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Antimicrobial peptides (AMPs) are increasingly recognized as potent therapeutic agents, with their selective affinity for pathological membranes, low toxicity profile, and minimal resistance development making them particularly attractive in the pharmaceutical landscape. This study offers a comprehensive analysis of the interaction between specific AMPs, including magainin-2, pleurocidin, CM15, LL37, and clavanin, with lipid bilayer models of very different compositions that have been ordinarily used as biological membrane models of healthy mammal, cancerous, and bacterial cells. Employing unbiased molecular dynamics simulations and metadynamics techniques, we have deciphered the intricate mechanisms by which these peptides recognize pathogenic and pathologic lipid patterns and integrate into lipid assemblies. Our findings reveal that the transverse component of the peptide's hydrophobic dipole moment is critical for membrane interaction, decisively influencing the molecule's orientation and expected therapeutic efficacy. Our approach also provides insight on the kinetic and dynamic dependence on the peptide orientation in the axial and azimuthal angles when coming close to the membrane. The aim is to establish a robust framework for the rational design of peptide-based, membrane-targeted therapies, as well as effective quantitative descriptors that can facilitate the automated design of novel AMPs for these therapies using machine learning methods.

Background: Antimicrobial peptides (AMPs) are a critical component of the innate immune system, playing a key role in defending against a variety of pathogenic microorganisms. While many AMPs act primarily on the cell membrane of target pathogens, leading to lysis and subsequent cell death, less is known about their nonlytic membrane activity. This nonlytic activity allows AMPs to target and disrupt bacterial cells without causing lysis, leading to bacterial death through alternative mechanisms.Understanding these nonlytic properties of AMPs is crucial, as they present a promising alternative to traditional antibiotics, which can induce bacterial resistance and have adverse effects on human health and the environment. The mechanisms by which AMPs exhibit nonlytic membrane activity are still being explored. However, it is believed that AMPs penetrate the bacterial membrane and interact directly with internal cellular components such as DNA, RNA, and various enzymes essential for microbial survival and replication. This interaction disrupts metabolic homeostasis, ultimately resulting in bacterial death.The nonlytic activity of AMPs also results in minimal damage to host cells and tissues, making them attractive candidates for the development of new, more effective antibiotics. This review emphasizes the mechanisms by which AMPs nonlytically target cellular components, including DNA, proteins, RNA, and other biomolecules, and discusses their clinical significance. Understanding these mechanisms may pave the way for developing alternatives to conventional antibiotics, offering a solution to the growing issue of antibiotic resistance.

The evolution of methicillin-resistant Staphylococcus aureus (MRSA) has required the development of new antimicrobial agents and new approaches to prevent and overcome drug resistance. AntiMicrobial Peptides (AMPs) represent promising alternatives due to their rapid bactericidal activity and their broad-spectrum of action against a wide range of microorganisms. The amphibian Temporins constitute a well-known family of AMPs with high antibacterial properties against both Gram-positive and Gram-negative bacteria. In this paper, we evaluated the in vivo effect of Temp-L on S. aureus performing morphological studies using Transmission Electron Microscopy (TEM) that revealed the occurrence of protrusions from the cell surface. The formation of vesicle-like structure was confirmed by Dynamic Light Scattering (DLS). The global effect of Temp-L on Staphylococcus aureus (S. aureus) was deeply investigated by differential proteomics leading to the identification of up-regulated proteins involved in the synthesis of the cell membrane and fatty acids, and down-regulated virulence factors. GC-MS analysis suggested a possible protective response mechanism implemented by the bacterium after treatment with Temp-L, as the synthesis of fatty acids was increased. Adhesion and invasion assays on eukaryotic cells confirmed a reduced virulence of S. aureus following treatment with Temp-L. These results suggested the targeting of virulence factors as novel strategy to replace traditional antimicrobial agents that can be used to treat infections, especially infections caused by the resistant pathogen S. aureus.

In addition to defensins, plants possess an array of defensin-like peptides that share many of their characteristics, as well as a role in plant’s innate immunity. Their involvement in the response to pathogens is well-known but the contribution in the plant response to abiotic stimuli is not fully understood. We have undertaken an in silico analysis to characterize all defensin family genes hitherto found in Arabidopsis, including genes encoding for defensin-like peptides, by detecting several peptides as candidates for further studies aiming to decipher specific responses to biotic and abiotic stresses, as well as to their crosstalk. We performed several analyses, including co-expression and cis-regulatory elements analyses, using transcriptomic data obtained from the ARS database, which integrates more than 20,000 Arabidopsis RNA-seq libraries.In silico analysis showed that jasmonates and ABA, together with transcription factors belonging to WRKY and AP2/EREBP families, modulate defensin and defensin-like gene expression. Indeed, the analysis performed in this study allowed to extract and organize omics data, which finally supported the inducible nature of defensins under both abiotic and biotic stresses. Moreover, in vivo expression analyses confirmed the heat and drought responsiveness of PDF1.4, ATTI1, PDF1.1, DEFL 206, defensin family genes selected for being upregulated by several abiotic conditions, at transcriptional level. Finally, the co-expression analysis provided information on other biological processes that may be correlated to the defensin induction, such as maintaining ROS homeostasis. Combining the comprehensive analysis of different transcriptional datasets with the integration of in vivo analyses emerged as a robust methodological approach to assess the proposed multi-stress responsive nature of defensin family genes.

Acinetobacter baumannii is a prominent hospital-associated bacterium whose eradication is increasingly challenging due to its remarkable ability to resist antibiotics. The most common illnesses caused by antibiotic-resistant A. baumannii are biofilm-associated. Therefore, novel methods to combat A. baumannii are urgently needed. Application of antimicrobial peptides (AMPs) is one of the avenues to be explored. Epsilon poly-l-lysine (ε-PL) is an antimicrobial peptide with low mammalian toxicity. It is commonly used as a food preservative and has advantages such as biodegradability, good water solubility, and thermal stability.Therefore, the antimicrobial activity of ε-PL against clinical isolates of A. baumannii was investigated. The effect of ε-PL on antimicrobial sensitivity was determined by broth dilution assay. The effect of ε-PL on biofilm formation and dispersion was studied using a crystal violet assay. The changes in the expression of quorum sensing related and virulence genes (abaI, csuE, pilT, bap, and luxI) were analyzed using qPCR by the Δ Δ CT method.All the A. baumannii clinical isolates (n = 28) tested, were resistant to multiple drugs. The treatment with ε-PL resulted in a significant (p

Acquired drug resistance is the major cause for disease recurrence in cancer patients, and this is particularly true for patients with metastatic melanoma that carry a BRAF V600E mutation. To address this problem, we investigated cyclic membrane-active peptides as an alternative therapeutic modality to kill drug-tolerant and resistant melanoma cells to avoid acquired drug resistance. We selected two stable cyclic peptides (cTI and cGm), previously shown to have anti-melanoma properties, and compared them with dabrafenib, a drug used to treat cancer patients with the BRAF V600E mutation. The peptides act via a fast membrane-permeabilizing mechanism and kill metastatic melanoma cells that are sensitive, tolerant, or resistant to dabrafenib. Melanoma cells do not become resistant to long-term treatment with cTI, nor do they evolve their lipid membrane composition, as measured by lipidomic and proteomic studies. In vivo studies in mice demonstrated that the combination treatment of cTI and dabrafenib resulted in fewer metastases and improved overall survival. Such cyclic membrane-active peptides are thus well suited as templates to design new anticancer therapeutic strategies.

Although antimicrobial peptides are considered one of the most promising alternatives to conventional antibiotics given the alarming increase in bacterial multidrug resistance, many aspects of their mechanism of action remain unclear, in particular the emergence and role of collective phenomena such as the spontaneous formation of nano-sized unstructured objects (clusters) and their effects on the biodynamics. We study this process using two novel peptides from the mucus of the garden snail Cornu aspersum as an example to reveal its dynamics and bioactivity implications through coordinated in silico and in vitro techniques — molecular dynamics simulations, UV–Vis and fluorescence spectroscopy, and antibacterial activity tests against two representative bacterial strains — one gram-negative (Escherichia coli 3458) and one gram-positive (Bacillus subtilis). The results obtained confirm the impact of the aggregation processes of the peptides on their biological activity and provide insight into possible synergies in their action.

Bacterial biofilm-associated infection was one of the most serious threats to human health. However, effective drugs for drug-resistance bacteria or biofilms remain rarely reported. Here, we propose an innovative strategy to develop a multifunctional antimicrobial agent with broad-spectrum antibacterial activity by coupling photosensitizers (PSs) with antimicrobial peptides (AMPs). This strategy capitalizes on the ability of PSs to generate reactive oxygen species (ROS) and the membrane-targeting property of AMPs (KRWWKWIRW, a peptide screened by an artificial neural network), synergistically enhancing the antimicrobial activity. In addition, unlike conventional aggregation-caused quenching (ACQ) photosensitizers, aggregation-induced emission (AIE) PSs show stronger fluorescence emission in the aggregated state to help visualize the antibacterial mechanism. In vitro antibacterial experiments demonstrated the excellent killing effects of the developed agent against both Gram-positive (G+) and Gram-negative (G–) bacteria. The bacterial-aggregations induced ability enhanced the photoactivatable antibacterial activity against G– bacteria. Notably, it exhibited a significant effect on destroying MRSA biofilms. Moreover, it also showed remarkable efficacy in treating wound infections in mice in vivo. This multifunctional antimicrobial agent holds significant potential in addressing the challenges posed by bacterial biofilm-associated infections and drug-resistant bacteria.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved multifunctional neuropeptide belonging to the secretin/glucagon/vasoactive intestinal peptide superfamily. In recent years, the role of PACAP in immunoregulation has been partially elucidated in mammals and teleost fishes, demonstrating that PACAP modulates both innate and acquired immunity. However, in invertebrates, little is known about its immunostimulant and antimicrobial properties. In previous research, studies performed in Litopenaeus (Penaeus) vannamei have revealed that Clarias gariepinus PACAP-38 improves immune functions like lectins, superoxide dismutase and nitric oxide synthase derived metabolites in postlarvae shrimp. In this study, the immunomodulatory effects of C. gariepinus PACAP-38 and four PACAP sequence modified peptides in juvenile L. vannamei were evaluated. The effect of several administrations of PACAP and its variants on shrimp immune effectors, as well as the effect of different doses was examined. Results revealed that PACAP can influence hemolymph immune parameters, and immune-related gene mRNA levels in the hemocytes, gill and hepatopancreas with different effects depending on the modification of the peptide sequence, the dose and the tissue assessed. Altogether, these findings provide valuable insights regarding the role of PACAP in crustacean innate immunity and highlight the need for further research into its potential application as an alternative to antibiotics in aquaculture.

The three-dimensional structure and network that compose naturally or synthetically derived polymers, such as hydrogels, allow for a wide variety in customization of the biomaterial characteristics, thus resulting in various applications. In medical care, hydrogels formed by intrinsic or exogenous antimicrobial components can act as effective vehicles for the administration of drugs and bioactive compounds, as alternatives to traditional wound dressings, and as antimicrobial coatings on implanted medical devices. In food safety, hydrogels with antimicrobial properties are desirable as food spoilage inhibitors. There has been a recent heightened focus on naturally derived hydrogels, due to their cost effectiveness and lack of concern for toxicity, which enhance their potential for a variety of food and biomedical applications. This concise review focuses on the recent advances of naturally derived peptide and polysaccharide antimicrobial hydrogels in the biomedical and food industries.

Background: Protozoal pathogens pose a considerable threat, leading to notable mortality rates and the ongoing challenge of developing resistance to drugs. This situation underscores the urgent need for alternative therapeutic approaches. Antimicrobial peptides stand out as promising candidates for drug development. However, there is a lack of published research focusing on predicting antimicrobial peptides specifically targeting protozoal pathogens. In this study, we introduce a successful machine learning-based framework designed to predict potential antiprotozoal peptides effective against protozoal pathogens. Objective: The primary objective of this study is to classify and predict antiprotozoal peptides using diverse negative datasets. Methods: A comprehensive literature review was conducted to gather experimentally validated antiprotozoal peptides, forming the positive dataset for our study. To construct a robust machine learning classifier, multiple negative datasets were incorporated, including (i) non-antimicrobial, (ii) antiviral, (iii) antibacterial, (iv) antifungal, and (v) antimicrobial peptides excluding those targeting protozoal pathogens. Various compositional features of the peptides were extracted using the pfeature algorithm. Two feature selection methods, SVC-L1 and mRMR, were employed to identify highly relevant features crucial for distinguishing between the positive and negative datasets. Additionally, five popular classifiers i.e. Decision Tree, Random Forest, Support Vector Machine, Logistic Regression, and XGBoost were used to build efficient decision models. Results: XGBoost was the most effective in classifying antiprotozoal peptides from each negative dataset based on the features selected by the mRMR feature selection method. The proposed machine learning framework efficiently differentiate the antiprotozoal peptides from (i) non-antimicrobial (ii) antiviral (iii) antibacterial (iv) antifungal and (v) antimicrobial with accuracy of 97.27 %, 93.64 %, 86.36 %, 90.91 %, and 89.09 % respectively on the validation dataset. Conclusion: The models are incorporated in a user-friendly web server (www.soodlab.com/appred) to predict the antiprotozoal activity of given peptides.

Antimicrobial peptides (AMPs) show promise in enhancing resistance against pathogens. Previously, we integrated two AMP genes, cathelicidin (Cath) from alligator (Alligator mississippiensis or Alligator sinensis) and cecropin (Cec) from moth (Hyalophora cecropia), into the channel catfish (Ictalurus punctatus) genome. This study examines the efficacy of exogenous AMP gene integration in improving bacterial resistance in transgenic channel catfish and assesses the direct and pleiotropic effects of gene replacement/knockout on survival and growth based on insertion site. Transgenic Cath- and Cec-expressing fish exhibited similar or higher survival rates (P ​> ​0.05) compared to controls during the initial culture. Integration of the Cec transgene doubled the survival rate when challenged with Edwardsiella ictaluri, with knock-in (KI) of Cath further increasing bacterial resistance. Coupling Cec KI with mstn knockout (KO) increased survival 3-fold after E. ictaluri infection and growth by 50% at 4 months post-fertilization (mpf). However, random integration of Cec had a minimal effect on disease resistance and did not enhance growth. Random integration of Cath increased survival 2.5-fold and 4-fold against E. ictaluri and Flavobacterium covae, respectively, without affecting growth. Cath KI at the lh locus increased survival 4-fold when challenged with F. covae and reduced growth by 10% (P ​> ​0.05) at 24 mpf, whereas Cath KI coupled with mc4r KO resulted in a 2.5-fold increase in survival following F. covae infection compared with controls, and increased growth by 80% at 3 mpf. Simultaneous KI of Cath and Cec, along with KO of mc4r and mstn, increased survival 4-fold against E. ictaluri, while increasing growth by 50% at 3 mpf. Dual insertion of AMP genes yielded the greatest resistance to disease. These direct and pleiotropic effects may increase comprehension and societal acceptance of genetic engineering in aquaculture.

Nicotine, a naturally occurring alkaloid found in tobacco, is a potent neurotoxin extensively used to control Nilaparvata lugens (Stål), a destructive insect pest of rice crops. The insect gut harbors a wide array of resident microorganisms that profoundly influence several biological processes, including host immunity. Maintaining an optimal gut microbiota and immune homeostasis requires a complex network of reciprocal regulatory interactions. However, the underlying molecular mechanisms driving these symbiotic exchanges, particularly between specific gut microbe and immunity, remain largely unknown in insects. Our previous investigations identified and isolated a nicotine-degrading Burkholderia cepacia strain (BsNLG8) with antifungal properties. Building on those findings, we found that nicotine intake significantly increased the abundance of a symbiotic bacteria BsNLG8, induced a stronger bacteriostatic effect in hemolymph, and enhanced the nicotine tolerance of N. lugens. Additionally, nicotine-induced antimicrobial peptides (AMPs) exhibited significant antibacterial effects against Staphylococcus aureus. We adopted RNA-seq to explore the underlying immunological mechanisms in nicotine-stressed N. lugens. Bioinformatic analyses identified numerous differentially expressed immune genes, including recognition/immune activation (GRPs and Toll) and AMPs (i.e., Defensin, Lugensin, lysozyme). Temporal expression profiling (12, 24, and 48 hours) of immune genes revealed pattern recognition proteins and immune effectors as primary responders to nicotine-induced stress. Defensin A, a broad-spectrum immunomodulatory cationic peptide, exhibited significantly high expression. RNA interference-mediated silencing of Defensin A reduced the survival, enhanced nicotine sensitivity of N. lugens to nicotine, and decreased the abundance of BsNLG8. The reintroduction of BsNLG8 improved the expression of immune genes, aiding nicotine resistance of N. lugens. Our findings indicate a potential reciprocal immunomodulatory interaction between Defensin A and BsNLG8 under nicotine stress. Moreover, this study offers novel and valuable insights for future research into enhancing nicotine-based pest management programs and developing alternative biocontrol methods involving the implication of insect symbionts.

The antimicrobial peptides (AMPs) have been regarded as the next generation antibiotics. This study aimed to explore the AMP inhibitors of β-lactamase enzyme employing computational biology methods. Protein-peptide docking of Acinetobacter baumannii OXA-24 class D β-lactamase with AMPs (melittin and RP-1) were performed using HawkDock webserver. The docked complexes were subjected to energy property analysis through MM/GBSA, and binding affinity (ΔG (kcal/mol)) and stability (dissociation constant, KD (M)) prediction using PRODIGY. Both the AMPs, melittin and RP-1, were well docked with A. baumannii OXA-24. The top ranked OXA-24-melittin and OXA-24-RP-1 complexes were detected on the basis of the HawkDock scores (-2974.08 and -2825.83, respectively), thereafter by rescoring with MM/GBSA-based binding free energy (BFE) of -33.85 and -29.29 kcal/mol, respectively. The PRODIGY-based respective BFE (-8.0 and -10.0 kcal/mol) and KD (1.4 × 10-6 and 5 × 10-8 M) of the complexes revealed excellent protein-peptide binding affinity and complex stability. The iMODS-based molecular dynamic simulation authenticated the stability and molecular motion flexibility of the protein-peptide complexes. The quantum mechanics and molecular mechanics energy estimated using density-functional theory with CP2K software, for the electrostatic interaction of OXA-24-RP-1 (-218862.95 kcal/mol), was more favourable than the H-bonded interaction of OXA-24-melittin (-200620.21 kcal/mol), nevertheless both the peptides were found effective to inhibit OXA-24. Both the AMPs had toxicity profiles within the acceptable limits as predicted through pkCSM. Current findings indicate the potential supplement or replacement of conventionally used antibiotics with melittin and RP-1 to treating or averting A. baumannii infection by escaping the β-lactamases-mediated resistance.

Antimicrobial peptides (AMPs) from natural sources represent promising candidates for novel antimicrobial agents instead of antibiotics in aquaculture. Moricin, a family of AMPs from Lepidoptera, has shown bactericidal activity against both gram-positive and gram-negative bacteria. However, it remains uncertain whether these peptides possess antimicrobial activity against Vibrio pathogens affecting various aquatic animals, including sea cucumbers. In this study, we investigated five moricin-like peptides (MLPs) from Antheraea pernyi (Anpe) and identified Anpe-MLP1 as a new member of the moricin family through bioinformatics analysis. Anpe-MLP1 was heterologously expressed using the AnpeNPV bacmid system and purified from A. pernyi pupae. Our findings revealed that recombinant Anpe-MLP1 (rAnpe-MLP1) exhibited antimicrobial activity against gram-positive Staphylococcus aureus (S. aureus ATCC 6538 P), gram-negative Escherichia coli (E. coli ATCC 25922) and three Vibrio species. Moreover, both in vitro and in vivo assays showed that rAnpe-MLP1 effectively inhibited wild-type Vibrio fluvialis infections in the sea cucumber (Apostichopus japonicus). These results suggest that Anpe-MLP1 holds great potential as a novel anti-infective agent and provide valuable insights for its future application in aquaculture.

Antimicrobial peptides (AMPs) constitute a group of small molecular peptides that exhibit a wide range of antimicrobial activity. These peptides are abundantly present in the innate immune system of various organisms. Given the rise of multidrug-resistant bacteria, microbiological studies have identified AMPs as potential natural antibiotics. In the context of antimicrobial resistance across various human pathogens, AMPs hold considerable promise for clinical applications. However, numerous challenges exist in the detection of AMPs, particularly by immunological and molecular biological methods, especially when studying of newly discovered AMPs in proteomics. This review outlines the current status of AMPs research and the strategies employed in their development, considering resent discoveries and methodologies. Subsequently, we focus on the advanced techniques of mass spectrometry for the quantification of AMPs in diverse samples, and analyzes their application, advantages, and limitations. Additionally, we propose suggestions for the future development of tandem mass spectrometry for the detection of AMPs.

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.

The impact of H. pylori resistance on patient's treatment failure is a major concern. Therefore, the development of novel or alternative therapies for H. pylori is urgently needed. The purpose of this study was to investigate the molecular interactions of various antimicrobial peptides (AMPs) to H. pylori proteins. We performed the peptide-protein molecular docking using HADDOCK 2.4 webserver. Fourteen AMPs were tested for their binding efficacy against four H. pylori proteins. Simulation of the peptide-protein complex was performed using molecular dynamic software package AMBER20. From molecular docking analysis, five peptides (LL-37, Tilapia piscidin 4, napin, snakin-1 and EcAMP1) showed strong binding interactions against H. pylori proteins. The strongest binding affinity was observed in the interactions between Snakin-1 and PBP2, TP4 and type I HopQ and EcAMP1 and type I HopQ with −11.1, −13.6 and −13.8 kcal/mol, respectively. The dynamic simulation was performed for two complexes (snakin1-PBP2 and EcAMP1-HopQ). Results of the dynamics simulation showed that EcAMP1 had stable interaction and binding to type I HopQ protein without significant structural changes. In conclusion, both results of docking and simulation showed that EcAMP1 might be useful as a potential therapeutic agent for H. pylori treatment. This molecular approach provides deep understanding of the interaction insights between AMPs and H. pylori proteins. It paves the way for the development of novel anti-H. pylori using antimicrobial peptides.

With over 39,000 students, and research expenditures in excess of $200 million, George Mason University (GMU) is the largest R1 (Carnegie Classification of very high research activity) university in Virginia. Mason scientists have been involved in the discovery and development of novel diagnostics and therapeutics in areas as diverse as infectious diseases and cancer. Below are highlights of the efforts being led by Mason researchers in the drug discovery arena.To enable targeted cellular delivery, and non-biomedical applications, Veneziano and colleagues have developed a synthesis strategy that enables the design of self-assembling DNA nanoparticles (DNA origami) with prescribed shape and size in the 10 to 100 nm range. The nanoparticles can be loaded with molecules of interest such as drugs, proteins and peptides, and are a promising new addition to the drug delivery platforms currently in use. The investigators also recently used the DNA origami nanoparticles to fine tune the spatial presentation of immunogens to study the impact on B cell activation. These studies are an important step towards the rational design of vaccines for a variety of infectious agents.To elucidate the parameters for optimizing the delivery efficiency of lipid nanoparticles (LNPs), Buschmann, Paige and colleagues have devised methods for predicting and experimentally validating the pKa of LNPs based on the structure of the ionizable lipids used to formulate the LNPs. These studies may pave the way for the development of new LNP delivery vehicles that have reduced systemic distribution and improved endosomal release of their cargo post administration.To better understand protein-protein interactions and identify potential drug targets that disrupt such interactions, Luchini and colleagues have developed a methodology that identifies contact points between proteins using small molecule dyes. The dye molecules noncovalently bind to the accessible surfaces of a protein complex with very high affinity, but are excluded from contact regions. When the complex is denatured and digested with trypsin, the exposed regions covered by the dye do not get cleaved by the enzyme, whereas the contact points are digested. The resulting fragments can then be identified using mass spectrometry. The data generated can serve as the basis for designing small molecules and peptides that can disrupt the formation of protein complexes involved in disease processes. For example, using peptides based on the interleukin 1 receptor accessory protein (IL-1RAcP), Luchini, Liotta, Paige and colleagues disrupted the formation of IL-1/IL-R/IL-1RAcP complex and demonstrated that the inhibition of complex formation reduced the inflammatory response to IL-1B.Working on the discovery of novel antimicrobial agents, Bishop, van Hoek and colleagues have discovered a number of antimicrobial peptides from reptiles and other species. DRGN-1, is a synthetic peptide based on a histone H1-derived peptide that they had identified from Komodo Dragon plasma. DRGN-1 was shown to disrupt bacterial biofilms and promote wound healing in an animal model. The peptide, along with others, is being developed and tested in preclinical studies. Other research by van Hoek and colleagues focuses on in silico antimicrobial peptide discovery, screening of small molecules for antibacterial properties, as well as assessment of diffusible signal factors (DFS) as future therapeutics.The above examples provide insight into the cutting-edge studies undertaken by GMU scientists to develop novel methodologies and platform technologies important to drug discovery.

Introduction: Odorant-binding proteins (OBPs) are a class of small molecular weight soluble proteins that exist as expanded gene families in all insects, acting as ligand carriers mediating olfaction and other physiological processes. During fungal infection, a subset of insect OBPs were shown to be differentially expressed. Objectives: We tested whether the altered expression of insect OBPs during pathogenic infection plays a role in behavioral or immune interactions between insect hosts and their pathogens. Methods: A wide range of techniques including RNAi-directed knockdown, heterologous protein expression, electrophysiological/behavioral analyses, transcriptomics, gut microbiome analyses, coupled with tandem mass spectrometry ion monitoring, were used to characterize the function of a locust OBP in host behavioral and immune responses. Results: The entomopathogenic fungus Metarhizium anisopliae produces the volatile compound phenylethyl alcohol (PEA) that causes behavioral avoidance in locusts. This is mediated by the locust odorant binding protein 11 (LmOBP11). Expression of LmOBP11 is induced by M. anisopliae infection and PEA treatment. LmOBP11 participates in insect detection of the fungal-produced PEA and avoidance of PEA-contaminated food, but the upregulation of LmOBP11 upon M. anisopliae infection negatively affects the insect immune responses to ultimately benefit successful mycosis by the pathogen. RNAi knockdown of LmOBP11 increases the production of antimicrobial peptides and enhances locust resistance to M. anisopliae infection, while reducing host antennal electrophysiological responses to PEA and locust avoidance of PEA treated food. Also, transcriptomic and gut microbiome analyses reveal microbiome dysbiosis and changes in host genes involved in behavior and immunity. These results are consistent with the elevated expression of LmOBP11 leading to enhanced volatile detection and suppression of immune responses. Conclusion: These findings suggest a crosstalk between olfaction and immunity, indicating manipulation of host OBPs as a novel target exploited by fungal pathogens to alter immune activation and thus promote the successful infection of the host.

The dataset profiled in this research is built on sequencing of lactic acid bacteria 16S rDNA mined from Nono (N4 and N5), Kunu (K4 and K1) and Garri. The 16S rDNA sequences files are accessible under the data identification numbers: OK017047, OK017046, OK017044, OK017043, OK017045 at the GenBank database, NCBI. Taxonomic identification and phylogenetic tree analysis were done using the online BLAST (blastn) and MEGA11 software, respectively. The effect of the bacteriocin produced by these organisms on spoilage bacteria associated with salad was evaluated using an agar well diffusion assay. Limosilactobacillus pontis strain EOINONO, Limosilactobacillus pontis strain OGENONO, Limosilactobacillus pontis strain SEOGARI, Lactiplantibacillus plantarum strain MJIKUNU and Limosilactobacillus pontis strain EEIKUNU were the identified bacteriocinogenic organisms while Bacillus tequilensis strain SEOABACHA, Bacillus tequilensis strain EEIABACHA, Achromobacter xylosoxidans strain IMABACHA and Achromobacter insolitus strain MJIABACHA were the identified spoilage organisms.

ABSTRACT: To evaluate the effect and its mechanism of heat-resistant antimicrobial peptide LLv on broilers, three hundred 1-day-old healthy AA+ female broilers were allocated into 5 groups with 6 replicates in each group and 10 birds in each replicate. Birds were given a basal diet, an antibiotic diet (10.2 mg/kg chlortetracycline hydrochloride), and the basal diet supplemented with 10, 50, and 100 mg/kg LLv for 42 d, respectively. Compared with the group which birds were fed an antibiotic-free basal diet (control group), supplementing 100 mg/kg LLv increased 21-day IgA, IgM, IL-4, AIV-Ab, IFN-γ levels and 42-day IgA, IgM, IL-4, AIV-Ab levels and reduced 42-day IL-1 levels in serum (P < 0.05). Compared with antibiotic group, the 10 and 50 mg/kg LLv decreased 42-day IgM levels in serum (P < 0.05). The 100 mg/kg LLv increased 21-day AIV-Ab levels and 42-day IL-4, AIV-Ab levels and reduced 42-day IL-1 levels in serum (P < 0.05). Compared with control group, the 100 mg/kg LLv increased the expression rate of sIgA secretory cells and sIgA content in jejunal mucosa at 21 d and 42 d (P < 0.05), which did not differ from antibiotic group (P > 0.05). Compared with antibiotic group, the 10 mg/kg LLv reduced 21-day sIgA content and the 50 mg/kg LLv reduced 42-d the expression rate of sIgA secretory cells in jejunal mucosa (P < 0.05). Compared with control group, the 100 mg/kg LLv increased the expression of TCR, IL-15, CD28, BAFF, CD86, CD83, MHC-II, and CD40 genes in jejunal mucosa at 21 d and 42 d (P < 0.05). Compared with antibiotic group, the 100 mg/kg LLv increased the expression of 21-day BAFF, CD40, MHC-II, CD83 genes and the expression of 42-day BAFF, TCR, IL-15, CD40, CD83 genes in jejunal mucosa (P < 0.05). The results showed that the addition of LLv to the ration had a promotional effect on the immune function of broiler chickens.

Insects are valuable models for studying innate immunity and its role in combating infections. The silkworm Bombyx mori L., a well-studied insect model, is susceptible to a range of pathogens, including bacteria, fungi, viruses, and microsporidia. Their susceptibility makes it a suitable model for investigating host-pathogen interactions and immune responses against infections and diseases. This review focuses on the humoral immune response and the production of antimicrobial peptides (AMPs), the phenoloxidase (PO) system, and other soluble factors that constitute the primary defense of silkworms against microbial pathogens. The innate immune system of silkworms relies on pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs), which then activate various immune pathways including Imd, Toll, JAK/STAT, and RNA interference (RNAi). Their activation triggers the secretion of AMPs, enzymatic defenses (lysozyme and PO), and the generation of reactive oxygen species (ROS). Collectively, these pathways work together to neutralize and eliminate pathogens, thereby contributing to the defense mechanism of silkworms. Understanding the innate immunity of silkworms can uncover conserved molecular pathways and key immune components shared between insects and vertebrates. Additionally, it can provide valuable insights for improving sericulture practices, developing strategies to control diseases affecting silk production, and providing a theoretical foundation for developing pest control measures.

Advances in implantable technologies results from the rapid improvements in research in the fields of biomedical engineering, biomaterial science, and chemistry. No doubt, implantable technologies have provided greater comfort against several illnesses and diseases; however, pathogens adherence and biofilm formation are still the issues that need to be taken into consideration for further implant modifications. Pathogens colonize implants in a surface-dependent manner, so chances of pathogens' adhesion can be minimized by creating anti-adhesive and antimicrobial surfaces. The ongoing research is focused on the design of appropriate multifunctional coatings with a broad antimicrobial spectrum and high biocompatibility. Antimicrobial peptides in conjugation with polymers, nanoparticles, and other biocompatible agents can be potentially used to develop multifunctional coatings. Antibiotics-based coatings are the ultimate approach; however, optimizing the controlled release of antibiotics will be a breakthrough in this decade. Polymers unveil the extraordinary potential in implant coatings by increasing biocompatibility of coatings, offering the possibility for a triggered release of antimicrobial agents. An in-depth review of effective and biocompatible antimicrobial implant coatings is provided herein. We thoroughly discuss the remarkable antimicrobial agents and their compatibility with the host, including seemingly underexplored stability issues and providing an overlook on different methods of fabrication. The ever-growing materials research field can use this review's highlights to develop multifunctional implant coatings to reduce implant-associated infections.

Food spoilage is a major issue in the food sector, posing financial and health risks. New preservation methods have emerged to prolong shelf life and prevent spoilage. Chemical preservatives have been widely used to inhibit spoilage but can adversely affect human health. In this scenario, searching for effective bio-preservatives is more urgent than ever. To address this, researchers are looking for effective bio-preservatives. Bacteriocins, a class of antimicrobial peptides synthesized by LAB (lactic acid bacteria), are used as bio-preservatives. Recently, researchers have emphasized using bacteriocins (e.g., nisin, pediocin) to target food spoilage and harmful microorganisms without causing notable adverse effects. Bacteriocins promote the generation of pores in targeted cell membranes, releasing molecules with low molecular weight and disturbing the proton motive force. However, bacteriocins have a limited antibacterial range and high dose demand. Bacteriocin-nanoconjugates have recently become a possible approach to overcome these barriers. Combining bacteriocins with silver nanoparticles (Ag-NPs) may show improved antimicrobial activity against food-spoiling bacteria. In this work, we have deliberated the various types of bacteriocin, their inherent properties, and their multifaceted mode of action against food pathogens. Moreover, the potential application of bacteriocin-silver nano-conjugation in the food sector is also discussed.

Plant defensins (PDs) display a CSαβ-fold that lacks a canonical hydrophobic core. They display almost all hydrophobic residues on the protein surface. The exposed hydrophobic residues form surface clusters stabilized by the vicinity of hydrophilic residues and the hydration shell. Here, we used Psd2 as a model to study the formation and stabilization of these local foldons named surface hydrophobic clusters (SHC). We characterized the temperature dependence of 15N CPMG relaxation dispersion profiles to describe the complex dynamics of Psd2 and indirectly study the thermodynamics of SHCs in PDs. We show a correlation between residues undergoing conformational exchange and the SHCs. Chemical shift changes between the native ground state and the first thermally accessible excited state enabled us to map the major conformational changes in Psd2 conformational equilibrium. The observation of a cold-driven excited state revealed that SHCs are stabilized by hydrophobic contacts, which are exposed at low temperatures, leading to a favorable decrease in enthalpy compensated by an unfavorable entropy reduction. At higher temperatures, we detected another excited conformer that may play a role in membrane-specific interaction, as previously described for other defensins.

Antimicrobial peptides (AMPs) are used to treat many infectious diseases. Nowadays, AMPs enjoy special attention due to their novel mode of action and kill various pathogens such as fungi, bacteria and viruses. Among insects, the ant genus can provide a potential antimicrobial peptide against various microorganisms. In the present study, AMPs compounds were isolated and purified from the ant species Trichomyrmex scabriceps using reversed-phase high performance liquid chromatography (RP-HPLC). The extract from abdomen showed the highest protein content (4.60 µg/µl) compared to head and thorax. The HPLC profile of the abdomen showed 14 different peaks, of which the peaks eluting at the 6th, 8th, 17th and 23rd minutes had a higher intensity. The number of peaks was comparatively lower in the extracts from the head and thorax. The antimicrobial activity of the head, thorax and abdomen extracts of ants was evaluated against Gram-positive Staphylococcus aureus (MTCC 3160) and Gram-negative Escherichia coli (MTCC 2692) bacterial strains. Among the extracts tested, the abdomen alone exhibited a distinct zone of inhibition against E. coli (176.98 ± 3.36 mm2). However, the extracts of the head and thorax showed no zone of inhibition. In addition, none of the extracts showed a zone of inhibition against S. aureus. To our knowledge, this is the first report observed AMPs in T. scabriceps ant and their antimicrobial property against these bacterial strains.

The phenomenon of antimicrobial resistance threatens our ability to treat common infections. The clinical pipeline for new antimicrobials is pretty much dry and hence, there is a need for the development of new antimicrobial agents with low toxicities to help fight resistant microorganisms. This work aimed to design antimicrobial peptides with low toxicities using a database filtering technology and evaluate their bioactivities. The physicochemical properties of the designed peptides were explored with molecular dynamics (MD) simulations. Microbroth dilution and hemolytic assays were used to assess the peptides' antimicrobial activity and toxicity. The activity of combinations of the peptides and some standard antibiotics was tested by the checkerboard method. In general, the designed peptides had a charge of +2, chain length of 13, and hydrophobicity of 61%. The predicted secondary structures of the peptides were either extended conformations or alpha-helices, and these structures were found to fluctuate during the MD simulations, where coils, bends, and helices dominated. , of the peptides, BRG003, BRG004 and BRG002 had the greatest aggregation propensities, whereas BRG001, BRG005, and BRG006 exhibited lower aggregation propensities. The minimum inhibitory concentration (MIC) of the peptides ranged from 0.015 to >1.879 μM, with BRGP-001 exhibiting high activity against MRSA with an MIC of 15 nM. BRGP-005 and BRGP-006 exhibited synergistic effects against Escherichia coliR when used in combination with erythromycin. At the minimum hemolytic concentration, the percentage of lysed erythrocytes was lower for all the peptides in comparison to the reference peptide, indicating low hemolytic activity. The study revealed the importance of peptide self-association in the antimicrobial activity of the peptides. These peptides provide a basis for the design of potent antimicrobial peptides that can further be developed for use in antimicrobial therapy.

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, stands as an immensely devastating and persistently relevant pathogen, claiming the lives of millions each year. This infectious bacterium remains a formidable global health challenge, necessitating urgent attention and comprehensive strategies to combat its profound impact on public health. MTB is a finicky bacterium that manages to sneak into macrophages and fibroblasts to avoid being eliminated. Current first-line treatments allow the control of the spread of an active MTB, but are not capable of effectively controlling when MTB is in its latent phase and struggle against MTB resistant strains. Lipid-based nanoparticles have gained significant attention in the field of tuberculosis nanotechnology treatments, owing to their compelling logical underpinnings, remarkable merits, and acknowledged demerits. These nanoparticles offer a rational approach by harnessing the unique properties of lipids, such as biocompatibility and stability, to encapsulate and protect anti-tuberculosis drugs. Their inherent ability to actively target infected macrophages holds immense promise for precise drug delivery to the infection site, enhancing therapeutic efficacy. However, it is crucial to consider potential limitations, such as the restricted payload capacity due to their small size and challenges in achieving sustained drug release. Despite these challenges, lipid-based nanotechnology represents an exciting frontier for combating drug resistance and advancing tuberculosis treatment strategies, warranting further exploration and development in this field. In addition, we emphasize the characteristics of lipid-based nanoparticles with the ability to improve the administration, stability, and dosage of these molecules. New modified systems are expected to be successful in the coming years as nanotechnology has improved various treatments in other diseases.

Abstract Background Invertebrates like crabs employ their own immune systems to fight against a number of invasive infections. Anti-lipopolysaccharide factors (ALFs) are an important class of antimicrobial peptides (AMPs) exhibiting binding and neutralizing activities against lipopolysaccharides. Results This study identified and characterized a novel homolog of ALF (Pp-ALF) from the blue swimmer crab Portunus pelagicus. Pp-ALF has a 369bp open-reading frame encoding a protein with 123 amino acids. The deduced protein featured an LPS-binding domain and a signal peptide. The predicted tertiary structure of Pp-ALF contains three α helices packed against four β sheets. The deduced amino acid sequence of Pp-ALF had a net positive charge of +10.75 and an isoelectric point of 9.8. Phylogenetic analysis revealed that Pp-ALF has a strong ancestral relationship with crab ALFs. Conclusion Antibacterial, antiviral, antifungal, anticancer, and antibiofilm activities of Pp-ALF could be revealed by in silico prediction tools. Recombinant expression of Pp-ALF was unsuccessful in the Escherichia coli Rosetta-gami expression system due to the cytotoxic effect of the peptide to the host. The toxic effect of Pp-ALF to the host was displayed by membrane permeabilization and death of the host cells by fluorescent staining with Syto9-Propidium Iodide and CTC-DAPI- FITC.

Background & Aims: Loss-of-function variants in the PTPN2 gene are associated with increased risk of inflammatory bowel disease. We recently showed that Ptpn2 is critical for intestinal epithelial cell (IEC) barrier maintenance, IEC–macrophage communication, and modulation of the gut microbiome in mice, restricting expansion of a small intestinal pathobiont associated with inflammatory bowel disease. Here, we aimed to identify how Ptpn2 loss affects ileal IEC subtypes and their function in vivo. Methods: Constitutive Ptpn2 wild-type, heterozygous, and knockout (KO) mice, as well as mice with inducible deletion of Ptpn2 in IECs, were used in the study. Investigation was performed using imaging techniques, flow cytometry, enteroid culture, and analysis of gene and protein levels of IEC markers. Results: Partial transcriptome analysis showed that expression of Paneth cell–associated antimicrobial peptides Lyz1, Pla2g2a, and Defa6 was down-regulated markedly in Ptpn2-KO mice compared with wild-type and heterozygous. In parallel, Paneth cell numbers were reduced, their endoplasmic reticulum architecture was disrupted, and the endoplasmic reticulum stress protein, C/EBP-homologous protein (CHOP), was increased in Ptpn2-KO mice. Despite reduced Paneth cell number, flow cytometry showed increased expression of the Paneth cell–stimulatory cytokines interleukin 22 and interferon γ+ in CD4+ T cells isolated from Ptpn2-KO ileum. Key findings in constitutive Ptpn2-KO mice were confirmed in epithelium-specific Ptpn2ΔIEC mice, which also showed impaired lysozyme protein levels in Paneth cells compared with Ptpn2fl/fl control mice. Conclusions: Constitutive Ptpn2 deficiency affects Paneth cell viability and compromises Paneth cell–specific antimicrobial peptide production. The observed effects may contribute to the increased susceptibility to intestinal infection and dysbiosis in these mice.

Objectives: Multidrug-resistant (MDR) bacteria are a continuously increasing threat for medicine, causing infections recalcitrant to antibiotics. Antimicrobial peptides (AMPs) were identified as alternatives to antibiotics, being naturally occurring short peptides and part of the innate immune system of a vast majority of organisms. However, the clinical application of AMPs is limited by suboptimal pharmacokinetic properties and relatively high toxicity. Combinatorial treatments using AMPs and classical antibiotics may decrease the concentrations of AMPs required for bacterial eradication, thus lowering the side effects of these peptides. Methods: Here, we investigate the in vitro efficiency of combinations of the recently described antimicrobial peptide TAT-RasGAP317-326 with a panel of commonly used antimicrobial agents against three Gram-negative bacteria, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii, using checkerboard and time-kill assays. Results: We identified synergistic combinations towards all three bacteria and demonstrated that these combinations had an increased bactericidal effect compared to individual drugs. Moreover, combinations were also effective against clinical isolates of A. baumannii. Finally, combination of TAT-RasGAP317-326 and meropenem had a promising antibiofilm effect towards A. baumannii. Conclusions: Taken together, our results indicate that combinations of TAT-RasGAP317-326 with commonly used antimicrobial agents may lead to the development of new treatment protocols against infections caused by MDR bacteria.

Objective: The growing microbial resistance to antibiotics is a global public concern, which creates serious needs for newer antimicrobial agents. Antimicrobial peptides (AMPs) are increasingly exploited in drug development as therapeutic candidates. Here, we aimed to design and characterize a novel peptide with broad spectrum antimicrobial activity. Methods: Hybridization and sequence modification approaches were used to design the novel peptide, named HAZ, aiming at optimizing the physicochemical parameters involved in antimicrobial activity. Peptide activities were assessed alone or combined with different selected antibiotics against various sensitive and drug-resistant bacterial strains. In addition, the hemolysis and the cytotoxic activities of HAZ peptide were evaluated on human red blood cells and epithelial adenocarcinoma cells (A549), respectively. Results: HAZ peptide was sequentially modified to result in favored physicochemical parameters (helicity 95.24 %, hydrophobic ratio 47 %, and net charge of 8 + ). Functional assessment of HAZ revealed significant antimicrobial activity, with MIC values of 15 – 20 µM against tested bacterial strains. It also exhibited biofilm eradication activity at slightly higher concentrations. HAZ-antibiotics combinations exhibited a synergistic action mode that led to dramatic decrease in the MIC values for both HAZ peptide and the antibiotic. Such efficacy was accompanied with minimal hemolytic toxicity on human erythrocytes. Importantly, HAZ displayed promising anticancer activity against human lung cancer cells. Conclusion: Rationally-designed antimicrobial peptides offer promising alternatives to the current antibiotics for management of infectious diseases. HAZ peptide is a broad-spectrum AMP, and a promising candidate for antimicrobial and anticancer drug development.

A feeding trial was carried out for 30 days to investigate the impacts of dietary immunity induced A. pernyi pupae powder (IPP) and A. pernyi cecropin (AC) on the growth, immune-related enzyme activities and disease resistance in Yesso scallop, Patinopecten yessoensis. A total of 320 scallops, with an average weight of 54.75 ± 0.07 g were randomly divided into four treatment groups: control group (provided with a diet of spirulina powder and microalgae), groups with basic diet supplemented with A. pernyi pupae powder (NPP, 2 % of the final concentration), immunity induced A. pernyi pupae powder (IPP, 2 % of the final concentration), and A. pernyi cecropin (AC, 2 % of the final concentration), respectively. The results showed that the specific growth rate (SGR), lysozyme (LSZ), acid phosphatase (ACP) and superoxide dismutase (SOD) were significantly higher in the scallops fed with AC than in the other groups (P

Acyldepsipeptides (ADEPs) are receiving more attention as prospective antimicrobial agents due to their unique mode of action and chemical properties. However, their therapeutic potential is limited by their poor pharmacokinetic properties. Chemical modifications have been successful in improving the biocompatibility and bioavailability of ADEPs. In the current study, ADEP1 was modified by introducing a disulphide linkage, replacement of the octa-2,4,6-trienoic acid (OTEA) with either adamantane (Ada) or palmitic acid (Pal), and lastly, comparing the use of D versus L amino acids. The antibacterial effects of the ADEP1 analogues were investigated in Gram-positive and Gram-negative strains using agar well diffusion and microdilution assays. Cytotoxicity was evaluated in human embryonic kidney (HEK)-293 and colon cancer (Caco-2) cells by the MTS assay. Using solid phase peptide synthesis (SPPS), the percentage yield of the synthetic peptides was increased to > 37% with > 96% purity. The anionic ADEP1 analogues demonstrated a broad-spectrum antibacterial activity. Although the ADEP1 analogues did not display the expected antibacterial activity in relation to the parent structure, they were not cytotoxic against the tested cell lines. This study proved that biocompatibility of natural ADEPs can be improved by modifying some of its chemical groups. Further studies are required to enhance the antibacterial activity of the ADEP1 analogues either by structural modifications or through supplementation of these anionic antimicrobial peptides (AMPs) with divalent metal ions.

ABSTRACT: Kawal is a fermented food condiment obtained by spontaneous fermentation of Senna obtusifolia leaves. Fermentative microbiota's consists of microorganisms producing a diversity of bioactive molecules. Thus, research of novel microbial products capable of inhibiting or eliminating plant and food pathogens is an absolute necessity. In this study, 126 bacterial strains were isolated from Kawal. Phenotypical, biochemical and genotypic methods were used to determine their genus or species. Forty-three (43) of them belonging to the genus Bacillus were selected for their antibacterial and antifungal properties. Their ability to produce lipopeptides (fengycins, surfactins, iturins, kurstakins) was investigated using HPLC and Matrix-assisted Laser Desorption Ionization-Time of Flight (MALDI-ToF-MS). Molecular characterization of 16S rRNA identified 10 species belonging to the genus Bacillus: B. subtilis, B. licheniformis, B. amyloliquefaciens, B. velezensis, B. spizizenii, B. wiedmannii, B. tropicus, B. cereus, B. tequilensis and B. safensis. MALDI-ToF-MS analysis revealed a diversity of antimicrobial molecules. Among all Bacillus strains tested, 84.44% produced surfactin, 22.22% produced fengycin, 11.11% produced iturin and 2.22% produced kurstakin. The results of this study demonstrated the high diversity of lipopeptides among kawal Bacillus strains and their antimicrobial capacity. In view of the potentialities that have been highlighted in these strains, some of them can be used in the bioconservation or in the bioprotection of plants.

Dysfunction of the intestinal epithelial barrier causes microbial invasion that would lead to inflammation in the gut. Antimicrobial peptides (AMPs) are essential components of the intestinal epithelial barrier, while the regulatory mechanisms of AMPs expression are not fully characterized. Here, we report that the ovarian tumor family deubiquitinase 4 (OTUD4) in Paneth cells restricts the expression of AMPs and thereby promotes experimental colitis and bacterial infection. OTUD4 is upregulated in the inflamed mucosa of ulcerative colitis patients and in the colon of mice treated with dextran sulfate sodium salt (DSS). Knockout of OTUD4 promotes the expression of AMPs in intestinal organoids after stimulation with lipopolysaccharide (LPS) or peptidoglycan (PGN) and in the intestinal epithelial cells (IECs) of mice after DSS treatment or Salmonella typhimurium (S.t.) infection. Consistently, Vil-Cre;Otud4fl/fl mice and Def-Cre;Otud4fl/fl mice exhibit hyper-resistance to DSS-induced colitis and S.t. infection compared to Otud4fl/fl mice. Mechanistically, knockout of OTUD4 results in hyper K63-linked ubiquitination of MyD88 and increases the activation of NF-κB and MAPKs to promote the expression of AMPs. These findings collectively highlight an indispensable role of OTUD4 in Paneth cells to modulate AMPs production and indicate OTUD4 as a potential target for gastrointestinal inflammation and bacterial infection.

Antimicrobial peptides (AMPs) are the natural antibiotics recognized for their broad-spectrum resistance to bacteria, fungi, viruses and parasites, and influencing the host immune responses. AMPs attributed to good biological effects have been used in various areas of human health, which are trying to completely replace antibiotics, owing to serious drug-resistance bacteria. However, limited bioactivity and potential biotoxicity of some AMPs was neglected, attributable to their hydrophobic structure with positive charges and nonspecific destruction of cell membranes. Various strategies have been used to design and biosynthetic optimized AMPs to improve their bioactivity, productivity, while lowering host toxicity and cost. Here, we focus on the progress made in understanding the AMPs, including biosynthesis (AMP-BioDesign 1.0 and 2.0), bioactivity (e.g. immune regulation and broad-spectrum or nonspecific actions against bacteria, viruses or parasites), and principle biotoxicity (e.g. hemolysis, acute toxicity and instability, ect). The application prospects of AMP for human health, clinical medicine (for novel drugs), tissue engineering and drug delivery system, respectively, are summarized in this review. Furthermore, future prospects and new strategy for the development of effective and low-toxic AMP formulations for human health are discussed.

Background: Proliferation of bacteria, such as Staphylococcus aureus and Staphylococcus epidermidis, on orthopedic implants has been a challenge in orthopedic surgery, highlighting the problem of the increasing antibiotic resistance and the necessity to develop new antimicrobial agents. In this sense, antimicrobial peptides are promising candidates, which can be attached to titanium surfaces in order to make them safer. Mytilus mussels are characterized by adhering efficiently to a wide variety of surfaces, especially metallic ones, through adhesive proteins with a high content of dihydroxyphenylalanine, which is a post-translational modification of tyrosine. Results: This work refers to the synthesis of a coating based on a bifunctional peptide that combines a sequence derived from the mussel foot protein-5 and the antimicrobial peptide pEM-2, derived from Bothrops asper snake venom. The adhesive properties of this bifunctional peptide were evaluated, as well as the adhesive sequence without the pEM-2, using a Quartz Crystal Microbalance. The results showed that the presence of the antimicrobial peptide improved the adhesion; however, a loss of the bactericidal activity was observed. Even so, the adhesive sequence by itself exhibited an important antifouling activity, preventing S. aureus and S. epidermidis adhesion to titanium by 75% and 45%, respectively, although the result against S. epidermidis was not significant. Conclusions: A 13-residue peptide derived from a natural and biocompatible source, like a Mytilus mussel adhesive protein, could be projected as a protective agent on titanium surfaces against S. aureus and S. epidermidis, being responsible for two thirds of the cases of orthopedic implant infection.How to cite: Gauna A, Mercado L, Guzmán F. Antimicrobial characterization of a titanium coating derived from mussel-glue and Bothrops asper snake venom for the prevention of implant-associated infections caused by Staphylococcus. Electron J Biotechnol 2022;56. https://doi.org/10.1016/j.ejbt.2022.02.001.

Abstract Background Increase of antibiotic resistance in pathogenic microbes necessitated novel molecules for curing infection. Antimicrobial peptides (AMPs) are the gene-encoded evolutionarily conserved small molecules with therapeutic value. AMPs are considered as an alternative drug for conventional antibiotics. Hepcidin, the cysteine-rich antimicrobial peptide, is an important component in innate immune response. In this study, we identified and characterized hepcidin gene from the fish, Catla catla (Indian major carp) and termed it as Cc-Hep. Results Open reading frame of Cc-Hep consists of 261 base pair that encodes 87 amino acids. Cc-Hep is synthesized as a prepropeptide consisting of 24 amino acid signal peptide, 36 amino acid propeptide, and 26 amino acid mature peptide. Sequence analysis revealed that Cc-Hep shared sequence similarity with hepcidin from Sorsogona tuberculata. Phylogenetic analysis indicated that Cc-Hep was grouped with HAMP2 family. Structure analysis of mature Cc-Hep identified two antiparallel beta sheets stabilized by four disulphide bonds and a random coil. The mature peptide region of Cc-Hep has a charge of + 2, isoelectric value 8.23 and molecular weight 2.73 kDa. Conclusion Functional characterization predicted antibacterial, antioxidant, and anticancer potential of Cc-Hep, which can be explored in aquaculture or human health care.

Antimicrobial peptides (AMPs), one of the most promising next-generation antibiotics to address the problem of antibiotic-resistance, have gained increasing attention in recent decades. However, some bottlenecks, such as high manufacturing costs and high toxicity, have greatly hindered their development. To overcome these problems, we developed an efficient modification approach to find the valid active-core fragments of AMPs by mimicking the cleavage process of trypsin-like specificity proteases in silico, and truncating the peptide. Herein, we used the structure of a novel AMP, palustrin-2LTb, as the template and synthesised a set of interceptive peptides using computer-aided design and prediction. Functional screening data indicated that truncated fragment 3 not only maintained and optimised antimicrobial efficacy of the parent peptide but also showed great in vivo therapeutic potential in an MRSA-infected insect larvae model. Overall, the demonstration of the therapeutic efficacy of fragment 3 showcases the efficiency of our approach for future modification of AMPs.

Host defense peptides (HDPs) are short cationic peptides that play a key role in the innate immune response of all living organisms. Their action mechanism does not depend on the presence of protein receptors, but on their ability to target and disrupt the membranes of a wide range of pathogenic and pathologic cells which are recognized by their specific compositions, typically with a relatively high concentration of anionic lipids. Lipid profile singularities have been found in cancer, inflammation, bacteria, viral infections, and even in senescent cells, enabling the possibility to use them as therapeutic targets and/or diagnostic biomarkers. Molecular dynamics (MD) simulations are extraordinarily well suited to explore how HDPs interact with membrane models, providing a large amount of qualitative and quantitative information that, nowadays, cannot be assessed by wet-lab methods at the same level of temporal and spatial resolution. Here, we present SuPepMem, an open-access repository containing MD simulations of different natural and artificial peptides with potential membrane lysis activity, interacting with membrane models of healthy mammal, bacteria, viruses, cancer or senescent cells. In addition to a description of the HDPs and the target systems, SuPepMem provides both the input files necessary to run the simulations and also the results of some selected analyses, including structural and MD-based quantitative descriptors. These descriptors are expected to be useful to train machine learning algorithms that could contribute to design new therapeutic peptides. Tools for comparative analysis between different HDPs and model membranes, as well as to restrict the queries to structural and time-averaged properties are also available. SuPepMem is a living project, that will continuously grow with more simulations including peptides of different sequences, MD simulations with different number of peptide units, more membrane models and also several resolution levels. The database is open to MD simulations from other users (after quality check by the SuPepMem team). SuPepMem is freely available under https://supepmem.com/.

The burgeoning menace of antimicrobial resistance across the globe has necessitated investigations into other chemotherapeutic strategies to combat infections. Antimicrobial peptides, or host defense peptides, are a set of promising therapeutic candidates in this regard. Most of them cause membrane permeabilization and are a key component of the innate immune response to pathogenic invasion. It has also been reported that peptide self-assembly is a driving factor governing the microbicidal activity of these peptide candidates. While efforts have been made to develop novel synthetic peptides against various microbes, many clinical trials of such peptides have failed due to toxicity and hemolytic activity to the host. A function-guided rational peptide engineering, based on evolutionary principles, physicochemical properties and activity determinants of AMP activity, is expected to help in targeting specific microbes. Furthermore, it is important to develop a unified understanding of the evolution of AMPs in order to fully appreciate their importance in host defense. This review seeks to explore the evolution of AMPs and the physicochemical determinants of AMP activity. The specific interactions driving AMP self-assembly have also been reviewed, emphasizing implications of this self-assembly on microbicidal and immunomodulatory activity.

The peptide MOp2 obtained from Moringa oleifera seeds showed good antimicrobial activity. However, the stability of its activity has not yet been studied. In the present study, MOp2-loaded thiolated chitosan-stabilized (CMOp2) Pickering emulsion was prepared and applied to prolong the shelf life of grass carp. The encapsulation rate of MOp2 was 57.7% in CMOp2. In addition, the effects of different concentrations of CMOp2 solid particles and pH on droplet size, zeta optional and storage stability of Pickering emulsions were evaluated; the best condition for preparing Pickering emulsion through experiment was 1.75% CMOp2 solid particles at pH 9.5. Moreover, morphological observations and rheological analysis indicated that Pickering emulsions were considered a water-in-oil emulsion with typical non-Newtonian fluid characteristics. Furthermore, the prepared Pickering emulsion could significantly inhibit the growth of Escherichia coli and Staphylococcus aureus. Besides, Pickering emulsion effectively prevented spoilage of grass carp, and the Pickering emulsion-treated group reduced its pH, TVB-N and color values, inhibited microbial growth, and extended shelf life to 9 day at the storage of 4 °C. Overall, the present findings provide a reference for the application of MOp2-loaded Pickering emulsions in food preservation.

According to WHO, to combat the resistant strains, new effective anti-microbial agents are needed on an urgent basis and global researchers should focus their efforts and discovery programs on developing them against antibiotic-resistant pathogens or priority pathogens like ESKAPE. In this context, Cationic antimicrobial peptides (AMPs) are being explored extensively as promising next-generation antimicrobials due to their broad range, fast kinetics and multifunctional role. Despite recent advances, it is still a daunting challenge to identify and design a potent AMP with no cytotoxicity, but with broad specific antimicrobial activity, stability and efficacy under in vivo conditions in a cost-effective and robust manner. In this work, as a proof of concept, we designed novel potent AMPs using artificial intelligence based in silico programs. Shortlisted peptide sequences were synthesized using the fmoc chemistry approach, assessed their antimicrobial activity, cell selectivity, mode of action and in vivo efficacy using a series of experiments. The synthesized peptide analogues demonstrated their antimicrobial activity (MIC in the range of 2.5–80 μM) against bacteria. The identified potential lead molecules showed antibacterial activity in physiological conditions with no signs of cytotoxicity. We further tested the antimicrobial activity of peptide analogues for treating wounds infected with Pseudomonas aeruginosa in the mice burn wound model. In drug-development programs, the identification of lead antimicrobial agents is always challenging and involves screening a large number of molecules which is time-consuming and expensive. This work demonstrates the utility of artificial intelligence based in silico analysis programs in discovering novel antimicrobial agents in an economical, robust way.

Antimicrobial peptides (AMPs) are natural biopolymers in the host defense system. AMPs display multiple functions against bacterial, fungal, and viral infection, as well as anticancer properties. AMPs are promising antiviral and anticancer agents. The average size, net charge, and hydrophobicity residue percentage of these AMPs in the Antimicrobial Peptide Database (APD) are 26 amino acids, 3.5 (+), and 48 %, respectively. However, pharmacokinetic disadvantages such as low proteolytic and chemical stability, high cytotoxicity and hemolytic activity, and salt sensitivity inhibit their clinical application. Given the promising effects of AMPs on antiviral and anticancer features, circumventing the drawbacks of AMPs and improving their efficacy are critically important for clinical application. The structure and amino acid components of AMPs can be modified by several methods, including stabilization, hybridization, cyclization, fragmentation, multimerization, alteration of amino acids, and conjugation or ligation. Furthermore, nanotechnologies are effective tools or strategies to improve their functions and reduce side effects. For example, melittin-nano conjugation can improve its therapeutic efficacy and minimize nonspecific cytotoxicity. Clinical trials are waiting to evaluate the efficacy of the delivery system and novel AMPs in antiviral and anticancer treatments.

Background: Dermaseptins (Drs) are peptides found in the skin secretions of a variety of Hylid frogs, particularly those belonging to the Agalychnis and Phyllomedusa families. Dermaseptin B2 (Drs B2), an amphipathic, α-helical polypeptide was reported as the most active of the Dermaseptin B family. We have previously shown that Drs B2 has strong anti-proliferative activities against RD cells in vitro and thus required further evaluations for future medical applications. Aim: The aim the study was to evaluate the 14-day sub-acute and 90-day sub-chronic toxicities Drs B2 in vivo. Materials and Methods: BALB/c mice were treated with increasing concentrations of 5–25 mg/kg of Drs B2. Rats were treated with 2, 4 and 10-fold concentrations of the calculated LD50 of Drs B2 following OECD recommendations. At the end of the experimentation periods, the animals were sacrificed and dissected to collect blood and selected organs for analysis of any effects caused by Drs B2 treatment on the biochemical, haematological, and histological parameters. Results: The 14-day sub-acute toxicity tests did not cause significant alteration in the biochemical, hematological and histological parameters. The 90-day sub-chronic toxicity study showed lower ALT and AST than control at doses 1.9 mg/kg and 4.6 mg/kg, respectively. Their haematology results also showed higher platelet count than the controls but the differences were not statistically significant. Histological analysis showed increased megakaryocytes in the spleen for both the mice and the rats. Conclusion: The results of this study indicate that short term treatment of Drs B2 could be safe to the animals, however, long-term treatment can have mild effects on the liver parameters and cause an inflammatory response in the spleen.

The number of infections caused by antibiotic-resistant pathogens is increasing alarmingly every year and will continue to grow. Antimicrobial peptides (AMPs) are considered new therapeutic agents for the effective combat against infectious diseases. We report the design of antimicrobial peptides derived from a database of leech metagenome proteins using a machine learning approach. Peptides with antimicrobial activity and reduced toxicity were identified by the CatBoost algorithm. Among them, Hm-AMP2 possesses the most promising application in practice, including antibacterial activity against both Gram-positive and Gram-negative bacteria, low cytotoxic and hemolytic effects. Hm-AMP2 kills various bacteria at low concentrations (4.6–18.5 μM) by the disruption of bacterial membranes. According to nuclear magnetic resonance analysis, the peptide adopts an α-helical structure in a membrane environment. Hm-AMP2 interacts with lipopolysaccharides of different bacteria according to microscale thermophoresis and CD spectroscopy analysis. This effect can play a role in the first defense against the organism’s bacterial invasion. The computational approach developed for the identification of AMP can be useful for the rational design of effective non-toxic peptide antibiotics.

Novel antimicrobial drugs are in high demand due to the increasing emergence of multi-drug resistant bacteria. In recent years, much attention has been given to natural occurring antimicrobial peptides (AMPs) such as indolicidin (ILPWKWPWWPWRR-NH2). In this study, 19 cyclic N-locked indolicidin analogues (3–21) were synthesized successfully by standard 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) combined with a convenient on-resin N-terminus to lysine side-chain cyclization via intramolecular halide substitution using bromoacetic acid. The effect of the ring size on antimicrobial activity and cytotoxicity was initially investigated and subsequently, the effect of fatty acid acylation was explored. We observed that a peptide macrocycle consisting of seven residues was optimal for Gram-positive antibacterial activity. Circular dichroism (CD) spectra of peptide macrocycles (3–13) showed that certain positions of ring-closure induced a conformation seen under sodium dodecyl sulfate (SDS) conditions where Trp side-chains can display exciton coupling. The far-UV CD was consistent with a backbone turn, a conformation with no evidence of helical structure. SAXS data on peptides mixed with lipid vesicles indicate a similar membrane interaction, when comparing the cyclic and linear indolicidin. The most potent analogue identified was 7, which showed antibacterial activity against Gram-positive bacteria (MIC against Staphylococcus aureus ATCC29213 ​= ​12.5 ​μg/mL and Staphylococcus epidermidis HJ56 ​= ​6.3 ​μg/mL). In addition, viable HaCaT cells after 200 ​μg/mL treatment with 7 was 84.3%. The results show that N-locked cyclization of linear antimicrobial peptides provides a method to reduce cytoxicity; although it may affect the antimicrobial activity.

Recently, the lactonase activity of several enzymes (lactonase AiiA, organophosphate hydrolase (His 6 -OPH) and New Delhi metallo-β-lactamase (NDM-1)) was revealed in the hydrolysis of lactone-containing fungal Quorum Sensing molecules (FQSM). This study was aimed at the investigation of possible use of these enzymes as components of antifungal combinations with antimicrobial peptides (AMPs) to increase their action efficiency against various fungi. For this, the interaction of various AMPs with AiiA, NDM-1 or His 6 -OPH, as well as the effect of AMPs on the catalytic characteristics of these enzymes in the hydrolysis of FQSM in enzyme/AMP combinations, were studied using in silico computer modeling methods. Enzymes combinations with 3 AMPs Bacitracin, Colistin and Polymyxin B were selected as the most rational in terms of maintaining the effectiveness of AMP and the catalytic activity of enzymes. The antifungal action of the selected combinations against cells of mycelial fungi and yeast was studied in vitro. It was found that combinations of the enzymes AiiA, His 6 -OPH and NDM-1 with Bacitracin, Colistin and Polymyxin B provide a significant increase in the action efficiency (up to 5000 times) of both AMPs and enzymes against fungi. The most effective variants were obtained for Polymyxin B in multicomponent combinations with enzymes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Elena Efremenko reports financial support was provided by Russian Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)