin A. In red, the amino acids implicated in the head-to-tail circularization of native pumilarin and altitudin A. In blue, the amino acids selected for the IV-CFPS coupled to the SIML procedure, and the in vivo production of pumilarin and altitudin A. (B) Schematic representation of the pUC-derived expression vectors encoding the gene products of interest, the recombinant peptides produced by IV-CFPS, and the SIML procedure where the Npu DnaE intein fragments, IC and IN, interact to form an active peptide that splices, including circularization of the mature peptides. (C) Antimicrobial activity against Pediococcus damnosus CECT 4797 by a spot-on-agar test (SOAT) using 5 µL fractions of pumilarin and altitudin A, produced individually by the IV-CFPS/SIML method. Full article ">Figure 5
MALDI-TOF MS analysis of fractions 12 (a) and 15 (b) after the second RP-HPLC round of purification of the CFS of B. altitudinis ECC22. The peptides with a molecular mass (m/z) of 6598.93 and 7089.15 are for altitudin A and pumilarin, respectively. The full amino acidic sequences of altitudin A and pumilarin are in the right side of the figures. In red, the sequences containing the circularization site, identified by LC-MS/MS after digestion of fractions 12 and 15. The black arrow shows the head-to-tail circularization of the native bacteriocins. Full article ">Figure 6
Three-dimensional (3D) structural model of pumilarin and altitudin A, created by RobettaFold. Each α-helix is numerated and shows a different color. Full article ">

17 pages, 6731 KiB

Open AccessArticle

New N-Terminal Fatty-Acid-Modified Melittin Analogs with Potent Biological Activity

by Sheng Huang, Guoqi Su, Shan Jiang, Li Chen, Jinxiu Huang and Feiyun Yang

Int. J. Mol. Sci. 2024, 25(2), 867; https://doi.org/10.3390/ijms25020867 - 10 Jan 2024

Cited by 6 | Viewed by 1414

Abstract

Melittin, a natural antimicrobial peptide, has broad-spectrum antimicrobial activity. This has resulted in it gaining increasing attention as a potential antibiotic alternative; however, its practical use has been limited by its weak antimicrobial activity, high hemolytic activity, and low proteolytic stability. In this study, N-terminal fatty acid conjugation was used to develop new melittin-derived lipopeptides (MDLs) to improve the characteristics of melittin. Our results showed that compared with native melittin, the antimicrobial activity of MDLs was increased by 2 to 16 times, and the stability of these MDLs against trypsin and pepsin degradation was increased by 50 to 80%. However, the hemolytic activity of the MDLs decreased when the length of the carbon chain of fatty acids exceeded 10. Among the MDLs, the newly designed analog Mel-C8 showed optimal antimicrobial activity and protease stability. The antimicrobial mechanism studied revealed that the MDLs showed a rapid bactericidal effect by interacting with lipopolysaccharide (LPS) or lipoteichoic acid (LTA) and penetrating the bacterial cell membrane. In conclusion, we designed and synthesized a new class of MDLs with potent antimicrobial activity, high proteolytic stability, and low hemolytic activity through N-terminal fatty acid conjugation. Full article

(This article belongs to the Special Issue Antimicrobial Peptides: Structure and Mechanism of Biological Activity: 3rd Edition)

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10 pages, 1530 KiB

Open AccessArticle

A Common Polymorphism in RNASE6 Impacts Its Antimicrobial Activity toward Uropathogenic Escherichia coli

by Raul Anguita, Guillem Prats-Ejarque, Mohammed Moussaoui, Brian Becknell and Ester Boix

Int. J. Mol. Sci. 2024, 25(1), 604; https://doi.org/10.3390/ijms25010604 - 3 Jan 2024

Cited by 1 | Viewed by 1415

Abstract

Human Ribonuclease (RNase) 6 is a monocyte and macrophage-derived protein with potent antimicrobial activity toward uropathogenic bacteria. The RNASE6 gene is heterogeneous in humans due to the presence of single nucleotide polymorphisms (SNPs). RNASE6 rs1045922 is the most common non-synonymous SNP, resulting in a G to A substitution that determines an arginine (R) to glutamine (Q) transversion at position 66 in the protein sequence. By structural analysis we observed that R66Q substitution significantly reduces the positive electrostatic charge at the protein surface. Here, we generated both recombinant RNase 6-R66 and -Q66 protein variants and determined their antimicrobial activity toward uropathogenic Escherichia coli (UPEC), the most common cause of UTI. We found that the R66 variant, encoded by the major SNP rs1045922 allele, exhibited superior bactericidal activity in comparison to the Q66 variant. The higher bactericidal activity of R66 variant correlated with an increase in the protein lipopolysaccharide binding and bacterial agglutination abilities, while retaining the same enzymatic efficiency. These findings encourage further work to evaluate RNASE6 SNP distribution and its impact in UTI susceptibility. Full article

(This article belongs to the Special Issue Antimicrobial Peptides: Structure and Mechanism of Biological Activity: 3rd Edition)

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Figure 1

Figure 1
RNASE6 rs1045922 SNP distribution among populations according to the 1000 Genomes Study [<a href="#B30-ijms-25-00604" class="html-bibr">30</a>]. Full article ">Figure 2
RNase 6-Q66 exhibits reduced antimicrobial activity toward laboratory and uropathogenic strains of E. coli, when compared with RNase 6-R66. Bacterial viability was performed by CFU counting taking the non-treated control as a 100% reference. Significant difference between the variants at each concentration is indicated (**** p < 0.0001; *** p < 0.0002; ** p < 0.002; * p < 0.03). The table below indicates the calculated Minimum Bactericidal Concentration (MBC) at which 100 or 75% of bacteria was eradicated. Horizontal dotted lines indicate the bacterial viability level taken to calculate MBC75 values. Full article ">Figure 3
Analysis on the impact of the R66Q substitution on the protein structure. (A) Details of sulphate and phosphate binding interactions of RNase 6-R66 in solved crystal structures (PDB ID: 4X09 and 5OAB respectively). RNase 6 main chain is drawn in cyan ribbon. Ligands and protein interacting side chains are drawn in ball-and-sticks and colored according to atom elements (B) The substitution of an arginine by a non-charged amino acid at the protein surface alters the cationic charge of this region, which in turn may disturb the ability of RNase 6 to bind to anionic bacterial wall components. The mutant structural prediction was obtained by AlphaFold2. Top: RNase 6-R66 is shown in cyan while the -Q66 variant is shown in orange. Bottom: Surface electrostatic charge prediction. The position corresponding to the amino acid change is indicated by the dotted black line. Pictures were drawn with PyMol 2.3.4. Full article ">

16 pages, 2517 KiB

Open AccessArticle

Novel Antimicrobial Peptides from Saline Environments Active against E. faecalis and S. aureus: Identification, Characterisation and Potential Usage

by Jakub Lach, Magdalena Krupińska, Aleksandra Mikołajczyk, Dominik Strapagiel, Paweł Stączek and Agnieszka Matera-Witkiewicz

Int. J. Mol. Sci. 2023, 24(14), 11787; https://doi.org/10.3390/ijms241411787 - 22 Jul 2023

Cited by 2 | Viewed by 1713

Abstract

Microorganisms inhabiting saline environments have been known for decades as producers of many valuable bioproducts. These substances include antimicrobial peptides (AMPs), the most recognizable of which are halocins produced by halophilic Archaea. As agents with a different modes of action from that of most conventionally used antibiotics, usually associated with an increase in the permeability of the cell membrane as a result of a formation of channels and pores, AMPs are a currently promising object of research focused on the investigation of antibiotics with non-standard modes of action. The aim of this study was to investigate antimicrobial activity against multidrug-resistant human pathogens of three peptides, which were synthetised based on sequences identified in metagenomes from saline environments. The investigations were performed against Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. Subsequently, the cytotoxicity and haemolytic properties of the tested peptides were verified. An in silico analysis of the interaction of the tested peptides with molecular targets for reference antibiotics was also carried out in order to verify whether or not they can act in a similar way. The P1 peptide manifested the growth inhibition of E. faecalis at a MIC₅₀ of 32 µg/mL and the P3 peptide at a MIC₅₀ of 32 µg/mL was shown to inhibit the growth of both E. faecalis and S. aureus. Furthermore, the P1 and P3 peptides were shown to have no cytotoxic or haemolytic activity against human cells. Full article

(This article belongs to the Special Issue Antimicrobial Peptides: Structure and Mechanism of Biological Activity: 3rd Edition)

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