Novel non-helical antimicrobial peptides insert into and fuse lipid model membranes.

This research addresses the growing menace of antibiotic resistance by exploring antimicrobial peptides (AMPs) as alternatives to conventional antibiotics. Specifically, we investigate two linear amphipathic AMPs, LE-53 (12-mer) and LE-55 (16-mer), finding that the shorter LE-53 exhibits greater bactericidal activity against both Gram-negative (G(-)) and Gram-positive (G(+)) bacteria. Remarkably, both AMPs are non-toxic to eukaryotic cells. The heightened effectiveness of LE-53 is attributed to its increased hydrophobicity (H) compared to LE-55. Circular dichroism (CD) reveals that LE-53 and LE-55 both adopt ß-sheet and random coil structures in lipid model membranes (LMMs) mimicking G(-) and G(+) bacteria, so secondary structure is not the cause of the potency difference. X-ray diffuse scattering (XDS) reveals increased lipid chain order in LE-53, a potential key distinction. Additionally, XDS study uncovers a significant link between LE-53's upper hydrocarbon location in G(-) and G(+) LMMs and its efficacy. Neutron reflectometry (NR) confirms the AMP locations determined using XDS. Solution small angle X-ray scattering (SAXS) demonstrates LE-53's ability to induce vesicle fusion in bacterial LMMs without affecting eukaryotic LMMs, offering a promising strategy to combat antibiotic-resistant strains while preserving human cell integrity, whereas LE-55 has a smaller ability to induce fusion.

Pap12-6: A host defense peptide with potent immunomodulatory activity in a chicken hepatic cell culture.

In the fight against antimicrobial resistance, host defense peptides (HDPs) are increasingly referred to as promising molecules for the design of new antimicrobial agents. In terms of their future clinical use, particularly small, synthetic HDPs offer several advantages, based on which their application as feed additives has aroused great interest in the poultry sector. However, given their complex mechanism of action and the limited data about the cellular effects in production animals, their investigation is of great importance in these species. The present study aimed to examine the immunomodulatory activity of the synthetic HDP Pap12-6 (PAP) solely and in inflammatory environments evoked by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (Poly I:C), in a primary chicken hepatocyte-non-parenchymal cell co-culture. Based on the investigation of the extracellular lactate dehydrogenase (LDH) activity, PAP seemed to exert no cytotoxicity on hepatic cells, suggesting its safe application. Moreover, PAP was able to influence the immune response, reflected by the decreased production of interleukin (IL)-6, IL-8, and "regulated on activation, normal T cell expressed and secreted"(RANTES), as well as the reduced IL-6/IL-10 ratio in Poly I:C-induced inflammation. PAP also diminished the levels of extracellular H2O2 and nuclear factor erythroid 2-related factor 2 (Nrf2) when applied together with Poly I:C and in both inflammatory conditions, respectively. Consequently, PAP appeared to display potent immunomodulatory activity, preferring to act towards the cellular anti-inflammatory and antioxidant processes. These findings confirm that PAP might be a promising alternative for designing novel antimicrobial immunomodulatory agents for chickens, thereby contributing to the reduction of the use of conventional antibiotics.

LL37 Microspheres Loaded on Activated Carbon-chitosan Hydrogel: Anti-bacterial and Anti-toxin Wound Dressing for Chronic Wound Infections.

Antimicrobial peptide LL37 is a promising antibacterial candidate due to its potent antimicrobial activity with no known bacterial resistance. However, intrinsically LL37 is susceptible to degradation in wound fluids limits its effectiveness. Bacterial toxins which are released after cell lysis are found to hinder wound healing. To address these challenges, encapsulating LL37 in microspheres (MS) and loading the MS onto activated carbon (AC)-chitosan (CS) hydrogel. This advanced wound dressing not only protects LL37 from degradation but also targets bacterial toxins, aiding in the healing of chronic wound infections. First, LL37 MS and LL37-AC-CS hydrogel were prepared and characterised in terms of physicochemical properties, drug release, and peptide-polymer compatibility. Antibacterial and antibiofilm activity, bacterial toxin elimination, cell migration, and cell cytotoxicity activities were investigated. LL37-AC-CS hydrogel was effective against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. LL37-AC-CS hydrogel bound more endotoxin than AC with CS hydrogel alone. The hydrogel also induced cell migration after 72 h and showed no cytotoxicity towards NHDF after 72 h of treatment. In conclusion, the LL37-AC-CS hydrogel was shown to be a stable, non-toxic advanced wound dressing method with enhanced antimicrobial and antitoxin activity, and it can potentially be applied to chronic wound infections to accelerate wound healing.

Multimodal binding and inhibition of bacterial ribosomes by the antimicrobial peptides Api137 and Api88.

Proline-rich antimicrobial peptides (PrAMPs) inhibit bacterial protein biosynthesis by binding to the polypeptide exit tunnel (PET) near the peptidyl transferase center. Api137, an optimized derivative of honeybee PrAMP apidaecin, inhibits protein expression by trapping release factors (RFs), which interact with stop codons on ribosomes to terminate translation. This study uses cryo-EM, functional assays and molecular dynamic (MD) simulations to show that Api137 additionally occupies a second binding site near the exit of the PET and can repress translation independently of RF-trapping. Api88, a C-terminally amidated (-CONH2) analog of Api137 (-COOH), binds to the same sites, occupies a third binding pocket and interferes with the translation process presumably without RF-trapping. In conclusion, apidaecin-derived PrAMPs inhibit bacterial ribosomes by multimodal mechanisms caused by minor structural changes and thus represent a promising pool for drug development efforts.

Antimicrobial Peptides towards Clinical Application-A Long History to Be Concluded.

Antimicrobial peptides (AMPs) are molecules with an amphipathic structure that enables them to interact with bacterial membranes. This interaction can lead to membrane crossing and disruption with pore formation, culminating in cell death. They are produced naturally in various organisms, including humans, animals, plants and microorganisms. In higher animals, they are part of the innate immune system, where they counteract infection by bacteria, fungi, viruses and parasites. AMPs can also be designed de novo by bioinformatic approaches or selected from combinatorial libraries, and then produced by chemical or recombinant procedures. Since their discovery, AMPs have aroused interest as potential antibiotics, although few have reached the market due to stability limits or toxicity. Here, we describe the development phase and a number of clinical trials of antimicrobial peptides. We also provide an update on AMPs in the pharmaceutical industry and an overall view of their therapeutic market. Modifications to peptide structures to improve stability in vivo and bioavailability are also described.

Lysine-homologue substitution: Impact on antimicrobial activity and proteolytic stability of cationic stapled heptapeptides.

Numerous natural antimicrobial peptides (AMPs) exhibit a cationic amphipathic helical conformation, wherein cationic amino acids, such as lysine and arginine, play pivotal roles in antimicrobial activity by aiding initial attraction to negatively charged bacterial membranes. Expanding on our previous work, which introduced a de novo design of amphipathic helices within cationic heptapeptides using an 'all-hydrocarbon peptide stapling' approach, we investigated the impact of lysine-homologue substitution on helix formation, antimicrobial activity, hemolytic activity, and proteolytic stability of these novel AMPs. Our results demonstrate that substituting lysine with ornithine enhances both the antimicrobial activity and proteolytic stability of the stapled heptapeptide AMP series, while maintaining low hemolytic activity. This finding underscores lysine-homologue substitution as a valuable strategy for optimizing the therapeutic potential of diverse cationic AMPs.

pDNA-tachyplesin treatment stimulates the immune system and increases the probability of apoptosis in MC4-L2 tumor cells.

Breast cancer is the most common cancer among women worldwide, and marine creatures are the most abundant reservoir of anticancer medicines. Tachyplesin peptides have shown antibacterial capabilities, but their potential to inhibit cancer growth and trigger cancer cell death has not been investigated. A synthetic tachyplesin nucleotide sequence was generated and inserted into the pcDNA3.1( +) Mammalian Expression Vector. PCR analysis and enzyme digesting procedures were used to evaluate the vectors' accuracy. The transfection efficiency of MCF-7 and MCF10-A cells was 57% and 65%, respectively. The proliferation of MCF-7 cancer cells was markedly suppressed. Administration of plasmid DNA (pDNA) combined with tachyplesin to mice with tumors did not cause any discernible morbidity or mortality throughout treatment. The final body weight curves revealed a significant reduction in weight among mice treated with pDNA/tachyplesin and tachyplesin at a dose of 100 µg/ml (18.4 ± 0.24 gr, P < 0.05; 11.4 ± 0.24 gr P < 0.01) compared to the control group treated with PBS (22 ± 0.31 gr). Animals treated with pDNA/tachyplesin and tachyplesin exhibited a higher percentage of CD4 + Foxp3 + Tregs, CD8 + Foxp3 + Tregs, and CD4 + and CD8 + T cell populations expressing CTLA-4 in their lymph nodes and spleen compared to the PBS group. The groups that received pDNA/tachyplesin exhibited a substantial upregulation in the expression levels of caspase-3, caspase-8, BAX, PI3K, STAT3, and JAK genes. The results offer new possibilities for treating cancer by targeting malignancies using pDNA/tachyplesin and activating the mTOR and NFκB signaling pathways.

An Antibacterial, Conductive Nanocomposite Hydrogel Coupled with Electrical Stimulation for Accelerated Wound Healing.

Background: Electrical stimulation (ES) can effectively promote skin wound healing; however, single-electrode-based ES strategies are difficult to cover the entire wound area, and the effectiveness of ES is often limited by the inconsistent mechanical properties of the electrode and wound tissue. The above factors may lead to ES treatment is not ideal. Methods: A multifunctional conductive hydrogel dressing containing methacrylated gelatin (GelMA), Ti3C2 and collagen binding antimicrobial peptides (V-Os) was developed to improve wound management. Ti3C2 was selected as the electrode component due to its excellent electrical conductivity, the modified antimicrobial peptide V-Os could replace traditional antibiotics to suppress bacterial infections, and GelMA hydrogel was used due to its clinical applicability in wound healing. Results: The results showed that this new hydrogel dressing (GelMA@Ti3C2/V-Os) not only has excellent electrical conductivity and biocompatibility but also has a durable and efficient bactericidal effect. The modified antimicrobial peptides V-Os used were able to bind more closely to GelMA hydrogel to exert long-lasting antibacterial effects. The results of cell experiment showed that the GelMA@Ti3C2/V-Os hydrogel dressing could enhance the effect of current stimulation and significantly improve the migration, proliferation and tissue repair related genes expression of fibroblasts. In vitro experiments results showed that under ES, GelMA@Ti3C2/V-Os hydrogel dressing could promote re-epithelialization, enhance angiogenesis, mediate immune response and prevent wound infection. Conclusion: This multifunctional nanocomposite hydrogel could provide new strategies for promoting infectious wound healing.

Orostachys malacophylla (pall.) fisch extracts alleviate intestinal inflammation in Drosophila.

ETHNOPHARMACOLOGICAL RELEVANCE: Orostachys malacophylla (Pall.) Fisch (O. malacophylla) is a succulent herbaceous plant that is the Orostachys genus of Crassulaceae family. O. malacophylla has been widely used as a traditional Chinese medicine with antioxidant, anti-inflammatory, anti-febrile, antidote, anti-Toxoplasma gondii properties. However, the biological function of alleviating intestinal inflammation and key bioactive compounds were still unknown. AIM OF THE STUDY: We used a Drosophila model to study the protective effects and bioactive compounds of O. malacophylla water extract (OMWE) and butanol extract (OMBE) on intestinal inflammation. MATERIALS AND METHODS: Drosophila intestinal inflammation was induced by oral invasion of dextran sodium sulfate (DSS) or Erwinia carotovora carotovora 15 (Ecc15). We revealed the protective effects of two extracts by determining intestinal reactive oxygen species (ROS) and antimicrobial peptide (AMP) levels and intestinal integrity, and using network pharmacology analysis to identify bioactive compounds. RESULTS: We demonstrated that both OMWE and OMBE could ameliorate the detrimental effects of DSS, including a decreased survival rate, elevated ROS levels, increased cell death, excessive proliferation of ISCs, acid-base imbalance, and disruption of intestinal integrity. Moreover, the overabundance of lipid droplets (LDs) and AMPs by Ecc15 infection is mitigated by these extracts, thereby enhancing the flies' resistance to adverse stimuli. In addition, we used widely targeted metabolomics and network pharmacology analysis to identify bioactive compounds associated with IBD healing that are present in OMWE and OMBE. CONCLUSIONS: In summary, our research indicates that OMWE and OMBE significantly mitigate intestinal inflammation and have the potential to be effective therapeutic agents for IBD in humans.

Marine-derived antimicrobial peptide piscidin-1 triggers extrinsic and intrinsic apoptosis in oral squamous cell carcinoma through reactive oxygen species production and inhibits angiogenesis.

Cancer of the head and neck encompasses a wide range of cancers, including oral and oropharyngeal cancers. Oral cancer is often diagnosed at advanced stages and has a dismal prognosis. Piscidin-1, a marine antimicrobial peptide (AMP) containing approximately 22 amino acids, also exhibits significant anticancer properties. We investigated the possible anti-oral cancer effects of piscidin-1 and clarified the mechanisms underlying these effects. We treated the oral squamous cell carcinoma cell lines OC2 and SCC4 with piscidin-1. Cell viability and the expression of different hallmark apoptotic molecules, including reactive oxygen species (ROS), were tested using the appropriate MTT assay, flow cytometry and western blotting assays, and human umbilical vein endothelial cell (HUVEC) wound healing, migration, and tube formation (angiogenesis) assays. Piscidin-1 increases cleaved caspase 3 levels to induce apoptosis. Piscidin-1 also increases ROS levels and intensifies oxidative stress in the endoplasmic reticulum and mitochondria, causing mitochondrial dysfunction. Additionally, it decreases the oxygen consumption rates and activity of mitochondrial complexes I-V. As expected, the antioxidants MitoTEMPOL and N-acetylcysteine reduce piscidin-1-induced ROS generation and intracellular calcium accumulation. Piscidin-1 also inhibits matrix metalloproteinase (MMP)-2/-9 expression in HUVECs, affecting migration and tube formation angiogenesis. We demonstrated that piscidin-1 can promote apoptosis via both intrinsic and extrinsic apoptotic pathways and findings indicate that piscidin-1 has anti-proliferative and anti-angiogenic properties in oral cancer treatment. Our study on piscidin-1 thus provides a basis for future translational anti-oral cancer drug research and a new theoretical approach for anti-oral cancer clinical research.

Processes and mechanisms underlying burst of giant unilamellar vesicles induced by antimicrobial peptides and compounds.

To clarify the damage of lipid bilayer region in bacterial cell membrane caused by antimicrobial peptides (AMPs) and antimicrobial compounds (AMCs), their interactions with giant unilamellar vesicles (GUVs) of various lipid compositions have been examined. The findings revealed two main causes for the leakage: nanopore formation in the membrane and burst of GUVs. Although GUV burst has been explained previously based on the carpet model, the supporting evidence is limited. In this review, to better clarify the mechanism of GUV burst by AMPs, AMCs, and other membrane-active peptides, we described current knowledge of the conditions, characteristics, and detailed processes of GUV burst and the changes in the shape of the GUVs during burst. We identified several physical factors that affect GUV burst, such as membrane tension, electrostatic interaction, structural changes of GUV membrane such as membrane folding, and oil in the membrane. We also clarified one of the physical mechanisms underlying the instability of lipid bilayers that are associated with leakage in the carpet model. Based on these results, we propose a mechanism underlying some types of GUV burst induced by these substances: the growth of a nanopore to a micropore, resulting in GUV burst.

Vesicle protrusion induced by antimicrobial peptides suggests common carpet mechanism for short antimicrobial peptides.

Short-cationic alpha-helical antimicrobial peptides (SCHAMPs) are promising candidates to combat the growing global threat of antimicrobial resistance. They are short-sequenced, selective against bacteria, and have rapid action by destroying membranes. A full understanding of their mechanism of action will provide key information to design more potent and selective SCHAMPs. Molecular Dynamics (MD) simulations are invaluable tools that provide detailed insights into the peptide-membrane interaction at the atomic- and meso-scale level. We use atomistic and coarse-grained MD to look into the exact steps that four promising SCHAMPs-BP100, Decoralin, Neurokinin-1, and Temporin L-take when they interact with membranes. Following experimental set-ups, we explored the effects of SCHAMPs on anionic membranes and vesicles at multiple peptide concentrations. Our results showed all four peptides shared similar binding steps, initially binding to the membrane through electrostatic interactions and then flipping on their axes, dehydrating, and inserting their hydrophobic moieties into the membrane core. At higher concentrations, fully alpha-helical peptides induced membrane budding and protrusions. Our results suggest the carpet mode of action is fit for the description of SCHAMPs lysis activity and discuss the importance of large hydrophobic residues in SCHAMPs design and activity.

Development of a defibrinated human blood hemolysis assay for rapid testing of hemolytic activity compared to computational prediction.

Cytotoxicity studies determining hemolytic properties of antimicrobial peptides or other drugs are an important step in the development of novel therapeutics for clinical use. Hemolysis is an affordable, accessible, and rapid method for initial assessment of cellular toxicity for all drugs under development. However, variability in species of red blood cells and protocols used may result in significant differences in results. AMPs generally possess higher selectivity for bacterial cells but can have toxicity against host cells at high concentrations. Knowing the hemolytic activity of the peptides we are developing contributes to our understanding of their potential toxicity. Computational approaches for predicting hemolytic activity of AMPs exist and were tested head-to-head with our experimental results. RESULTS: Starting with an observation of high hemolytic activity of LL-37 peptide against human red blood cells that were collected in EDTA, we explored alternative approaches to develop a more robust, accurate and simple hemolysis assay using defibrinated human blood. We found significant differences between the sensitivity of defibrinated red blood cells and EDTA treated red blood cells. SIGNIFICANCE: Accurately determining the hemolytic activity using human red blood cells will allow for a more robust calculation of the therapeutic index of our potential antimicrobial compounds, a critical measure in their pre-clinical development. CONCLUSION: We introduce a standardized, more accurate protocol for assessing hemolytic activity using defibrinated human red blood cells. This approach, facilitated by the increased commercial availability of de-identified human blood and defibrination methods, offers a robust tool for evaluating toxicity of emerging drug compounds, especially AMPs.

Antibacterial and Hemolytic Activity of Antimicrobial Hydrogels Utilizing Immobilized Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are viewed as potential compounds for the treatment of bacterial infections. Nevertheless, the successful translation of AMPs into clinical applications has been impeded primarily due to their low stability in biological environments and potential toxicological concerns at higher concentrations. The covalent attachment of AMPs to a material's surface has been sought to improve their stability. However, it is still an open question what is required to best perform such an attachment and the role of the support. In this work, six different AMPs were covalently attached to a long-ranged ordered amphiphilic hydrogel, with their antibacterial efficacy evaluated and compared to their performance when free in solution. Among the tested AMPs were four different versions of synthetic end-tagged AMPs where the sequence was altered to change the cationic residue as well as to vary the degree of hydrophobicity. Two previously well-studied AMPs, Piscidin 1 and Omiganan, were also included as comparisons. The antibacterial efficacy against Staphylococcus aureus remained largely consistent between free AMPs and those attached to surfaces. However, the activity pattern against Pseudomonas aeruginosa on hydrogel surfaces displayed a marked contrast to that observed in the solution. Additionally, all the AMPs showed varying degrees of hemolytic activity when in solution. This activity was entirely diminished, and all the AMPs were non-hemolytic when attached to the hydrogels.

[Progress of researches on the antiparasitic activity of antimicrobial peptide LL-37].

Parasitic diseases caused by protozoan and helminth infections are still widespread across the world, notably in tropical and subtropical areas, which threaten the children and adult health. Long-term use of anti-parasitic drugs may result in reduced drug susceptibility and even drug resistance. Antimicrobial peptides have been demonstrated to inhibit parasite growth and development, which has potential antiparasitic values. LL-37, the only human antimicrobial peptide in the cathelicidin family, has been widely investigated. This paper reviews the progress of researches on the antiparasitic activity of LL-37, and discusses the prospects of LL-37 in the research of parasites.

Fur-mediated regulation of hydrogen sulfide synthesis, stress response, and virulence in Edwardsiella piscicida.

The production of endogenous hydrogen sulfide (H2S) is an important phenotype of bacteria. H2S plays an important role in bacterial resistance to ROS and antibiotics, which significantly contributes to bacterial pathogenicity. Edwardsiella piscicida, the Gram-negative pathogen causing fish edwardsiellosis, has been documented to produce hydrogen sulfide. In the study, we revealed that Ferric uptake regulator (Fur) controlled H2S synthesis by activating the expression of phsABC operon. Besides, Fur participated in the bacterial defense against ROS and cationic antimicrobial peptides and modulated T3SS expression. Furthermore, the disruption of fur exhibited a significant in vivo colonization defect. Collectively, our study demonstrated the regulation of Fur in H2S synthesis, stress response, and virulence, providing a new perspective for better understanding the pathogenesis of Edwardsiella.

Vitamin D triggers hCAP18/LL-37 production: Implications for LL-37-induced human osteoblast cytotoxicity.

The human cathelicidin LL-37 shows activity against microorganisms, but it is also cytotoxic to host cells. The CAMP gene codes for the LL-37 precursor hCAP18 which is processed extracellularly to active LL-37. It has previously been shown that vitamin D stimulates CAMP gene activity, but less information is available demonstrating that vitamin D also can increase hCAP18/LL-37 protein production. Here, we show with RT-qPCR that a physiological concentration of vitamin D (50 nM) enhances CAMP mRNA levels by about 170 times in human THP-1 monocyte cells. Stimulation with 50 nM vitamin D increases hCAP18/LL-37 protein contents 3-4 times in THP-1 cell lysates demonstrated by both dot blot analysis and ELISA applying two different hCAP18/LL-37 antibodies. Treatment with the proteasome inhibitor MG132 enhances hCAP18/LL-37 levels, suggesting that turnover of hCAP18/LL-37 protein is regulated by the proteasome. The hCAP18/LL-37 concentration in vitamin D-stimulated THP-1 cells corresponds to 1.04 µM LL-37. Interestingly, synthetic LL-37, at this concentration, reduces viability of human osteoblast-like MG63 cells, whereas the THP-1 cells are less sensitive as demonstrated by the MTT assay. In summary, we show that vitamin D enhances hCAP18/LL-37 production, and that this effect can be of physiological/pathophysiological relevance for LL-37-induced human osteoblast toxicity.

Broad-spectrum activity of membranolytic cationic macrocyclic peptides against multi-drug resistant bacteria and fungi.

The emergence of multidrug-resistant (MDR) strains causes severe problems in the treatment of microbial infections owing to limited treatment options. Antimicrobial peptides (AMPs) are drawing considerable attention as promising antibiotic alternative candidates to combat MDR bacterial and fungal infections. Herein, we present a series of small amphiphilic membrane-active cyclic peptides composed, in part, of various nongenetically encoded hydrophilic and hydrophobic amino acids. Notably, lead cyclic peptides 3b and 4b showed broad-spectrum activity against drug-resistant Gram-positive (MIC = 1.5-6.2 µg/mL) and Gram-negative (MIC = 12.5-25 µg/mL) bacteria, and fungi (MIC = 3.1-12.5 µg/mL). Furthermore, lead peptides displayed substantial antibiofilm action comparable to standard antibiotics. Hemolysis (HC50 = 230 µg/mL) and cytotoxicity (>70 % cell viability against four different mammalian cells at 100 µg/mL) assay results demonstrated the selective lethal action of 3b against microbes over mammalian cells. A calcein dye leakage experiment substantiated the membranolytic effect of 3b and 4b, which was further confirmed by scanning electron microscopy. The behavior of 3b and 4b in aqueous solution and interaction with phospholipid bilayers were assessed by employing nuclear magnetic resonance (NMR) spectroscopy in conjunction with molecular dynamics (MD) simulations, providing a solid structural basis for understanding their membranolytic action. Moreover, 3b exhibited stability in human blood plasma (t1/2 = 13 h) and demonstrated no signs of resistance development against antibiotic-resistant S. aureus and E. coli. These findings underscore the potential of these newly designed amphiphilic cyclic peptides as promising anti-infective agents, especially against Gram-positive bacteria.

An Overview of Frog Skin-Derived Esc Peptides: Promising Multifunctional Weapons against Pseudomonas aeruginosa-Induced Pulmonary and Ocular Surface Infections.

Antimicrobial resistance is a silent pandemic harming human health, and Pseudomonas aeruginosa is the most common bacterium responsible for chronic pulmonary and eye infections. Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics. In this review, the in vitro/in vivo activities of the frog skin-derived AMP Esc(1-21) are shown. Esc(1-21) rapidly kills both the planktonic and sessile forms of P. aeruginosa and stimulates migration of epithelial cells, likely favoring repair of damaged tissue. However, to undertake preclinical studies, some drawbacks of AMPs (cytotoxicity, poor biostability, and limited delivery to the target site) must be overcome. For this purpose, the stereochemistry of two amino acids of Esc(1-21) was changed to obtain the diastereomer Esc(1-21)-1c, which is more stable, less cytotoxic, and more efficient in treating P. aeruginosa-induced lung and cornea infections in mouse models. Incorporation of these peptides (Esc peptides) into nanoparticles or immobilization to a medical device (contact lens) was revealed to be an effective strategy to ameliorate and/or to prolong the peptides' antimicrobial efficacy. Overall, these data make Esc peptides encouraging candidates for novel multifunctional drugs to treat lung pathology especially in patients with cystic fibrosis and eye dysfunctions, characterized by both tissue injury and bacterial infection.

An esculentin-1 homolog from a dark-spotted frog (Pelophylax nigromaculatus) possesses antibacterial and immunoregulatory properties.

BACKGROUND: Esculentin-1, initially discovered in the skin secretions of pool frogs (Pelophylax lessonae), has demonstrated broad-spectrum antimicrobial activity; however, its immunomodulatory properties have received little attention. RESULTS: In the present study, esculentin-1 cDNA was identified by analysing the skin transcriptome of the dark-spotted frog (Pelophylax nigromaculatus). Esculentin-1 from this species (esculentin-1PN) encompasses a signal peptide, an acidic spacer peptide, and a mature peptide. Sequence alignments with other amphibian esculentins-1 demonstrated conservation of the peptide, and phylogenetic tree analysis revealed its closest genetic affinity to esculentin-1P, derived from the Fukien gold-striped pond frog (Pelophylax fukienensis). Esculentin-1PN transcripts were observed in various tissues, with the skin exhibiting the highest mRNA levels. Synthetic esculentin-1PN demonstrated antibacterial activity against various pathogens, and esculentin-1PN exhibited bactericidal activity by disrupting cell membrane integrity and hydrolyzing genomic DNA. Esculentin-1PN did not stimulate chemotaxis in RAW264.7, a murine leukemic monocyte/macrophage cell line. However, it amplified the respiratory burst and augmented the pro-inflammatory cytokine gene (TNF-α and IL-1ß) expression in RAW264.7 cells. CONCLUSIONS: This novel finding highlights the immunomodulatory activity of esculentin-1PN on immune cells.