Potent Antimicrobial Peptides Research Articles

Hyaluronic acid based nanoparticles that mediate sustained thanatin release protect against NDM-1–resistant bacterial infections in a murine model

Thanatin, a potent cationic antimicrobial peptide, has demonstrated remarkable efficacy against new NDM-1 producing bacteria. However, its clinical application is hampered by suboptimal stability in circulation and limited bioavailability in the human body. To overcome these challenges, a novel thanatin nanomedicine has been developed, which encapsulated thanatin in nanoparticles formed by electrostatic interactions between negatively charged HA and PLGA. The obtained ThaNPs demonstrated good stability, low cytotoxicity, and good metabolic ratio. ThaNPs significantly improve the stability of thanatin in the circulation, increasing its half-life in 50 % serum from 0.6 h to 3.2 h. Notably, the protective effect of ThaNPs against sepsis induced by NDM-1–producing Escherichia coli. was 10-fold higher than that of unencapsulated thanatin. These findings suggest that hyaluronic acid-based nanoparticles have the potentiality to overcome the clinical limitations associated with cationic antimicrobial peptides, thereby providing a novel and effective strategy for treating severe infections caused by antibiotic-resistant bacteria.

Unlocking the Potential of Antimicrobial Maximin Peptides From Bombina maxima Against Staphylococcus aureus: Deciphering Their Mode of Action Through a Mimetic Bacterial Membrane Environment

ABSTRACTAntimicrobial peptides (AMPs) offer a promising strategy to address bacterial resistance by targeting bacterial membranes, bypassing the limitations of receptor site‐based approaches. This study focuses on combating the notorious multidrug resistance of Staphylococcus aureus using AMPs, particularly maximin peptides derived from Bombina maxima. Previous research suggested that maximin peptides could disrupt bacterial membranes among anuran AMPs. This prompted us to screen these maximin peptides to identify those with strong membrane‐targeting abilities against S. aureus. Initially, stability and activity assessments on all 89 peptides involved analyzing hydrogen bond dilution, peptide permeation, and hemolytic activity predictions, leading to the rationalization of four promising candidates: Max_5, Max_13, Max_21, and Max_45. When subjected to membrane simulations, the monomeric state of these peptides displayed partial helix‐coil transitions with significant structural interactions that disrupted the membrane, particularly for Max_5 and Max_13. Additionally, the multimeric states of these two peptides were examined through membrane simulations to elucidate their mechanisms of action. Analyses focusing on membrane thickness, lipid distortions, and curvature revealed that both Max_5 and Max_13 exerted strong membrane‐rupturing effects. These peptides seemed to operate by forming pores, facilitating lipid diffusion, creating cavities, and affecting membrane thickness, which allowed water penetration due to increased membrane fluidity, indicating the barrel‐stave pore model. Despite structural differences between Max_5 and Max_13, both peptides demonstrated similar outcomes, emphasizing their potential for future therapeutic applications. This study highlights the efficacy of computational methods in accelerating the identification of potent antimicrobial peptides, providing a pathway for developing novel antimicrobial therapies.

Antibacterial peptides from black soldier fly (Hermetia illucens) larvae: mode of action and characterization

Antibacterial peptides from black soldier fly larvae extract were prepared using Flash column chromatography. Three out of five fractions (F2, F3 and F4) showed antibacterial activity against Listeria monocytogenes DMST 17303 with a minimum inhibitory concentration (MIC) of 1 mM, followed by Salmonella enterica Enteritidis DMST 15679 and Escherichia coli O157:H7 DMST 12743 with a MIC ranging from 4 to 8 mM. Due to the higher yield, F2 and F3 were further analyzed on their mode of action against L. monocytogenes DMST 17303. Both fractions, particularly F2, exerted antibacterial activity through inducing bacterial cell membrane disintegration and interaction with intracellular compounds including fatty acids, proteins, and nucleic acids. The F3 did not show significant hemolytic activity up to 4 mM, while F2 showed lower than 5% hemolysis up to 8 mM. Time-to-kill analysis revealed that F2 was more effective and exerted a sustainable killing effect after 2 and 4 h, depending on the concentration of 1 and 2×MIC, respectively, while the F3 at 2×MIC could completely kill the test bacteria within 24 h. Among the identified peptides in the fractions, those with charged, either positively or negatively, and moderate hydrophobicity of ranging 6.68–15.70, namely CGPPRQGPFPR, HLEEELK, LEEAEERAD, TEELEEAKKK, and KGNSELEEAKKK, are potential antimicrobial peptides.

Proteomic analysis of psychedelic mushroom isolate and exploring potential antimicrobial peptides against bacteria

Psychedelic mushrooms belonging to basidiomycota have gained prominence in research due to the range of hallucinogenic compounds. To combat the challenge of antimicrobial resistance, exploring alternative antimicrobial peptides has become crucial. In this study, we present the proteomic analysis of psychedelic mushroom. Psilocybe cubensis was identified by molecular characterisation using the ITS1 and ITS4 regions. Subsequently, LC-MS/MS and gene ontology analyses were used to characterise the proteome of P. cubensis. The proteomic analysis unveiled several antimicrobial peptides. The results revealed favourable binding scores, suggesting the potential efficacy of these peptides against Staphylococcus aureus. Hence the inhibition of bacterial growth, supporting the antimicrobial properties of the identified peptides. In our findings, the individual peptides from P. cubensis against S. aureus suggest a promising avenue for the discovery of novel antimicrobial peptides.

Dimerization and lysine substitution of melittin have differing effects on bacteria.

Melittin is a potent antimicrobial peptide from bee venom that is effective against both Gram-positive and Gram-negative bacteria. However, it is extremely toxic to mammalian cells and, as yet, has no clinical use. Modifications to its amino acid sequence, cyclization, truncation, and dimerization have been attempted in order to reduce its toxicity whilst maintaining its antimicrobial activity. In this study, we targeted the three lysine residues present in melittin and substituted them with lysine homologs containing shorter side chains (ornithine, Orn, diaminobutyric acid, Dab, and diaminopropanoic acid, Dap) and made both parallel and antiparallel melittin dimers to observe how lysine substitution and dimerization affects its activity and toxicity. The antibacterial activity of melittin and its analogs was tested against S. aureus (Gram-positive bacteria) and E. coli (Gram-negative bacteria), and cytotoxicity was tested against the mammalian cell lines HEK293 and H4IIE. Overall, dimerization and lysine substitution exhibited improved antimicrobial activity toward E. coli and limited improvement toward S. aureus. However, mammalian cell toxicity was only marginally reduced compared to native melittin. Interestingly, the parallel dimer was found to be marginally more active than the antiparallel dimer, indicating orientation maybe important for activity, although both dimers were less effective than the native and Lys-analog peptides toward S. aureus. Of the Lys substitutions, Dab and Dap improved melittin's activity toward E. coli. Dimerization and Lys substitution of melittin improved the antimicrobial activity toward Gram-negative bacteria but did not significantly improve its activity toward Gram-positive bacteria. Some analogs also displayed reduced toxicity toward HEK293 and H4IIE cells but overall remained toxic at bactericidal concentrations. Our data indicates that although highly antibacterial, melittin's toxicity is the major drawback in its potential use.

Influence of enzymatic hydrolysis conditions on antimicrobial activities and peptide profiles of milk protein-derived hydrolysates from white wastewater

The focus of our investigation lies in the hydrolysis of milk proteins found in the substantial wastewater generated by the dairy industry, particularly in the white and cleaning wastewater resulting from rinsing and cleaning-in-place processes in order to enhance the value of dairy constituents by producing a diverse population of peptides, including potential antimicrobial peptides generated by 4 different enzymes: pepsin, trypsin, pronase E, and thermolysin. The protein/peptide content was influenced by the degree of hydrolysis values ranging from 2 % to 13 %, and UPLC-MS/MS characterization reveals distinct peptide sequences in enzymatic hydrolysates. The impact of hydrolysis time was also examined, revealing significant differences between 30 minutes and 240 minutes, with 555 peptides identified at 30 minutes, increasing to 693 at 240 minutes. After 4 hours, grouping showed variations: 181 peptides (thermolysin), 153 (pepsin), 126 (trypsin), and 83 (pronase E). A comparative analysis of the characterized sequences with antimicrobial peptides databases identified 17 antimicrobial peptides after 240 minutes pronase E and thermolysin hydrolysis, though they were insufficient to inhibit strains like Clostridium tyrobutyricum and Pseudomonas aeruginosa. Antimicrobial assays also revealed that peptides from Pronase T30, Pronase T240, and Thermolysine T240 exhibited antifungal activity against Mucor racemosus (MIC 2.5 mg/mL), but none against Penicillium commune. The effet of enzymatic hydrolysis demonstrated in this study highlights, for the first time, the potential for valorizing dairy white wastewater within the context of a circular economy framework.

A Foundation Model Identifies Broad-Spectrum Antimicrobial Peptides against Drug-Resistant Bacterial Infection

Development of potent and broad-spectrum antimicrobial peptides (AMPs) could help overcome the antimicrobial resistance crisis. We develop a peptide language-based deep generative framework (deepAMP) for identifying potent, broad-spectrum AMPs. Using deepAMP to reduce antimicrobial resistance and enhance the membrane-disrupting abilities of AMPs, we identify, synthesize, and experimentally test 18 T1-AMP (Tier 1) and 11 T2-AMP (Tier 2) candidates in a two-round design and by employing cross-optimization-validation. More than 90% of the designed AMPs show a better inhibition than penetratin in both Gram-positive (i.e., S. aureus) and Gram-negative bacteria (i.e., K. pneumoniae and P. aeruginosa). T2-9 shows the strongest antibacterial activity, comparable to FDA-approved antibiotics. We show that three AMPs (T1-2, T1-5 and T2-10) significantly reduce resistance to S. aureus compared to ciprofloxacin and are effective against skin wound infection in a female wound mouse model infected with P. aeruginosa. In summary, deepAMP expedites discovery of effective, broad-spectrum AMPs against drug-resistant bacteria.

Immunometabolic interplay in Edwardsiella tarda-infected crucian carp (Carassius auratus) and in vitro identification of the antimicrobial activity of apolipoprotein D (ApoD) by utilization of multiomics analyses

Edwardsiella tarda is an intracellular pathogenic bacteria that can imperil the health of farmed fish. However, the interactive networks of immune regulation and metabolic response in E. tarda-infected fish are still unclear. In this investigation, we aimed to explore immunometabolic interplay in crucian carp after E. tarda infection by utilizing multiomics analyses. Crucian carp (Carassius auratus) receiving E. tarda infection showed increased levels of tissue damage and oxidative injury in liver. Multiomics analyses suggested that carbon and amino acid metabolism may be considered as crucial metabolic pathways in liver of crucian carp following E. tarda infection, while spaglumic acid, isocitric acid and tetrahydrocortisone were the crucial liver biomarkers. After that, a potential antimicrobial peptide (AMP) sequence called apolipoprotein D (ApoD) was identified from omics study. Then, tissue-specific analysis indicated that liver CaApoD showed the highest expression among isolated tissues. After Aeromonas hydrophila stimulated, CaApoD expressions increased sharply in immune-related tissues. Moreover, CaApoD fusion protein could mediate the in vitro binding to A. hydrophila and E. tarda, attenuate bacterial growth as well as diminish bacterial biofilm forming activity. These findings may have a comprehensive implication for understanding immunometabolic response in crucian carp upon infection.

Protein Language Models and Machine Learning Facilitate the Identification of Antimicrobial Peptides.

Peptides are bioactive molecules whose functional versatility in living organisms has led to successful applications in diverse fields. In recent years, the amount of data describing peptide sequences and function collected in open repositories has substantially increased, allowing the application of more complex computational models to study the relations between the peptide composition and function. This work introduces AMP-Detector, a sequence-based classification model for the detection of peptides' functional biological activity, focusing on accelerating the discovery and de novo design of potential antimicrobial peptides (AMPs). AMP-Detector introduces a novel sequence-based pipeline to train binary classification models, integrating protein language models and machine learning algorithms. This pipeline produced 21 models targeting antimicrobial, antiviral, and antibacterial activity, achieving average precision exceeding 83%. Benchmark analyses revealed that our models outperformed existing methods for AMPs and delivered comparable results for other biological activity types. Utilizing the Peptide Atlas, we applied AMP-Detector to discover over 190,000 potential AMPs and demonstrated that it is an integrative approach with generative learning to aid in de novo design, resulting in over 500 novel AMPs. The combination of our methodology, robust models, and a generative design strategy offers a significant advancement in peptide-based drug discovery and represents a pivotal tool for therapeutic applications.

Peptide PaDBS1R6 has potent antibacterial activity on clinical bacterial isolates and integrates an immunomodulatory peptide fragment within its sequence

BackgroundResistant infectious diseases caused by gram-negative bacteria are among the most serious worldwide health problems. Antimicrobial peptides (AMPs) have been explored as promising antibacterial, antibiofilm, and anti-infective candidates to address these health challenges. Major conclusionsHere we report the potent antibacterial effect of the peptide PaDBS1R6 on clinical bacterial isolates and identify an immunomodulatory peptide fragment incorporated within it. PaDBS1R6 was evaluated against Acinetobacter baumannii and Escherichia coli clinical isolates and had minimal inhibitory concentration (MIC) values from 8 to 32 μmol L−1. It had a rapid bactericidal effect, with eradication showing within 3 min of incubation, depending on the bacterial strain tested. In addition, PaDBS1R6 inhibited biofilm formation for A. baumannii and E. coli and was non-toxic toward healthy mammalian cells. These findings are explained by the preference of PaDBS1R6 for anionic membranes over neutral membranes, as assessed by surface plasmon resonance assays and molecular dynamics simulations. Considering its potent antibacterial activity, PaDBS1R6 was used as a template for sliding-window fr agmentation studies (window size = 10 residues). Among the sliding-window fragments, PaDBS1R6F8, PaDBS1R6F9, and PaDBS1R6F10 were ineffective against any of the bacterial strains tested. Additional biological assays were conducted, including nitric oxide (NO) modulation and wound scratch assays, and the R6F8 peptide fragment was found to be active in modulating NO levels, as well as having strong wound healing properties. General significanceThis study proposes a new concept whereby peptides with different biological properties can be derived by the screening of fragments from within potent AMPs.

A Novel Bacitracin-like Peptide from Mangrove-Isolated Bacillus paralicheniformis NNS4-3 against MRSA and Its Genomic Insights.

The global rise of antimicrobial resistance (AMR) presents a critical challenge necessitating the discovery of novel antimicrobial agents. Mangrove microbes are valuable sources of new antimicrobial compounds. This study reports the discovery of a potent antimicrobial peptide (AMP) from Bacillus paralicheniformis NNS4-3, isolated from mangrove sediment, exhibiting significant activity against methicillin-resistant Staphylococcus aureus (MRSA). The AMP demonstrated a minimum inhibitory concentration ranging from 1 to 16 µg/mL in the tested bacteria and exhibited bactericidal effects at higher concentrations. Structural analysis revealed a bacitracin-like configuration and the peptide acted by disrupting bacterial membranes in a time- and concentration-dependent manner. The AMP maintained stability under heat, proteolytic enzymes, surfactants, and varying pH treatments. The ten biosynthetic gene clusters (BGCs) of secondary metabolites were found in the genome. Detailed sequence comparison of the predicted bacitracin BGC indicated distinct DNA sequences compared to previously reported strains. Although the antibiotic resistance genes were found, this strain was susceptible to antibiotics. Our findings demonstrated the potential of Bacillus paralicheniformis NNS4-3 and its AMP as a promising agent in combating AMR. The genetic information could be pivotal for future applications in the healthcare industry, emphasizing the need for continued exploration of marine microbial diversity in drug discovery.

Identification and Design of Novel Potential Antimicrobial Peptides Targeting Mycobacterial Protein Kinase PknB

Antimicrobial peptides have gradually gained advantages over small molecule inhibitors for their multifunctional effects, synthesising accessibility and target specificity. The current study aims to determine an antimicrobial peptide to inhibit PknB, a serine/threonine protein kinase (STPK), by binding efficiently at the helically oriented hinge region. A library of 5626 antimicrobial peptides from publicly available repositories has been prepared and categorised based on the length. Molecular docking using ADCP helped to find the multiple conformations of the subjected peptides. For each peptide served as input the tool outputs 100 poses of the subjected peptide. To maintain an efficient binding for relatively a longer duration, only those peptides were chosen which were seen to bind constantly to the active site of the receptor protein over all the poses observed. Each peptide had different number of constituent amino acid residues; the peptides were classified based on the length into five groups. In each group the peptide length incremented upto four residues from the initial length form. Five peptides were selected for Molecular Dynamic simulation in Gromacs based on higher binding affinity. Post-dynamic analysis and the frame comparison inferred that neither the shorter nor the longer peptide but an intermediate length of 15 mer peptide bound well to the receptor. Residual substitution to the selected peptides was performed to enhance the targeted interaction. The new complexes considered were further analysed using the Elastic Network Model (ENM) for the functional site’s intrinsic dynamic movement to estimate the new peptide’s role. The study sheds light on prospects that besides the length of peptides, the combination of constituent residues equally plays a pivotal role in peptide-based inhibitor generation. The study envisages the challenges of fine-tuned peptide recovery and the scope of Machine Learning (ML) and Deep Learning (DL) algorithm development. As the study was primarily meant for generation of therapeutics for Tuberculosis (TB), the peptide proposed by this study demands meticulous invitro analysis prior to clinical applications.Graphical

Characterising the Tasmanian devil (Sarcophilus harrisii) pouch microbiome in lactating and non-lactating females

Wildlife harbour a diverse range of microorganisms that affect their health and development. Marsupials are born immunologically naïve and physiologically underdeveloped, with primary development occurring inside a pouch. Secretion of immunological compounds and antimicrobial peptides in the epithelial lining of the female’s pouch, pouch young skin, and through the milk, are thought to boost the neonate’s immune system and potentially alter the pouch skin microbiome. Here, using 16S rRNA amplicon sequencing, we characterised the Tasmanian devil pouch skin microbiome from 25 lactating and 30 non-lactating wild females to describe and compare across these reproductive stages. We found that the lactating pouch skin microbiome had significantly lower amplicon sequence variant richness and diversity than non-lactating pouches, however there was no overall dissimilarity in community structure between lactating and non-lactating pouches. The top five phyla were found to be consistent between both reproductive stages, with over 85% of the microbiome being comprised of Firmicutes, Proteobacteria, Fusobacteriota, Actinobacteriota, and Bacteroidota. The most abundant taxa remained consistent across all taxonomic ranks between lactating and non-lactating pouch types. This suggests that any potential immunological compounds or antimicrobial peptide secretions did not significantly influence the main community members. Of the more than 16,000 total identified amplicon sequence variants, 25 were recognised as differentially abundant between lactating and non-lactating pouches. It is proposed that the secretion of antimicrobial peptides in the pouch act to modulate these microbial communities. This study identifies candidate bacterial clades on which to test the activity of Tasmanian devil antimicrobial peptides and their role in pouch young protection, which in turn may lead to future therapeutic development for human diseases.

Molecular Dynamics Simulations Help Determine the Molecular Mechanisms of Lasioglossin-III and Its Variant Peptides' Membrane Interfacial Interactions.

Lasioglossin-III (LL-III) is a potent broad-spectrum antimicrobial peptide used in diverse antimicrobial applications. In this work, coarse-grained and all-atom molecular dynamics simulation strategies were used in tandem to interpret the molecular mechanisms involved in the interfacial dynamics of LL-III and its recombinant variants during interactions with diverse cell membrane systems. Our results indicate that the membrane charges act as the driving force for initiating the membrane-peptide interactions, while the hydrophobic or van der Waals forces help to reinforce the membrane-peptide bindings. The optimized charge-hydrophobicity ratio of the LL-III peptides helps ensure their high specificity toward bacterial membranes compared to mammalian membrane systems, which also helps explain our experimental observations. Overall, we hope that our work gives new insight into the antimicrobial action of LL-III peptides and that the adopted simulation strategy will help other scientists and engineers extract maximal information from complex molecular simulations using minimal computational power.

Facile Halogenation of Antimicrobial Peptides As Demonstrated by Producing Bromotryptophan-Labeled Nisin Variants with Enhanced Antimicrobial Activity.

Antimicrobial peptides (AMPs) have raised significant interest, forming a potential new class of antibiotics in the fight against multi-drug-resistant bacteria. Various AMPs are ribosomally synthesized and post-translationally modified peptides (RiPPs). One post-translational modification found in AMPs is the halogenation of Trp residues. This modification has, for example, been shown to be critical for the activity of the potent AMP NAI-107 from Actinoallomurus. Due to the importance of organohalogens, establishing methods for facile and selective halogen atom installation into AMPs is highly desirable. In this study, we introduce an expression system utilizing the food-grade strain Lactococcus lactis, facilitating the efficient incorporation of bromo-Trp (BrTrp) into (modified) peptides, exemplified by the lantibiotic nisin with a single Trp residue or analogue incorporated at position 1. This provides an alternative to the challenges posed by halogenase enzymes, such as poor substrate selectivity. Our method yields expression levels comparable to that of wild-type nisin, while BrTrp incorporation does not interfere with the post-translational modifications of nisin (dehydration and cyclization). One brominated nisin variant exhibits a 2-fold improvement in antimicrobial activity against two tested pathogens, including a WHO priority pathogen, while maintaining the same lipid II binding and bactericidal activity as wild-type nisin. The work presented here demonstrates the potential of this methodology for peptide halogenation, offering a new avenue for the development of diverse antimicrobial products labeled with BrTrp.

Isolation of peptide mixtures with potential biological activities from the protein hydrolysate of Arsenura armida larval meal

Abstract This study explores using insects as a high-quality protein source rich in bioactive peptides for human nutrition. The objectives include establishing optimal conditions for peptide extraction, identifying their sequences, and assessing their potential as antioxidants and antimicrobial agents in Arsenura armida flour. The protein content in the A. armida flour extract was 54%. To obtain potential antioxidant and antimicrobial peptides, a 10:1 and 1:1 enzyme (pepsin)/substrate ratio was employed, with digestion times of 2 h (A2) and 1 h (A4), respectively. The IC50 value for the antioxidant assay DPPH (2,2-Difenil-1-Picrilhidrazilo) was 22.5 μg/μL for A2. In the antioxidant in vivo assay with Caenorhabditis elegans, A2 peptides at concentrations of 0.75 μg/μL and 1.5 μg/μL exhibited a survival percentage ranging from 32.6% to 35.3%. The A4 sample demonstrated the highest antimicrobial activity, inhibiting the growth of Gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus) with MIC (minimum inhibitory concentration) values of 0.11 μg/mL and 0.23 μg/mL and MBC (minimum bactericidal concentration) of 0.23 μg/mL for both. Peptide analysis revealed that A2 contained peptides with identified antihypertensive (37%) and antioxidant (58%) activities, while A4 comprised peptides with antihypertensive (62%), antioxidant (31%), and antimicrobial (2%) properties. The antimicrobial peptides identified through the BIOPEP database were LFGF and FLLF. This investigation underscores the potential of A. armida peptides as valuable components in functional foods or nutraceuticals, offering health benefits beyond basic nutrition.

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.