Antimicrobial spectrum against wound pathogens and cytotoxicity of star-arranged poly-l-lysine-based antimicrobial peptide polymers.

Introduction. As growing numbers of patients are at higher risk of infection, novel topical broad-spectrum antimicrobials are urgently required for wound infection management. Robust pre-clinical studies should support the development of such novel antimicrobials.Gap statement. To date, evidence of robust investigation of the cytotoxicity and antimicrobial spectrum of activity of antimicrobial peptides (AMP)s is lacking in published literature. Using a more clinical lens, we address this gap in experimental approach, building on our experience with poly-l-lysine (PLL)-based AMP polymers.Aim. To evaluate the in vitro bactericidal activity and cytotoxicity of a PLL-based 16-armed star AMP polymer, designated 16-PLL10, as a novel candidate antimicrobial.Methods. Antimicrobial susceptibilities of clinical isolates and reference strains of ESKAPE (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) pathogens, to 16-PLL10 were investigated. Human erythrocyte haemolysis and keratinocyte viability assays were used to assess toxicity. Modifications were made to 16-PLL10 and re-evaluated for improvement.Results. Minimum bactericidal concentration of 16-PLL10 ranged from 1.25 µM to ≥25 µM. At 2.5 µM, 16-PLL10 was broadly bactericidal against ESKAPE strains/wound isolates. Log-reduction in colony forming units (c.f.u.) per millilitre after 1 h, ranged from 0.3 (E. cloacae) to 5.6 (K. pneumoniae). At bactericidal concentrations, 16-PLL10 was toxic to human keratinocyte and erythrocytes. Conjugates of 16-PLL10, Trifluoroacetylated (TFA)-16-PLL10, and Poly-ethylene glycol (PEG)ylated 16-PLL10, synthesised to address toxicity, only moderately reduced cytotoxicity and haemolysis.Conclusions. Due to poor selectivity indices, further development of 16-PLL10 is unlikely warranted. However, considering the unmet need for novel topical antimicrobials, the ease of AMP polymer synthesises/modification is attractive. To support more rational development, prioritising clinically relevant pathogens and human cells, to establish selective toxicity profiles in vitro, is critical. Further characterisation and discovery utilising artificial intelligence and computational screening approaches can accelerate future AMP nanomaterial development.

Thanatin: A Promising Antimicrobial Peptide Targeting the Achilles' Heel of Multidrug-Resistant Bacteria.

Antimicrobial resistance poses an escalating threat to human health, necessitating the development of novel antimicrobial agents capable of addressing challenges posed by antibiotic-resistant bacteria. Thanatin, a 21-amino acid ß-hairpin insect antimicrobial peptide featuring a single disulfide bond, exhibits broad-spectrum antibacterial activity, particularly effective against multidrug-resistant strains. The outer membrane biosynthesis system is recognized as a critical vulnerability in antibiotic-resistant bacteria, which thanatin targets to exert its antimicrobial effects. This peptide holds significant promise for diverse applications. This review begins with an examination of the structure-activity relationship and synthesis methods of thanatin. Subsequently, it explores thanatin's antimicrobial activity, detailing its various mechanisms of action. Finally, it discusses prospective clinical, environmental, food, and agricultural applications of thanatin, offering valuable insights for future research endeavors.

The Archetypal Gamma-Core Motif of Antimicrobial Cys-Rich Peptides Inhibits H+-ATPases in Target Pathogens.

Human lactoferrin (hLf) is an innate host defense protein that inhibits microbial H+-ATPases. This protein includes an ancestral structural motif (i.e., γ-core motif) intimately associated with the antimicrobial activity of many natural Cys-rich peptides. Peptides containing a complete γ-core motif from hLf or other phylogenetically diverse antimicrobial peptides (i.e., afnA, SolyC, PA1b, PvD1, thanatin) showed microbicidal activity with similar features to those previously reported for hLf and defensins. Common mechanistic characteristics included (1) cell death independent of plasma membrane (PM) lysis, (2) loss of intracellular K+ (mediated by Tok1p K+ channels in yeast), (3) inhibition of microbicidal activity by high extracellular K+, (4) influence of cellular respiration on microbicidal activity, (5) involvement of mitochondrial ATP synthase in yeast cell death processes, and (6) increment of intracellular ATP. Similar features were also observed with the BM2 peptide, a fungal PM H+-ATPase inhibitor. Collectively, these findings suggest host defense peptides containing a homologous γ-core motif inhibit PM H+-ATPases. Based on this discovery, we propose that the γ-core motif is an archetypal effector involved in the inhibition of PM H+-ATPases across kingdoms of life and contributes to the in vitro microbicidal activity of Cys-rich antimicrobial peptides.

Antibacterial and Anti-Inflammatory Activity of Branched Peptides Derived from Natural Host Defense Sequences.

Antibiotic resistance is a major global health threat, necessitating the development of new treatments and diverse molecules to combat severe infections and preserve the efficacy of existing drugs. Antimicrobial peptides (AMPs) offer a versatile arsenal against bacteria, and peptide structure branching can enhance their resistance to proteases and improve their overall efficacy. A small library of peptides derived from natural host defense peptides and synthesized in a tetrabranched form was selected against E. coli. Six selected branched peptides were further studied for antibacterial activity against a panel of strains, biofilm inhibition, protease resistance, and cytotoxicity. Their structure was predicted computationally and their mechanism of action was investigated by electron microscopy and by using fluorescent dyes. The peptide BAMP2 showed promise in a mouse skin infection model, indicating the potential for local infection treatment.

Antimicrobial peptide-based strategies to overcome antimicrobial resistance.

Antibiotic resistance has emerged as a global threat, rendering the existing conventional treatment strategies ineffective. In view of this, antimicrobial peptides (AMPs) have proven to be potent alternative therapeutic interventions with a wide range of applications in clinical health. AMPs are small peptides produced naturally as a part of the innate immune responses against a broad range of bacterial, fungal and viral pathogens. AMPs present a myriad of advantages over traditional antibiotics, including their ability to target multiple sites, reduced susceptibility to resistance development, and high efficacy at low doses. These peptides have demonstrated notable potential in inhibiting microbes resistant to traditional antibiotics, including the notorious ESKAPE pathogens, recognized as the primary culprits behind nosocomial infections. AMPs, with their multifaceted benefits, emerge as promising candidates in the ongoing efforts to combat the escalating challenges posed by antibiotic resistance. This in-depth review provides a detailed discussion on AMPs, encompassing their classification, mechanism of action, and diverse clinical applications. Focus has been laid on combating newly emerging drug-resistant organisms, emphasizing the significance of AMPs in mitigating this pressing challenge. The review also illuminates potential future strategies that may be implemented to improve AMP efficacy, such as structural modifications and using AMPs in combination with antibiotics and matrix-inhibiting compounds.

Antimicrobial peptides derived from food byproducts: Sources, production, purification, applications, and challenges.

Food wastes can be a valuable reservoir of bioactive substances that can serve as natural preservatives in foods or as functional ingredients with potential health benefits. The antimicrobial properties of protein hydrolysates, especially antimicrobial peptides (AMPs) derived from food byproducts (FBs), have been extensively explored. These protein fragments are defined by their short length, low molecular weight, substantial content of hydrophobic and basic amino acids, and positive net charge. The intricate mechanisms by which these peptides exert their antimicrobial effects on microorganisms and pathogens have been elaborately described. This review also focuses on techniques for producing and purifying AMPs from diverse FBs, including seafood, livestock, poultry, plants, and dairy wastes. According to investigations, incorporating AMPs as additives and alternatives to chemical preservatives in food formulations and packaging materials has been pursued to enhance both consumer health and the shelf life of foods and their products. However, challenges associated with the utilization of AMPs derived from food waste depend on their interaction with the food matrix, acceptability, and commercial viability. Overall, AMPs can serve as alternative safe additives, thereby ensuring the safety and prolonging the storage duration of food products based on specific regulatory approvals as recommended by the respective safety authorities.

Recent approaches in the application of antimicrobial peptides in food preservation.

Antimicrobial peptides (AMPs) are small peptides existing in nature as an important part of the innate immune system in various organisms. Notably, the AMPs exhibit inhibitory effects against a wide spectrum of pathogens, showcasing potential applications in different fields such as food, agriculture, medicine. This review explores the application of AMPs in the food industry, emphasizing their crucial role in enhancing the safety and shelf life of food and how they offer a viable substitute for chemical preservatives with their biocompatible and natural attributes. It provides an overview of the recent advancements, ranging from conventional approaches of using natural AMPs derived from bacteria or other sources to the biocomputational design and usage of synthetic AMPs for food preservation. Recent innovations such as structural modifications of AMPs to improve safety and suitability as food preservatives have been discussed. Furthermore, the active packaging and creative fabrication strategies such as nano-formulation, biopolymeric peptides and casting films, for optimizing the efficacy and stability of these peptides in food systems are summarized. The overall focus is on the spectrum of applications, with special attention to potential challenges in the usage of AMPs in the food industry and strategies for their mitigation.

Synergistic Antimicrobial Mechanism of the Ultrashort Antimicrobial Peptide R3W4V with a Tadpole-like Conformation.

Antimicrobial peptides (AMPs) are promising candidates in combating multidrug-resistant microorganisms because of their unique mode of action. Among these peptides, ultrashort AMPs (USAMPs) possess sequences containing less than 10 amino acids and have some advantages over traditional AMPs. However, one of the main limitations of designing novel and highly active USAMPs is that their mechanism of action at the molecular level is not well-known. In this article, we report the antimicrobial mechanism of the USAMP verine (R3W4V) with high antibacterial activity against Escherichia coli. Here, by using well-tempered bias-exchange metadynamics simulations and long-time conventional molecular dynamics simulations, we evaluated whether verine exhibits the same antimicrobial mode of action as that of traditional AMPs. The single verine-membrane system exhibited a relatively flat surface with multiple shallow minima separated by very small energy barriers and adopted highly dynamic structural ensembles. Although the verine sequence is very short, it can still exist briefly in the center of the cell membrane in a transmembrane state. As the concentration of verine increased, the transmembrane conformation was relatively stabilized in the membrane center or proceeded toward the membrane bottom. The lipid bilayer membrane showed relatively large deformation, including the phospholipid head groups embedded inside the lipid hydrophobic center, accompanied by a flip-flop of some lipids. Simulation results indicated that verine has a specific mechanism of action different from that of traditional AMPs. Based on this antimicrobial mechanism of verine, we can design new high-potential USAMPs by enhancing the structural stability of the transmembrane state.

Potential therapeutic use of dermaseptin S4 from the frog Phyllomedusa sauvagii and its derivatives against bacterial pathogens in fish.

AIM: Dermaseptins are one of the main families of antimicrobial peptides (AMPs) derived from the skin secretions of Hylidae frogs. Among them, dermaseptin S4 (DS4) is characterized by its broad-spectrum of activity against bacteria, protozoa, and fungi. In this study, the physicochemical properties of the native peptide DS4 (1-28) and two derivatives [DS4 (1-28)a and DS4 (1-26)a] isolated from the skin of the frog Phyllomedusa sauvagii were investigated and their antimicrobial properties against two marine pathogenic bacteria (Vibrio harveyi and Vibrio anguillarum) were examined. METHODS AND RESULTS: The results indicate that the peptide DS4 (1-26)a has high-antibacterial activity against the tested strains and low-hemolytic activity (<30% lysis at the highest tested concentration of 100 µg/mL) compared to the other two peptides tested. In addition, all three peptides affect the membrane and cell wall integrity of both pathogenic bacteria, causing leakage of cell contents, with DS4 (1-26)a having the most severe impact. These skills were corroborated by transmission electron microscopy and by the variation of cations in their binding sites due to the effects caused by the AMPs. CONCLUSIONS: These results suggest that DS4 and its derivatives, in particular the truncated and amidated peptide DS4 (1-26)a could be effective in the treatment of infections caused by these marine pathogenic bacteria. Future studies are required to validate the use of DS4  in vivo for the prevention of bacterial diseases in fish.

Antibacterial efficacy of antimicrobial peptide-functionalized hydrogel particles combined with vancomycin and oxacillin antibiotics.

The rise of antibiotic resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), requires novel approaches to combat infections. Medical devices like implants and wound dressings are frequently used in conjunction with antibiotics, motivating the development of antibacterial biomaterials capable of exhibiting combined antibacterial effects with conventional antibiotics. This study explores the synergistic antibacterial effects of combining antimicrobial peptide (AMP) functionalized hydrogel particles with conventional antibiotics, vancomycin (VCM) and oxacillin (OXA), against Staphylococcus aureus and MRSA. The AMP employed, RRPRPRPRPWWWW-NH2, has previously demonstrated broad-spectrum activity and enhanced stability when attached to hydrogel substrates. Here, checkerboard assays revealed additive and synergistic interactions between the free AMP and both VCM and OXA against Staphylococcus aureus and MRSA. Notably, the AMP-OXA combination displayed a significant synergistic effect against MRSA, with a 512-fold reduction in OXA's minimum inhibitory concentration (MIC) when combined with free AMP. The observed synergism against MRSA was retained upon covalent AMP immobilization onto the hydrogel particles; however, at a lower rate with a 64-fold reduction in OXA MIC. Despite this, the OXA-AMP hydrogel particle combinations retained considerable synergistic potential against MRSA, a strain resistant to OXA, highlighting the potential of AMP-functionalized materials for enhancing antibiotic efficacy. These findings underscore the importance of developing antimicrobial biomaterials for future medical devices to fight biomaterial-associated infections and reverse antimicrobial resistance.

Venom: A Promising Avenue for Antimicrobial Therapeutics.

Venom in medicine is well documented in the chronicles of ancient Greece and the Roman Empire and persisted into the Renaissance and even into the modern era. Venoms were not always associated with detrimental consequences. Since ancient times, the curative capacity of venom has been recognized, portraying venom as a metaphor for pharmacy and medicine. Venom proteins and peptides' antimicrobial potential has not undergone systematic exploration despite the huge literature on natural antimicrobials. In light of the escalating challenge of antimicrobial resistance and the diminishing effectiveness of antibiotics, there is a pressing need for innovative antimicrobials capable of effectively addressing illnesses caused by multidrug-resistant microorganisms. This review adds to our understanding of the effectiveness of different venom components against a host of pathogenic microorganisms. The aim is to illuminate the various antimicrobials present in venom and venom peptides, thereby emphasizing the unexplored medicinal potential for antimicrobial properties. We have presented a concise summary of the molecular examination of the venom peptides' functioning processes, as well as the current clinical and preclinical progress of venom antimicrobial peptides.

High Therapeutic Index α-Helical AMPs and Their Therapeutic Potential on Bacterial Lung and Skin Wound Infections.

Antimicrobial peptides (AMPs) possess strong antibacterial activity and low drug resistance, making them ideal candidates for bactericidal drugs for addressing the issue of traditional antibiotic resistance. In this study, a template (G(XXKK)nI, G = Gly; X = Leu, Ile, Phe, or Trp; n = 2, 3, or 4; K = Lys; I = Ile.) was employed for the devised of a variety of novel α-helical AMPs with a high therapeutic index. The AMP with the highest therapeutic index, WK2, was ultimately chosen following a thorough screening process. It demonstrates broad-spectrum and potent activity against both standard and multidrug-resistant bacteria, while also showing low hemolysis and rapid and efficient time-kill kinetics. Additionally, WK2 exhibits excellent efficacy in treating mouse models of Klebsiella pneumonia-induced lung infections and methicillin-resistant Staphylococcus aureus (MRSA)-induced skin wound infections while demonstrating good safety profiles in vivo. In conclusion, the template-based design methodology for novel AMPs with high therapeutic indices offers new insights into addressing antibiotic resistance problems. WK2 represents a promising antimicrobial agent.

Cocktail Approach with Polyserotonin Nanoparticles and Peptides for Treatment of Streptococcus mutans.

Dental plaque, formed by a Streptococcus mutans biofilm, is a major contributor to cavity formation. While antimicrobial strategies exist, the growing risk of antibiotic resistance necessitates alternative therapeutic solutions. Polyserotonin nanoparticles (PSeNPs), recently recognized for their photothermal property and promising biomedical applications, open up a new avenue for antimicrobial use. Here, we introduced a UV-initiated synthetic route for PSeNPs with improved yield. Using these PSeNPs, a cocktail treatment to reduce the viability of this cavity-causing bacteria was developed. This cocktail comprises an S. mutans-targeting antimicrobial peptide (GH12), an intraspecies competence-stimulating peptide that triggers altruistic cell death in S. mutans, and laser-activated heating of PSeNPs. The "peptide + PSeNP + laser" combination effectively inhibits S. mutans growth in both planktonic and biofilm states. Moreover, the cocktail approach remains effective in reducing the viability of S. mutans in a more virulent dual-species biofilm with Candida albicans. Overall, our results reinforce the utility of a multipronged therapeutic strategy to reduce cariogenic bacteria in the complex model oral biofilm.

Molecular characterization of histone gene in golden pompano (Trachinotus ovatus) and antimicrobial activity of its derived peptides.

In addition to controlling gene expression, mediating DNA folding into chromatin, and responding to immunological stimuli, histones are also thought to have antimicrobial effects. This study identified the molecular characteristics of core Histone MacroH2A2 (TOMacroH2A2) and Histone H2B 1/2 (TOH2B) from Trachinotus ovatus, and the antimicrobial potential of their derived peptides (To.mh2a and To. h2b). The open reading frames (ORFs) of TOMacroH2A2 and TOH2B from T. ovatus were 1010 bp and 375 bp, encoding polypeptides of 369 and 124 amino acids, respectively. The TOMacroH2A2 included an H2A domain and an A1pp domain, while TOH2B included an H2B domain. The amino acid sequences of TOMacroH2A2 and TOH2B demonstrated high homology with other teleost's sequences of histone macroh2a2 and histone h2b, with homologies exceeding 90 %. Expression analysis showed high expression of TOMacroH2A2 in brain, stomach, heart, and skin tissues and TOH2B in gill, brain, and skin tissues. In addition, the histone-derived peptides To. mh2a and To. h2b, synthesized based on two histone sequences from T. ovatus, exhibited typical physical characteristics of antimicrobial peptides, including positive charges, amphipathicity, hydrophobicity, and rich α-helix structure. Crucially, the vitro antibacterial results demonstrated that To. mh2a and To. h2b can inhibit the growth of various aquatic pathogens including Streptococcus agalactiae, Staphylococcus aureus, Bacillus subtilis, Acinetobacter baumannii, Aeromonas hydrophila, and Escherichia coli to varying degrees. Specifically, To. mh2a and To. h2b were capable of disrupting the cell surface structures of S. aureus and penetrating the cell membrane, leading to the leakage of cellular contents, thereby exerting their antibacterial effects. Furthermore, gel electrophoresis migration assays showed that To. mh2a and To. h2b participated in antimicrobial activity by binding to bacterial genomic DNA and reducing the migration rate of gDNA in a dose-dependent manner. The minimum effective concentration for binding to DNA was approximately 50 µM. In conclusion, our study suggested that To. mh2a and To. h2b can act as antimicrobial peptides, providing a potential strategy for controlling bacterial diseases in T. ovatus.

Mining human microbiomes reveals an untapped source of peptide antibiotics.

Drug-resistant bacteria are outpacing traditional antibiotic discovery efforts. Here, we computationally screened 444,054 previously reported putative small protein families from 1,773 human metagenomes for antimicrobial properties, identifying 323 candidates encoded in small open reading frames (smORFs). To test our computational predictions, 78 peptides were synthesized and screened for antimicrobial activity in vitro, with 70.5% displaying antimicrobial activity. As these compounds were different compared with previously reported antimicrobial peptides, we termed them smORF-encoded peptides (SEPs). SEPs killed bacteria by targeting their membrane, synergizing with each other, and modulating gut commensals, indicating a potential role in reconfiguring microbiome communities in addition to counteracting pathogens. The lead candidates were anti-infective in both murine skin abscess and deep thigh infection models. Notably, prevotellin-2 from Prevotella copri presented activity comparable to the commonly used antibiotic polymyxin B. Our report supports the existence of hundreds of antimicrobials in the human microbiome amenable to clinical translation.

A positive loop between relish and cuticle proteins and their roles in regulating AMPs expression during bacterial infection in Eriocheir sinensis.

Cuticle proteins (CPs) are the vital components of the cuticle and chitin lining covering the digestive tract of crustaceans. In this study, four new CP genes (designated as EsCP3, EsCP4, EsCP5, and EsCP8) were initially cloned and identified from the Chinese mitten crab Eriocheir sinensis. EsCP3/4/5/8 included 375, 411, 381, and 570 bp open reading frame encoding 124, 136, 126, and 189 amino acid proteins, respectively. Except for EsCP8, EsCP3/4/5 all contained a Chitin_bind_4 domain. EsCP3/4/5/8 were clustered into different groups in the phylogenetic tree. Quantitative real-time PCR results indicated that four EsCP genes have different patterns of tissue distribution. Changes in the expression levels of these four EsCP genes were observed in the intestine of crabs under Vibrio parahaemolyticus challenge. RNA interference assay showed that the knockdown of EsCPs in the intestine could inhibit the expression of antimicrobial peptides (AMPs), including crustins and anti-lipopolysaccharide factors. In addition, the knockdown of EsRelish in the intestine decreased the expression levels of these four EsCP genes. These results indicated that EsCPs were involved in regulating the expression of AMPs, and EsCPs were regulated by EsRelish.

Synergistic collaboration between AMPs and non-direct antimicrobial cationic peptides.

Non-direct antimicrobial cationic peptides (NDACPs) are components of the animal innate immune system. But their functions and association with antimicrobial peptides (AMPs) are incompletely understood. Here, we reveal a synergistic interaction between the AMP AW1 and the NDACP AW2, which are co-expressed in the frog Amolops wuyiensis. AW2 enhances the antibacterial activity of AW1 both in vitro and in vivo, while mitigating the development of bacterial resistance and eradicating biofilms. AW1 and AW2 synergistically damage bacterial membranes, facilitating cellular uptake and interaction of AW2 with the intracellular target bacterial genomic DNA. Simultaneously, they trigger the generation of ROS in bacteria, contributing to cell death upon reaching a threshold level. Moreover, we demonstrate that this synergistic antibacterial effect between AMPs and NDACPs is prevalent across diverse animal species. These findings unveil a robust and previously unknown correlation between AMPs and NDACPs as a widespread antibacterial immune defense strategy in animals.

Repurposing an antimicrobial peptide for the development of a dual ion channel/molecular receptor-like platform for metal ion detection.

The presence of non-essential metals in the environment as contaminants is prone to cause hazardous health problems following accumulation in the human body and the ensuing toxic effects. This calls for continuous discovery and innovation in the realm of developing easy-to-operate, cheap and sensitive sensors. Herein, we describe the proof of concept approach for designing a molecular receptor-like, chimeric sensor based on the pore-forming peptide alamethicin (Alm), tethered via a linker with an ultrashort peptide nucleic acid (PNA) moiety, capable of generating functional ion channel oligomers in planar lipid membranes. The working principle of the sensor exploits the ability of Hg2+ ions to complex mismatching thymine-thymine sequences between the PNA receptor moiety on Alm oligomers and free, thymine-based, single-stranded DNAs (ssDNAs) in solution, thus creating a stable base pair at the oligomer entrance. This generates a transducing mechanism which converts the metal ion complexation into a specific electrical signature of the self-assembled Alm oligomers, enabling selective Hg2+ ion detection. The platform is programmable, whereby the simple exchange of the PNA sequence and its ssDNA counterpart in solution rendered the system selective for Cu2+ ion detection. With further optimization, the presented solution has the potential to translate into miniaturized, cost-effective biosensors suitable for the real-time, label-free and continuous detection of metal ions or other biomolecules.

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.

Whole-genome sequencing and identification of antimicrobial peptide coding genes in parsley (Petroselinum crispum), an important culinary and medicinal Apiaceae species.

Parsley is a commonly cultivated Apiaceae species of culinary and medicinal importance. Parsley has several recognized health benefits and the species has been utilized in traditional medicine since ancient times. Although parsley is among the most commonly cultivated members of Apiaceae, no systematic genomic research has been conducted on parsley. In the present work, parsley genome was sequenced using the long-read HiFi (high fidelity) sequencing technology and a draft contig assembly of 1.57 Gb that represents 80.9% of the estimated genome size was produced. The assembly was highly repeat-rich with a repetitive DNA content of 81%. The assembly was phased into a primary and alternate assembly in order to minimize redundant contigs. Scaffolds were constructed with the primary assembly contigs, which were used for the identification of AMP (antimicrobial peptide) genes. Characteristic AMP domains and 3D structures were used to detect and verify antimicrobial peptides. As a result, 23 genes (PcAMP1-23) representing defensin, snakin, thionin, lipid transfer protein and vicilin-like AMP classes were identified. Bioinformatic analyses for the characterization of peptide physicochemical properties indicated that parsley AMPs are extracellular peptides, therefore, plausibly exert their antimicrobial effects through the most commonly described AMP action mechanism of membrane attack. AMPs are attracting increasing attention since they display their fast antimicrobial effects in small doses on both plant and animal pathogens with a significantly reduced risk of resistance development. Therefore, identification and characterization of AMPs is important for their incorporation into plant disease management protocols as well as medicinal research for the treatment of multi-drug resistant infections.