Potent Antimicrobial Peptides Research Articles

Bacteriocin diversity, function, discovery and application as antimicrobials.

Bacteriocins are potent antimicrobial peptides that are produced by bacteria. Since their discovery almost a century ago, diverse peptides have been discovered and described, and some are currently used as commercial food preservatives. Many bacteriocins exhibit extensively post-translationally modified structures encoded on complex gene clusters, whereas others have simple linear structures. The molecular structures, mechanisms of action and resistance have been determined for a number of bacteriocins, but most remain incompletely characterized. These gene-encoded peptides are amenable to bioengineering strategies and heterologous expression, enabling metagenomic miningand modification of novel antimicrobials. The ongoing global antimicrobial resistance crisis demands that novel therapeutics be developed to combat infectious pathogens. New compounds that are target-specific and compatible with the resident microbiota would be valuable alternatives to current antimicrobials. As bacteriocins can be broad or narrow spectrum in nature, they are promising tools for this purpose. However, few bacteriocins have gone beyond preclinical trials and none is currently used therapeutically in humans. In this Review, we explore the broad diversity in bacteriocin structure and function, describe identification and optimization methods and discuss the reasons behind the lack of translation beyond the laboratory of these potentially valuable antimicrobials.

Unveiling thymosin antimicrobial peptide from pearl spot, Etroplus suratensis: Molecular characterization, phylogenetic analysis, and functional implications

BackgroundAntimicrobial peptides are small, cationic, amphipathic peptides composed of 10–50 amino acids, playing a crucial role in innate immunity. While they are found in both vertebrates and invertebrates, they are particularly effective in invertebrates. This study presents the identification and molecular characterization of the potential antimicrobial peptide thymosin from Pearl spot, Etroplus suratensis (Es-Thyβ). Phylogenetic analysis and structural characterization of the identified peptide are analyzed and substantiated through the study. ResultsThe sequence analysis of Es-Thyβ revealed an Open Reading Frame (ORF) of 132 nucleotides, encoding a 43-amino acid peptide. The hydrophobicity of the peptide, determined using PepDraw, was found to be +68.93 Kcal*mol−1. Through in silico analysis the identified Thymosin was determined to be Es-Thyβ. The structure and characteristics of the peptide sequence were further analyzed using multiple in silico software tools, providing confirmation of its identity as Es-Thyβ. ConclusionThe study identified and described a potential antimicrobial peptide Es-Thyβ, a member of the thymosin β4 family, with conserved sequences and evolutionary links across teleost fishes.

First vertebrate BRICHOS antimicrobial peptides: β-hairpin host defense peptides in limbless amphibia lung resemble those of marine worms

Innate immunity of invertebrates offers potent antimicrobial peptides (AMPs) against drug-resistant infections. To identify new worm β-hairpin AMPs, we explored the sequence diversity of proteins with a BRICHOS domain, which comprises worm AMP precursors. Strikingly, we discovered new BRICHOS AMPs not in worms, but in caecilians, the least studied clade of vertebrates. Two precursor proteins from Microcaecilia unicolor and Rhinatrema bivittatum resemble SP-C lung surfactants and bear worm AMP-like peptides at C-termini. The analysis of M. unicolor tissue transcriptomes shows that the AMP precursor is highly expressed in the lung along with regular SP-C, suggesting a different, protective function. The peptides form right-twisted β-hairpins, change conformation upon lipid binding, and rapidly disrupt bacterial membranes. Both peptides exhibit broad-spectrum activity against multidrug-resistant ESKAPE pathogens with 1–4 μM MICs and remarkably low toxicity, giving 40–70-fold selectivity towards bacteria. These BRICHOS AMPs, previously unseen in vertebrates, reveal a novel lung innate immunity mechanism and offer a promising antibiotics template.

New insight into the biological activity of Salmo salar NK-lysin antimicrobial peptides.

NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of nk-lysin from Atlantic salmon (Salmo salar) using EST sequences. In the present study, we reported and characterized two additional transcripts of NK-lysin from S. salar. Besides, we evaluated the tissue distribution of three NK-lysins from S. salar and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against Piscirickettsia salmonis (LF-89) and Flavobacterium psychrophilum. These peptides induced the expression of immune genes related to innate and adaptive immune responses in vitro and in vivo. The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun N-terminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NK-lysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture.

Identification of Indigenous Lactobacillus fermentum by 16S rRNA Gene Sequencing Isolated from Two Different Ecosystems: Newborn Infants and Bee’s Intestine

Background: The consumption of functional ferments that have functional characteristic sand contributes enormously to the safety and quality of food by offering several nutritional, technological and health properties. Lactobacillus fermentum (Lb. fermentum) is a Gram-positive bacterium belonging to the genus Lactobacillus and many are said to have the effect of preventing gastro-intestinal infections and improving immunological response. Additionally, Lb. fermentum produces diverse and potent antimicrobial peptides which can be applied as alternatives to antibiotics. Methods: In this study, samples were isolated from two different ecological niches, the first was feces of newborn infants (0-3 months) and the second from bee’s intestine, from south of Algeria : Ghardaïa 32°29'00" North, 3°41'00" East. Two indigenous lactic acid bacteria were isolated and evaluated for their probiotic properties. The isolate identification by sequencing 16S r DNA was performed using the universal primers 27F and 1492R. The NCBI Gen Bank database was used to have similarity of species. Result: The cultures showed good survival in simulated transit fluids as well as high acid and bile tolerance. The isolates also demonstrated antibacterial activity against Escherichia coli and Staphylococcus aureus, the inhibition zone was measured between 10 mm and 20 mm of diameter respectively. Comparing the sequences of the isolates with the Gen Bank database (NCBI), a high percentage with species of Lactobacillus fermentum was shown.

Pore-forming peptide C14R exhibits potent antifungal activity against clinical isolates of Candida albicans and Candida auris.

Invasive candidiasis is a global public health problem as it poses a significant threat in hospital-settings. The aim of this study was to evaluate C14R, an analog derived from peptide BP100, as a potential antimicrobial peptide against the prevalent opportunistic yeast Candida albicans and the emergent multidrug-resistant yeast Candida auris. Antifungal susceptibility testing of C14R against 99C. albicans and 105C. auris clinical isolates from Colombia, was determined by broth microdilution. Fluconazole was used as a control antifungal. The synergy between C14R and fluconazole was assessed in resistant isolates. Assays against fungal biofilm and growth curves were also carried out. Morphological alterations of yeast cell surface were evaluated by scanning electron microscopy. A permeability assay verified the pore-forming ability of C14R. C. albicans and C. auris isolates had a geometric mean MIC against C14R of 4.42 µg/ml and 5.34 µg/ml, respectively. Notably, none of the isolates of any species exhibited growth at the highest evaluated peptide concentration (200 µg/ml). Synergistic effects were observed when combining the peptide and fluconazole. C14R affects biofilm and growth of C. albicans and C. auris. Cell membrane disruptions were observed in both species after treatment with the peptide. It was confirmed that C14R form pores in C. albicans' membrane. C14R has a potent antifungal activity against a large set of clinical isolates of both C. albicans and C. auris, showing its capacity to disrupt Candida membranes. This antifungal activity remains consistent across isolates regardless of their clinical source. Furthermore, the absence of correlation between MICs to C14R and resistance to fluconazole indicates the peptide's potential effectiveness against fluconazole-resistant strains. Our results suggest the potential of C14R, a pore-forming peptide, as a treatment option for fungal infections, such as invasive candidiasis, including fluconazole and amphotericin B -resistant strains.

Novel design of simplified β-hairpin antimicrobial peptide as a potential food preservative based on Trp-pocket backbone

Food contamination from microbial deterioration requires the development of potent antimicrobial peptides (AMPs). The deployment of approved AMPs as dietary preservatives is limited due to barriers such as instability, toxicity, and high synthetic costs. This exploration utilizes the primary structural elements of the Trp-pocket backbone to engineer a series of β-hairpin AMPs (XWRWRPGXKXXR-NH2, X representing I, V, F, and/or L). Peptides WpLF, with Phe as X and Leu arranged at the 11th position, demonstrated exceptional selectivity index (SI = 123.08) and sterilization effects both in vitro and in vivo. WpLF consistently exhibited stable bacteriostasis, regardless of physiological salts, serum, and extreme pH. Mechanistic analysis indicated that the peptide penetrates microbial cell membranes, inducing membrane disruption, thereby impeding drug resistance evolution. Conclusively, AMPs engineered by the Trp-pocket skeleton hold substantial potential as innovative biological preservatives in food preservation, providing valuable insights for sustainable and safe peptide-based food preservatives.

Antimicrobial peptides designed by computational analysis of proteomes.

Antimicrobial peptides (AMPs) are promising cationic and amphipathic molecules to fight antibiotic resistance. To search for novel AMPs, we applied a computational strategy to identify peptide sequences within the organisms' proteome, including in-house developed software and artificial intelligence tools. After analyzing 150.450 proteins from eight proteomes of bacteria, plants, a protist, and a nematode, nine peptides were selected and modified to increase their antimicrobial potential. The 18 resulting peptides were validated by bioassays with four pathogenic bacterial species, one yeast species, and two cancer cell-lines. Fourteen of the 18 tested peptides were antimicrobial, with minimum inhibitory concentrations (MICs) values under 10µM against at least three bacterial species; seven were active against Candida albicans with MICs values under 10µM; six had a therapeutic index above 20; two peptides were active against A549 cells, and eight were active against MCF-7 cells under 30µM. This study's most active antimicrobial peptides damage the bacterial cell membrane, including grooves, dents, membrane wrinkling, cell destruction, and leakage of cytoplasmic material. The results confirm that the proposed approach, which uses bioinformatic tools and rational modifications, is highly efficient and allows the discovery, with high accuracy, of potent AMPs encrypted in proteins.

A novel antimicrobial peptide with broad-spectrum and exceptional stability derived from the natural peptide Brevicidine

The global issue of antibiotic resistance is increasingly severe, highlighting the urgent necessity for the development of new antibiotics. Brevicidine, a natural cyclic lipopeptide, exhibits remarkable antimicrobial activity against Gram-negative bacteria. In this study, a comprehensive structure-activity relationship of Brevicidine was investigated through 20 newly synthesized cyclic lipopeptide analogs, resulting in the identification of an optimal linear analog 22. The sequence of analog 22 consisted of five d-amino acids and four non-natural amino acid 2,5-diaminovaleric acid (Orn) and conjugated with decanoic acid at N-terminal. Compared to Brevicidine, analog 22 was easier to synthesize, and exerted broad spectrum antimicrobial activity and excellent stability (t1/2 = 40.98 h). Additionally, analog 22 demonstrated a rapid bactericidal effect by permeating non-specifically through the bacterial membranes, thereby minimizing the likelihood of inducing resistance. Moreover, it exhibited remarkable efficacy in combating bacterial biofilms and reversing bacterial resistance to conventional antibiotics. Furthermore, it effectively suppressed the growth of bacteria in vital organs of mice infected with S. aureus ATCC 25923. In conclusion, analog 22 may represent a potential antimicrobial peptide for further optimization.

SRAGE-binding and antimicrobial bioactivities of soy and pea protein after heating and in vitro infant digestion

During infant formula production, proteins are always heated, potentially affecting their digestibility and the bioactivities of resulting peptides. Although plant proteins are a promising dairy alternative for infant formula, they remain understudied, necessitating further investigations. Therefore, this research aimed to fill this gap by assessing the impact of different heating modes on soy protein (SP) and pea protein (PP), focusing on glycation levels, peptide formation during in vitro infant digestion, and immune protection potential (sRAGE-binding and antimicrobial activities) of the resulting peptides. Consequently, dry heating led to increased glycation and glycated peptide production, particularly with higher glycation in PP than SP. Moreover, PP exhibited an overall stronger sRAGE-binding capacity than SP, regardless of heating and digestion conditions. Regarding antimicrobial activity, both SP and PP-derived peptides displayed reduced effectiveness against Enterobacter cloacae after dry heating. Additionally, Staphylococcus epidermidis was differently inhibited, where PP-derived peptides showed inherent inhibition. The primary determinant of sRAGE-binding and antimicrobial potential in digestion-derived peptides was the protein source. Subsequent bioinformatics analysis predicted 519 and 133 potential antimicrobial peptides in SP and PP, respectively. This study emphasises the importance of protein source for infant formula to ensure infant health.

Waste to resource: Mining antimicrobial peptides in sludge from metagenomes using machine learning

The emergence of antibiotic-resistant bacteria poses a huge threat to the treatment of infections. Antimicrobial peptides are a class of short peptides that widely exist in organisms and are considered as potential substitutes for traditional antibiotics. Here, we use metagenomics combined with machine learning to find antimicrobial peptides from environmental metagenomes and successfully obtained 16,044,909 predicted AMPs. We compared the abundance of potential antimicrobial peptides in natural environments and engineered environments, and found that engineered environments also have great potential. Further, we chose sludge as a typical engineered environmental sample, and tried to mine antimicrobial peptides from it. Through metaproteome analysis and correlation analysis, we mined 27 candidate AMPs from sludge. We successfully synthesized 25 peptides by chemical synthesis, and experimentally verified that 21 peptides had antibacterial activity against the 4 strains tested. Our work highlights the potential for mining new antimicrobial peptides from engineered environments and demonstrates the effectiveness of mining antimicrobial peptides from sludge.

Evaluation of the synergistic potential and mechanisms of action for de novo designed cationic antimicrobial peptides

Antimicrobial peptides (AMPs) have emerged as promising candidates in combating antimicrobial resistance – a growing issue in healthcare. However, to develop AMPs into effective therapeutics, a thorough analysis and extensive investigations are essential. In this study, we employed an in silico approach to design cationic AMPs de novo, followed by their experimental testing. The antibacterial potential of de novo designed cationic AMPs, along with their synergistic properties in combination with conventional antibiotics was examined. Furthermore, the effects of bacterial inoculum density and metabolic state on the antibacterial activity of AMPs were evaluated. Finally, the impact of several potent AMPs on E. coli cell envelope and genomic DNA integrity was determined. Collectively, this comprehensive analysis provides insights into the unique characteristics of cationic AMPs.

SYNTHESIS AND BIOLOGICAL COMPARISON OF TWO ANTIMICROBIAL PEPTIDES ORIGINATED FROM THE VENOM OF VESPA CRABRO AND POLYBIA PAULISTA

Bee venom is abundant in potential antimicrobial peptides, AMPs, . Among them, Mastoparan C and Polybia-MP1 are two well-known AMPs that were found in the European Hornet Vespa crabro and social wasp Polybia paulista, respectively. Due to the potent antimicrobial activity toward a wide range of microbial pathogens, including Gram-negative, Gram-positive and fungal species, they are promising to tackle the global antimicrobial resistance crisis. Thus, understanding the structure and activity relationships (SARs) is essential for the development of potential therapeutic applications. In this study, we synthesized and compared the differences in biological properties of these two antimicrobial peptides. The preliminary data suggests various important information regarding the structural requirements for optimizing the pharmacological properties of antimicrobial peptides.

Anionic liposome formulation for oral delivery of thuricin CD, a potential antimicrobial peptide therapeutic

Thuricin CD is a two-peptide antimicrobial produced by Bacillus thuringiensis. Unlike previous antibiotics, it has shown narrow spectrum activity against Clostridioides difficile, a bacterium capable of causing infectious disease in the colon. However, peptide antibiotics have stability, solubility, and permeability problems that can affect their performance in vivo. This work focuses on the bioactivity and bioavailability of thuricin CD with a view to developing a formulation for delivery of active thuricin CD peptides through the gastrointestinal tract (GIT) for local delivery in the colon. The results indicate that thuricin CD is active at low concentrations only when both peptides are present. While thuricin CD was degraded by proteases and was unstable and poorly soluble in gastric fluid, it showed increased solubility in intestinal fluid, probably due to micelle encapsulation. Based on this, thuricin CD was encapsulated in anionic liposomes, which showed increased activity compared to the free peptide, maintained activity after exposure to pepsin in gastric fluid and intestinal fluid, was stable in suspension for over 21 days at room temperature and for 60 days at 4 °C, and exhibited no toxicity to epithelial intestinal cells. These findings suggest that an anionic lipid-based nano formulation may be a promising approach for local oral delivery of thuricin CD.

Accelerating Antimicrobial Peptide Discovery for WHO Priority Pathogens through Predictive and Interpretable Machine Learning Models.

The escalating menace of multidrug-resistant (MDR) pathogens necessitates a paradigm shift from conventional antibiotics to innovative alternatives. Antimicrobial peptides (AMPs) emerge as a compelling contender in this arena. Employing in silico methodologies, we can usher in a new era of AMP discovery, streamlining the identification process from vast candidate sequences, thereby optimizing laboratory screening expenditures. Here, we unveil cutting-edge machine learning (ML) models that are both predictive and interpretable, tailored for the identification of potent AMPs targeting World Health Organization's (WHO) high-priority pathogens. Furthermore, we have developed ML models that consider the hemolysis of human erythrocytes, emphasizing their therapeutic potential. Anchored in the nuanced physical-chemical attributes gleaned from the three-dimensional (3D) helical conformations of AMPs, our optimized models have demonstrated commendable performance-boasting an accuracy exceeding 75% when evaluated against both low-sequence-identified peptides and recently unveiled AMPs. As a testament to their efficacy, we deployed these models to prioritize peptide sequences stemming from PEM-2 and subsequently probed the bioactivity of our algorithm-predicted peptides vis-à-vis WHO's priority pathogens. Intriguingly, several of these new AMPs outperformed the native PEM-2 in their antimicrobial prowess, thereby underscoring the robustness of our modeling approach. To elucidate ML model outcomes, we probe via Shapley Additive exPlanations (SHAP) values, uncovering intricate mechanisms guiding diverse actions against bacteria. Our state-of-the-art predictive models expedite the design of new AMPs, offering a robust countermeasure to antibiotic resistance. Our prediction tool is available to the public at https://ai-meta.chem.ncu.edu.tw/amp-meta.

Insights into the Adsorption Mechanisms of the Antimicrobial Peptide CIDEM-501 on Membrane Models.

CIDEM-501 is a hybrid antimicrobial peptide rationally designed based on the structure of panusin and panulirin template peptides. The new peptide exhibits significant antibacterial activity against multidrug-resistant pathogens (MIC = 2-4 μM) while conserving no toxicity in human cell lines. We conducted molecular dynamics (MD) simulations using the CHARMM-36 force field to explore the CIDEM-501 adsorption mechanism with different membrane compositions. Several parameters that characterize these interactions were analyzed to elucidate individual residues' structural and thermodynamic contributions. The membrane models were constructed using CHARMM-GUI, mimicking the bacterial and eukaryotic phospholipid compositions. Molecular dynamics simulations were conducted over 500 ns, showing rapid and highly stable peptide adsorption to bacterial lipids components rather than the zwitterionic eucaryotic model membrane. A predominant peptide orientation was observed in all models dominated by an electric dipole. The peptide remained parallel to the membrane surface with the center loop oriented to the lipids. Our findings shed light on the antibacterial activity of CIDEM-501 on bacterial membranes and yield insights valuable for designing potent antimicrobial peptides targeting multi- and extreme drug-resistant bacteria.

Rational Design of a Potent Antimicrobial Peptide Based on the Active Region of a Gecko Cathelicidin.

The emergence of multidrug-resistant (MDR) bacteria presents a significant challenge to public health, increasing the risk of infections that are resistant to current antibiotic treatment. Antimicrobial peptides (AMPs) offer a promising alternative to conventional antibiotics in the prevention of MDR bacterial infections. In the present study, we identified a novel cathelicidin AMP from Gekko japonicus, which exhibited broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria, with minimal inhibitory concentrations ranging from 2.34 to 4.69 μg/mL. To improve its potential therapeutic application, a series of peptides was synthesized based on the active region of the gecko-derived cathelicidin. The lead peptide (RH-16) showed an antimicrobial activity comparable to that of the parent peptide. Structural characterization revealed that RH-16 adopted an amphipathic α-helical conformation. Furthermore, RH-16 demonstrated neither hemolytic nor cytotoxic activity but effectively killed a wide range of clinically isolated, drug-resistant bacteria. The antimicrobial activity of RH-16 was attributed to the nonspecific targeting of bacterial membranes, leading to rapid bacterial membrane permeabilization and rupture. RH-16 also retained its antibacterial activity in plasma and exhibited mild toxicity in vivo. Notably, RH-16 offered robust protection against skin infection in a murine model. Therefore, this newly identified cathelicidin AMP may be a strong candidate for future pharmacological development targeting multidrug resistance. The use of a rational design approach for isolating the minimal antimicrobial unit may accelerate the transition of natural AMPs to clinically applicable antibacterial agents.

Computational Study of Antimicrobial Peptides for Promising Therapeutic Applications Against Methicillin-resistant Staphylococcus Aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a causative agent for multiple drug-resistant diseases and is a prime health concern. Currently, antibiotics like vancomycin, daptomycin, fluoroquinolones, linezolid, fifth-generation cephalosporin and others are available in the market for the treatment of MRSA infection. With the increasing prevalence of drug-resistant cases, researchers are actively investigating alternative strategies to combat MRSA, including the exploration of peptide therapeutics. This study employed computational methods to prospect for potential Antimicrobial Peptides (AMPs). A total of One hundred and fifty antimicrobial peptides were explored based on physicochemical properties. The results showed that Clavanin B was the most appropriate candidate. Molecular Docking and Molecular Dynamics Simulation results showed the protein-peptide interaction of the MRSA target proteins, Penicillin Binding Protein 2a and Panton-Valentine Leukocidin Toxin, with the Antimicrobial Peptide Clavanin B. Currently, the antimicrobial peptide database highlights Clavanin B's role as an anti-HIV peptide. Moreover, this investigation proposes Clavanin B as a viable repurposed drug for treating MRSA, underscoring its potential deployment in the management of MRSA infections.

Reducing Self-Assembly by Increasing Net Charge: Effect on Biological Activity of Mastoparan C.

The ability of amphipathic peptides to arrange themselves in aqueous solutions, known as self-assembly, has been found to reduce the effectiveness of these peptides in interacting with cell membranes. Therefore, minimizing their tendency to self-assemble could be a potential strategy for enhancing the pharmacological properties of antimicrobial peptides (AMPs). To explore this idea, this study prepared a series of natural peptides mastoparan C (MPC) with increased net charge and hydrophilicity via alanine-to-lysine substitution and investigated the impact on the biological activity. The preliminary data suggested the influence of both the overall positive charge and the position of a lysine residue on the self-assembly of MPC and its derivatives. Besides, the analogue MPC-A5K,A8K displayed higher anticancer activity and comparable antimicrobial activity with significantly lower hemolysis than MPC. Hence, reducing self-assembly by expanding the cationic area could be a promising approach for developing potent and selective AMPs.

Mimicking Nonribosomal Peptides from the Marine Actinomycete Streptomyces sp. H-KF8 Leads to Antimicrobial Peptides.

Microorganisms within the marine environment have been shown to be very effective sources of naturally produced antimicrobial peptides (AMPs). Several nonribosomal peptides were identified based on genome mining predictions of Streptomyces sp. H-KF8, a marine Actinomycetota isolated from a remote Northern Chilean Patagonian fjord. Based on these predictions, a series of eight peptides, including cyclic peptides, were designed and chemically synthesized. Six of these peptides showed antimicrobial activity. Mode of action studies suggest that two of these peptides potentially act on the cell membrane via a novel mechanism allowing the passage of small ions, resulting in the dissipation of the membrane potential. This study shows that though structurally similar peptides, determined by NMR spectroscopy, the incorporation of small sequence mutations results in a dramatic influence on their bioactivity including mode of action. The qualified hit sequence can serve as a basis for more potent AMPs in future studies.