Majority Of Antimicrobial Peptides Research Articles

Structural and Functional Characterization of a Novel Recombinant Antimicrobial Peptide from Hermetia illucens.

Antibiotics are commonly used to treat pathogenic bacteria, but their prolonged use contributes to the development and spread of drug-resistant microorganisms raising the challenge to find new alternative drugs. Antimicrobial peptides (AMPs) are small/medium molecules ranging 10–60 residues synthesized by all living organisms and playing important roles in the defense systems. These features, together with the inability of microorganisms to develop resistance against the majority of AMPs, suggest that these molecules might represent effective alternatives to classical antibiotics. Because of their high biodiversity, with over one million described species, and their ability to live in hostile environments, insects represent the largest source of these molecules. However, production of insect AMPs in native forms is challenging. In this work we investigate a defensin-like antimicrobial peptide identified in the Hermetia illucens insect through a combination of transcriptomics and bioinformatics approaches. The C-15867 AMP was produced by recombinant DNA technology as a glutathione S-transferase (GST) fusion peptide and purified by affinity chromatography. The free peptide was then obtained by thrombin proteolysis and structurally characterized by mass spectrometry and circular dichroism analyses. The antibacterial activity of the C-15867 peptide was evaluated in vivo by determination of the minimum inhibitory concentration (MIC). Finally, crystal violet assays and SEM analyses suggested disruption of the cell membrane architecture and pore formation with leaking of cytosolic material.

In-Cell FRET Indicates Magainin Peptide Induced Permeabilization of Bacterial Cell Membranes at Lower Peptide-to-Lipid Ratios Relevant to Liposomal Studies.

Antimicrobial peptides (AMPs) are promising candidates for anti-infective drugs. The majority of AMPs are considered to disrupt the lipid matrix of bacterial membranes, exerting bactericidal activity. A number of biophysical studies have been carried out to elucidate the underlying molecular mechanisms. However, the fact that the number of peptide molecules bound to a bacterial cell under bactericidal conditions is much larger than that expected from liposomal studies raises the question of whether membrane permeabilization mechanisms proposed by liposomal studies are relevant to bacteria. In this study, the peptide-to-lipid molar ratio needed for an antimicrobial magainin peptide to permeabilize the cell membrane of the Gram-positive bacterium Bacillus megaterium was estimated by random fluorescence resonance energy transfer from a BODIPY FL-labeled lipid to a Texas Red-labeled peptide. The comparison of the observed energy transfer efficiency with the two-dimensional energy transfer theory estimated that the leakage of the calcein dye from bacterial cells occurred at a peptide-to-lipid molar ratio of 0.025. At this ratio, the peptide induced dye leakage from liposomes mimicking the bacterial membrane, indicating that the lipid matrix is a target of membrane-acting AMPs and that liposomes are a useful model system to investigate their mechanisms of action. Furthermore, a binding assay suggested that most peptide molecules were bound to cellular components other than cell membranes.

Antimicrobial Peptides from Black Soldier Fly (Hermetia illucens) as Potential Antimicrobial Factors Representing an Alternative to Antibiotics in Livestock Farming.

Simple SummaryMicrobial resistance to antibiotics is a constant threat to livestock farming, and unreasonable use of antibiotics has increased the prevalence of infectious diseases in humans and animals. Antimicrobial peptides derived from black soldier fly, Hermetia illucens (Diptera: Stratiomyidae), have great potential as alternatives to antibiotics for prophylaxis and treatment of diseases in animals because they have extensive antimicrobial properties and a lower tendency to induce resistance. Additionally, several studies have shown that Hermetia illucens larvae can participate in a circular economy by digesting organic waste alone and then promoting the growth performance of domestic animals fed the larvae. Therefore, antimicrobial peptides from Hermetia illucens are promising candidate for replacement of antibiotics in livestock farming.Functional antimicrobial peptides (AMPs) are an important class of effector molecules of innate host immune defense against pathogen invasion. Inability of microorganisms to develop resistance against the majority of AMPs has made them alternatives to antibiotics, contributing to the development of a new generation of antimicrobials. Due to extensive biodiversity, insects are one of the most abundant sources of novel AMPs. Notably, black soldier fly insect (BSF; Hermetia illucens (Diptera: Stratiomyidae)) feeds on decaying substrates and displays a supernormal capacity to survive under adverse conditions in the presence of abundant microorganisms, therefore, BSF is one of the most promising sources for identification of AMPs. However, discovery, functional investigation, and drug development to replace antibiotics with AMPs from Hermetia illucens remain in a preliminary stage. In this review, we provide general information on currently verified AMPs of Hermetia illucens, describe their potential medical value, discuss the mechanism of their synthesis and interactions, and consider the development of bacterial resistance to AMPs in comparison with antibiotics, aiming to provide a candidate for substitution of antibiotics in livestock farming or, to some extent, for blocking the horizontal transfer of resistance genes in the environment, which is beneficial to human and animal welfare.

Shedding Light on the Antimicrobial Peptide Arsenal of Terrestrial Isopods: Focus on Armadillidins, a New Crustacean AMP Family.

In crustaceans, antimicrobial peptides (AMPs) are clustered into four major groups according to their amino acid composition and structure: (1) single-domain peptides containing cysteine residues such as anti-lipopolysaccharide-factor (ALF), (2) multi-domain or chimeric AMPs such as crustins, (3) non-conventional AMPs, and (4) linear single-domain AMPs. The majority of AMPs has been described in commercially exploited crustaceans, particularly decapods living in aquatic environments (crab, shrimp, lobster, and crayfish). Here, we aimed at establishing the AMPs repertoire of terrestrial isopods (Oniscidea), an original suborder of crustaceans adapted to life outside of the aquatic environment. Using transcriptomic data from 21 species, we identified 110 ALF and 73 crustin sequences. We also characterized the full-length sequence of armadillidins from 17 species, similar to the AMP previously described in the terrestrial isopod Armadillidium vulgare. Furthermore, we tested the antimicrobial activity of three armadillidin peptides characterized from three distantly related species. This analysis revealed similar activity spectra against pathogens, despite extensive structural variation among the tested peptides. In addition to conventional crustacean AMPs, our work highlights armadillidins as a new and independent family of AMPs specific to the Oniscidea, thus opening new perspectives concerning the study of the immune system of terrestrial isopods.

Worms' Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are natural antibiotics produced by all living organisms. In metazoans, they act as host defense factors by eliminating microbial pathogens. But they also help to select the colonizing bacterial symbionts while coping with specific environmental challenges. Although many AMPs share common structural characteristics, for example having an overall size between 10–100 amino acids, a net positive charge, a γ-core motif, or a high content of cysteines, they greatly differ in coding sequences as a consequence of multiple parallel evolution in the face of pathogens. The majority of AMPs is specific of certain taxa or even typifying species. This is especially the case of annelids (ringed worms). Even in regions with extreme environmental conditions (polar, hydrothermal, abyssal, polluted, etc.), worms have colonized all habitats on Earth and dominated in biomass most of them while co-occurring with a large number and variety of bacteria. This review surveys the different structures and functions of AMPs that have been so far encountered in annelids and nematodes. It highlights the wide diversity of AMP primary structures and their originality that presumably mimics the highly diverse life styles and ecology of worms. From the unique system that represents marine annelids, we have studied the effect of abiotic pressures on the selection of AMPs and demonstrated the promising sources of antibiotics that they could constitute.

Effects of Hydrophobic Amino Acid Substitutions on Antimicrobial Peptide Behavior.

Antimicrobial peptides (AMPs) are naturally occurring components of the immune system that act against bacteria in a variety of organisms throughout the evolutionary hierarchy. There have been many studies focused on the activity of AMPs using biophysical and microbiological techniques; however, a clear and predictive mechanism toward determining if a peptide will exhibit antimicrobial activity is still elusive, in addition to the fact that the mechanism of action of AMPs has been shown to vary between peptides, targets, and experimental conditions. Nonetheless, the majority of AMPs contain hydrophobic amino acids to facilitate partitioning into bacterial membranes and a net cationic charge to promote selective binding to the anionic surfaces of bacteria over the zwitterionic host cell surfaces. This study explores the role of hydrophobic amino acids using the peptide C18G as a model system. These changes were evaluated for the effects on antimicrobial activity, peptide-lipid interactions using Trp fluorescence spectroscopy, peptide secondary structure formation, and bacterial membrane permeabilization. The results show that while secondary structure formation was not significantly impacted by the substitutions, antibacterial activity and binding to model lipid membranes were well correlated. The variants containing Leu or Phe as the sole hydrophobic groups bound bilayers with highest affinity and were most effective at inhibiting bacterial growth. Peptides with Ile exhibited intermediate behavior while those with Val or α-aminoisobutyric acid (Aib) showed poor binding and activity. The Leu, Phe, and Ile peptides demonstrated a clear preference for anionic bilayers, exhibiting significant emission spectrum shifts upon binding. Similarly, the Leu, Phe, and Ile peptides demonstrated greater ability to disrupt lipid vesicles and bacterial membranes. In total, the data indicate that hydrophobic moieties in the AMP sequence play a significant role in the binding and ability of the peptide to exhibit antibacterial activity.

Free Energies of Molecular Bound States in Lipid Bilayers: Lethal Concentrations of Antimicrobial Peptides

The lipid matrix, or the lipid bilayer, of cell membranes is a natural binding site for amphipathic molecules, including antimicrobial peptides, pore-forming proteins, and many drugs. The unique property of pore-forming antimicrobial peptides is that they exhibit a threshold concentration (called the lethal concentration or the minimum inhibitory concentration) for activity, below which no effect is seen. Without this property, antimicrobial peptides would not be effective self-defense weapons, because they would have harmed all cells at any concentration. The question is what gives rise to this unique property? This study provides a free energy description for the origin of a threshold concentration. The same free energy applied differently also explains the binding of drugs that shows no threshold concentrations. The idea is compared with theories of micellar solutions that require a large oligomer size (n ⪞ 15) to achieve a threshold concentration. The elasticity of lipid bilayers makes the phenomena in membranes different. The majority of antimicrobial peptides have a large negative binding energy to the bilayer interface, but the binding causes an expansion in the membrane area, or equivalently a thinning in the membrane thickness. This elastic energy of membrane thinning elevates the energy level of interfacial binding with the peptide concentration, hence gives rise to a threshold concentration for forming pores containing as few as four peptides.

Advances in antimicrobial peptide immunobiology

Antimicrobial peptides are ancient components of the innate immune system and have been isolated from organisms spanning the phylogenetic spectrum. Over an evolutionary time span, these peptides have retained potency, in the face of highly mutable target microorganisms. This fact suggests important coevolutionary influences in the host-pathogen relationship. Despite their diverse origins, the majority of antimicrobial peptides have common biophysical parameters that are likely essential for activity, including small size, cationicity, and amphipathicity. Although more than 900 different antimicrobial peptides have been characterized, most can be grouped as belonging to one of three structural classes: (1) linear, often of alpha-helical propensity; (2) cysteine stabilized, most commonly conforming to beta-sheet structure; and (3) those with one or more predominant amino acid residues, but variable in structure. Interestingly, these biophysical and structural features are retained in ribosomally as well as nonribosomally synthesized peptides. Therefore, it appears that a relatively limited set of physicochemical features is required for antimicrobial peptide efficacy against a broad spectrum of microbial pathogens. During the past several years, a number of themes have emerged within the field of antimicrobial peptide immunobiology. One developing area expands upon known microbicidal mechanisms of antimicrobial peptides to include targets beyond the plasma membrane. Examples include antimicrobial peptide activity involving structures such as extracellular polysaccharide and cell wall components, as well as the identification of an increasing number of intracellular targets. Additional areas of interest include an expanding recognition of antimicrobial peptide multifunctionality, and the identification of large antimicrobial proteins, and antimicrobial peptide or protein fragments derived thereof. The following discussion highlights such recent developments in antimicrobial peptide immunobiology, with an emphasis on the biophysical aspects of host-defense polypeptide action and mechanisms of microbial resistance.

Antimicrobial peptides from marine invertebrates.

Marine invertebrates lack an acquired, memory-type immunity based on T-lymphocyte subsets and clonally derived immunoglobulins (72). This differs from the vertebrate immune system, which is characterized by somatic gene rearrangement, clonal selection, and expansion and a discriminative ability that includes lymphocytes, among other factors, which impart specificity and memory (71). Marine invertebrates rely solely on innate immune mechanisms that include both humoral and cellular responses. Humoral immunity in marine invertebrates is characterized by antimicrobial agents present in the blood cells and plasma (92), along with reactions such as hemolymph coagulation or melanization (79, 85). Cellular immunity in marine invertebrates is based on cell defense reactions, including encapsulation, nodule formation, and phagocytosis (92). The cellular component of marine invertebrate immunity is mediated by hemocytes, motile cells that phagocytize microbes and secrete soluble antimicrobial and cytotoxic substances into the hemolymph (53). This differs from insects, especially Drosophila melanogaster, which rely largely on the challenge-induced synthesis of antimicrobial peptides by the fat body (30, 88) and use exclusion, via a tough exoskeleton, as their major antimicrobial defense. The circulating hemolymph in marine invertebrates contains biologically active substances such as complement, lectins, clotting factors, and antimicrobial peptides (57). All of these factors contribute to a self-defense system in marine invertebrates against invading microorganisms, which can number up to 106 bacteria/ml and 109 virus/ml of seawater (2). The survival of marine invertebrates in this environment suggests that their innate immune system is effective and robust (52). Antimicrobial peptides are a major component of the innate immune defense system in marine invertebrates. They are defined as molecules less than 10 kDa in mass which show antimicrobial properties (12) and provide an immediate and rapid response to invading microorganisms (8). The major classes of antimicrobial peptides include (i) α-helices, (ii) β-sheet and small proteins, (iii) peptides with thio-ether rings, (iv) peptides with an overrepresentation of one or two amino acids, (v) lipopeptides, and (vi) macrocyclic cystine knot peptides (24). There is evidence that antimicrobial peptides are widespread in invertebrates (15), especially in tissues such as the gut and respiratory organs in marine invertebrates, where exposure to pathogenic microorganisms is likely. In spite of variations in structure and size, the majority of antimicrobial peptides are amphiphilic, displaying both hydrophilic and hydrophobic surfaces. These peptides generally act by forming pores in microbial membranes or otherwise disrupting membrane integrity (82), which is facilitated by their amphiphilic structure. This mode of action is unlikely to lead to the development of resistance (9, 58), although it must be noted that this presumption is debatable (10). Recently, cationic antimicrobial peptides have been reported to be involved in many aspects of innate host defenses, associated with processes such as acute inflammation (25). The value of antimicrobial peptides in innate immunity lies in their ability to function without either high specificity or memory, and their small size makes them easy to synthesize (72). In addition, many antibacterial peptides show remarkable specificity for prokaryotes with low toxicity for eukaryotic cells (97). This is a characteristic that has favored their investigation and exploitation as potential new antibiotics (97). The recent appearance of a growing number of bacteria resistant to conventional antibiotics has become a serious medical problem. To overcome this resistance, the development of antibiotics with novel mechanisms of action is a pressing issue (48). Endogenous antimicrobial peptides are exciting candidates as new antibacterial agents due to their broad antimicrobial spectra, highly selective toxicities, and the difficulty for bacteria to develop resistance to these peptides (11, 26, 47). The ocean covers 71% of the surface of the earth and contains approximately half of the total global biodiversity, with estimates ranging between 3 and 500 × 106 different species (28). Marine macrofauna alone comprise 0.5 to 10 × 106 species (23). Therefore, the marine environment, especially marine invertebrates that rely solely on innate immune mechanisms for host defense, is a spectacular resource for the development of new antimicrobial compounds. This minireview will encompass what is known about gene-encoded antimicrobial peptides from marine invertebrates, covering the phyla Arthropoda, Chordata, and Mollusca (Table ​(Table11). TABLE 1. Antimicrobial peptides from marine invertebrates