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Self-assembled peptides are used for diverse applications in the biomedical and technological fields. The morphology and function of the assembled systems are dictated by the peptide sequence and length. In this work, a supramolecular catalyst was obtained upon self-assembly of the diphenylalanine peptide conjugated to a triphenylphosphine Au(I) complex in acetonitrile. The assembled molecules were characterized by spectroscopic techniques and by scanning electron microscopy. The activity of the catalyst was tested on two substrates in cyclization reactions. The morphology and the dimensions of the assembled systems vary depending on the presence of a carboxyl versus an amide C-terminal end. The catalyst efficiently promotes intramolecular cyclization reactions. Results obtained encourage the use of self-assembled peptides for the obtainment of new and efficient catalysts.
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The in-depth studies over the years on the defence barriers by tomato plants have shown that the Systemin peptide controls the response to a wealth of environmental stress agents. This multifaceted stress reaction seems to be related to the intrinsic disorder of its precursor protein, Prosystemin (ProSys). Since latest findings show that ProSys has biological functions besides Systemin sequence, here we wanted to assess if this precursor includes peptide motifs able to trigger stress-related pathways. Candidate peptides were identified in silico and synthesized to test their capacity to trigger defence responses in tomato plants against different biotic stressors. Our results demonstrated that ProSys harbours several repeat motifs which triggered plant immune reactions against pathogens and pest insects. Three of these peptides were detected by mass spectrometry in plants expressing ProSys, demonstrating their effective presence in vivo. These experimental data shed light on unrecognized functions of ProSys, mediated by multiple biologically active sequences which may partly account for the capacity of ProSys to induce defense responses to different stress agents.
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Peptídeos , Proteínas de Plantas , Peptídeos/metabolismo , Proteínas de Plantas/metabolismoRESUMO
Peptide nucleic acids and their conjugates to peptides can self-assemble and generate complex architectures. In this work, we explored the self-assembly of PNA dimers conjugated to the dipeptide WW. Our studies suggest that the indole ring of tryptophan promotes aggregation of the conjugates. The onset of fluorescence is observed upon self-assembly. The structure of self-assembled WWgc is concentration-dependent, being spherical at low concentrations and fibrous at high concentrations. As suggested by molecular modeling studies, fibers are stabilized by stacking interactions between tryptophans and Watson-Crick hydrogen bonds between nucleobases.
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Ácidos Nucleicos Peptídicos , Triptofano , Ácidos Nucleicos Peptídicos/química , Dipeptídeos/química , Peptídeos , Modelos MolecularesRESUMO
Temporins are short peptides secreted by frogs from all over the world. They exert antimicrobial activity, mainly against Gram-positive bacteria, including resistant pathogens; recent studies highlight other possible applications of these peptides as anticancer or antiviral agents. This review is meant to describe the main features of temporins produced by different ranid genera. Due to the abundance of published papers, we focus on the most widely investigated peptides. We report studies on their mechanism of action and three-dimensional structure in model systems mimicking bacterial membranes or in the presence of cells. The design and the antimicrobial activity of peptide analogues is also described, with the aim of highlighting elements that are crucial to improve the bioactivity of peptides while reducing their toxicity. Finally, a short section is dedicated to the studies aimed at applying these peptides as drugs, to produce new antimicrobial materials or in other technological uses.
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Anti-Infecciosos , Ranidae , Animais , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Anuros , PeleRESUMO
Surface functionalization of nanoparticles (NPs) with tumor-targeting peptides is an emerging approach with a huge potential to translate in the clinic and ameliorate the efficacy of nano-oncologicals. One major challenge is to find straightforward strategies for anchoring peptides on the surface of biodegradable NPs and ensuring their correct exposure and orientation to bind the target receptor. Here, we propose a non-covalent strategy to functionalize polyester aminic NPs based on the formation of either electrostatic or lipophilic interactions between NPs and the peptide modified with an anchoring moiety. We selected an iNGRt peptide containing a CendR motif (CRNGR) targeting neuropilin receptor 1 (NRP-1), which is upregulated in several cancers. iNGRt was linked with either a short poly(glutamic acid) chain (polyE) or a palmitoyl chain (Palm) and used to functionalize the surface of NPs made of a diamine poly(ε-caprolactone). iNGRt-PolyE was adsorbed on preformed cationic NPs through electrostatic interaction, whereas iNGRt-Palm was integrated into the forming NPs through interactions. In both cases, peptides were strongly associated with NPs of â¼100 nm, low polydispersity indexes, and positive zeta potential values. NPs entered MDA-MB231 breast cancer cells overexpressing NRP-1 via receptor-mediated endocytosis and showed a different cell localization depending on the mode of peptide anchoring. When loaded with the lipophilic anticancer drug docetaxel (DTX), NPs functionalized with the iNGRt-Palm variant exerted a time- and dose-dependent cytotoxicity similar to DTX in MDA-MB-231 cells but were less toxic than DTX toward control MRC-5 human fibroblasts, not expressing NRP-1. In a heterotopic mouse model of triple negative breast cancer, iNGRt-Palm NPs were tolerated better than free DTX and demonstrated superior anticancer activity and survival compared to both free DTX and NPs without peptide functionalization. We foresee that the functionalization strategy with palmitoylated peptides proposed here can be extended to other biodegradable NPs and peptide sequences designed for therapeutic or targeting purposes.
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Antineoplásicos , Nanopartículas , Neoplasias de Mama Triplo Negativas , Camundongos , Animais , Humanos , Docetaxel , Antineoplásicos/farmacologia , Polímeros , Peptídeos , Linhagem Celular Tumoral , Portadores de FármacosRESUMO
Temporin B (TB) is a 13-amino-acid-long, cationic peptide secreted by the granular glands of the European frog Rana temporaria. We recently showed that the modified TB peptide analog TB_KKG6K rapidly killed planktonic and sessile Candida albicans at low micromolar concentrations and was neither hemolytic nor cytotoxic to mammalian cells in vitro. The present study aimed to shed light into its mechanism of action, with a focus on its fungal cell membrane activity. We utilized different fluorescent dyes to prove that it rapidly induces membrane depolarization and permeabilization. Studies on model membrane systems revealed that the TB analog undergoes hydrophobic and electrostatic membrane interactions, showing a preference for anionic lipids, and identified phosphatidylinositol and cardiolipin as possible peptide targets. Fluorescence microscopy using fluorescein isothiocyanate-labeled TB_KKG6K in the presence of the lipophilic dye FM4-64 indicated that the peptide compromises membrane integrity and rapidly enters C. albicans cells in an energy-independent manner. Peptide-treated cells analyzed by cryo-based electron microscopy exhibited no signs of cell lysis; however, subcellular structures had disintegrated, suggesting that intracellular activity may form part of the killing mechanism of the peptide. Taken together, this study proved that TB_KKG6K compromises C. albicans membrane function, which explains the previously observed rapid, fungicidal mode of action and supports its great potential as a future anti-Candida therapeutic. IMPORTANCE Fungal infections with the opportunistic human pathogen C. albicans are associated with high mortality rates in immunocompromised patients. This is partly due to the yeast's ability to rapidly develop resistance toward currently available antifungals. Small, cationic, membrane-active peptides are promising compounds to fight against resistance development, as many of them effectuate rapid fungal cell death. This fast killing is believed to hamper the development of resistance, as the fungi do not have sufficient time to adapt to the antifungal compound. We previously reported that the synthetic variant of the amphibian TB peptide, TB_KKG6K, rapidly kills C. albicans. In the current study, the mechanism of action of the TB analog was investigated. We show that this TB analog is membrane-active and impairs cell membrane function, highlighting its potential to be developed as an attractive alternative anti-C. albicans therapeutic that may hinder the development of resistance.
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Antifúngicos , Candida albicans , Animais , Anfíbios , Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Candida albicans/efeitos dos fármacos , Cardiolipinas , Fluoresceínas , Corantes Fluorescentes , Isotiocianatos , Fosfatidilinositóis , RanidaeRESUMO
Self-assembly of biomolecules such as peptides, nucleic acids or their analogues affords supramolecular objects, exhibiting structures and physical properties dependent on the amino-acid or nucleobase composition. Conjugation of the peptide diphenylalanine (FF) to peptide nucleic acids triggers formation of self-assembled structures, mainly stabilized by interactions between FF. In this work we report formation of homogeneous chiral fibers upon self-assembly of the hybrid composed of the tetraphenylalanine peptide (4F) conjugated to the PNA dimer adenine-thymine (at). In this case nucleobases seem to play a key role in determining the morphology and chirality of the fibers. When the PNA "at" is replaced by guanine-cytosine dimer "gc", disordered structures are observed. Spectroscopic characterization of the self-assembled hybrids, along with AFM and SEM studies is reported. Finally, a structural model consistent with the experimental evidence has also been obtained, showing how the building blocks of 4Fat arrange to give helical fibers.
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Nanoestruturas , Ácidos Nucleicos Peptídicos , Nanoestruturas/química , Ácidos Nucleicos Peptídicos/química , Peptídeos/química , Fenilalanina/química , Polímeros , TiminaRESUMO
A biodegradable engineered nanoplatform combining anti-angiogenic activity and targeting of cancer cells to improve the anticancer activity of docetaxel (DTX) is here proposed. Indeed, we have developed biodegradable nanoparticles (NPs) of poly(ethylene glycol)-poly(ε-caprolactone), exposing on the surface both folate motifs (Fol) for recognition in cells overexpressing Folate receptor-α (FRα) and the anti-angiogenic hexapeptide aFLT1. NPs showed a size around 100 nm, the exposure of 60% of Fol moieties on the surface, and the ability to entrap DTX and sustain its release with time. NPs were stable in simulated biological fluids and slightly interacted with Fetal Bovine serum, especially in the formulation decorated with Fol and aFLT1. The presence of Fol on NPs did not impair the anti-angiogenic activity of aFLT1, as assessed by in vitro tube formation assay in HUVEC endothelial cells. In both 2D and 3D KB cell cultures in vitro, the cytotoxicity of DTX loaded in NPs was not significantly affected by Fol/aFLT1 double decoration compared to free DTX. Remarkably, NPs distributed differently in 3D multicellular spheroids of FRα-positive KB cancer cells depending on the type of ligand displayed on the surface. In particular, NPs unmodified on the surface were randomly distributed in the spheroid, whereas the presence of Fol promoted the accumulation in the outer rims of the spheroid. Finally, NPs with Fol and aFLT1 gave a uniform distribution throughout the spheroid structure. When tested in zebrafish embryos xenografted with KB cells, NPs displaying Fol/aFLT1 reduced DTX systemic toxicity and inhibited the growth of the tumor mass and associated vasculature synergistically. Overall, nanotechnology offers excellent ground for combining therapeutic concepts in cancer, paving the way to novel multifunctional nanopharmaceuticals decorated with bioactive elements that can significantly improve therapeutic outcomes.
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Antineoplásicos , Nanopartículas , Neoplasias , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Docetaxel/farmacologia , Portadores de Fármacos/química , Células Endoteliais , Ácido Fólico/química , Nanopartículas/química , Neoplasias/tratamento farmacológico , Peixe-ZebraRESUMO
The analysis of the forces governing helix formation and stability in peptides and proteins has attracted considerable interest in order to shed light on folding mechanism. We analyzed the role of hydrophobic interaction, steric hindrance and chain length on i, i + 3 position in QK peptide, a VEGF mimetic helical peptide. We focused on position 10 of QK, occupied by a leucine, as previous studies highlighted the key role of the Leu7-Leu10 interaction in modulating the helix formation and inducing an unusual thermodynamic stability. Leu10 has been replaced by hydrophobic amino acids with different side-chain length, hydrophobicity and steric hindrance. Ten peptides were, hence, synthesized and analyzed combining circular dichroism, calorimetry and NMR spectroscopy. We found that helical content and thermal stability of peptide QK changed when Leu10 was replaced. Interestingly, we observed that the changes in the helical content and thermal stability were not always correlated and they depend on the type of interaction (strength and geometry) that could be established between Leu7 and the residue in position 10.
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Peptídeos/química , Fatores de Crescimento do Endotélio Vascular/química , Interações Hidrofóbicas e Hidrofílicas , Conformação ProteicaRESUMO
Temporin B (TB) is a short, positively charged peptide secreted by the granular glands of the European frog Rana temporaria. While the antibacterial and antiviral efficacy of TB and some of its improved analogs are well documented, nothing is known about their antifungal potency so far. We dedicated this study to characterize the antifungal potential of the TB analog TB_KKG6K and the newly designed D-Lys_TB_KKG6K, the latter having the L-lysines replaced by the chiral counterpart D-lysines to improve its proteolytic stability. Both peptides inhibited the growth of opportunistic human pathogenic yeasts and killed planktonic and sessile cells of the most prevalent human pathogen, Candida albicans. The anti-yeast efficacy of the peptides coincided with the induction of intracellular reactive oxygen species. Their thermal, cation, pH and serum tolerance were similar, while the proteolytic stability of D-Lys_TB_KKG6K was superior to that of its template peptide. Importantly, both peptides lacked hemolytic activity and showed minimal in vitro cytotoxicity in primary human keratinocytes. The tolerance of both peptides in a reconstructed human epidermis model further supports their potential for topical application. Our results open up an exciting field of research for new anti-Candida therapeutic options based on amphibian TB analogs.
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Although a plethora of chemistries have been developed to selectively decorate protein molecules, novel strategies continue to be reported with the final aim of improving selectivity and mildness of the reaction conditions, preserve protein integrity, and fulfill all the increasing requirements of the modern applications of protein conjugates. The targeting of the protein N-terminal alpha-amine group appears a convenient solution to the issue, emerging as a useful and unique reactive site universally present in each protein molecule. Herein, we provide an updated overview of the methodologies developed until today to afford the selective modification of proteins through the targeting of the N-terminal alpha-amine. Chemical and enzymatic strategies enabling the selective labeling of the protein N-terminal alpha-amine group are described.
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Aminas/química , Azidas/química , Proteínas/química , Sítios de Ligação , Química Click/métodos , Técnicas de Sonda Molecular , Domínios ProteicosRESUMO
Peptides and nucleic acids can self-assemble to give supramolecular structures that find application in different fields, ranging from the delivery of drugs to the obtainment of materials endowed with optical properties. Forces that stabilize the "suprastructures" typically are hydrogen bonds or aromatic interactions; in case of nucleic acids, Watson-Crick pairing drives self-assembly while, in case of peptides, backbone hydrogen bonds and interactions between aromatic side chains trigger the formation of structures, such as nanotubes or ribbons. Molecules containing both aromatic peptides and nucleic acids could in principle exploit different forces to self-assemble. In this work we meant to investigate the self-assembly of mixed systems, with the aim to understand which forces play a major role and determine formation/structure of aggregates. We therefore synthesized conjugates of the peptide FF to the peptide nucleic acid dimer "gc" and characterized their aggregates by different spectroscopic techniques, including NMR, CD and fluorescence.
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Ácidos Nucleicos Peptídicos , Ligação de Hidrogênio , Peptídeos , FenilalaninaRESUMO
The chemical structure of oligonucleotide analogues dictates the conformation of oligonucleotide analogue oligomers, their ability to hybridize complementary DNA and RNA, their stability to degradation and their pharmacokinetic properties. In a study aimed at investigating new analogues featuring a neutral backbone, we explored the ability of oligomers containing a morpholino-peptide backbone to bind oligonucleotides. Circular Dichroism studies revealed the ability of our oligomers to interact with DNA, molecular modelling studies revealed the interaction responsible for complex stabilization.
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DNA/metabolismo , Morfolinos/síntese química , Morfolinos/metabolismo , Peptídeos/síntese química , Peptídeos/metabolismo , Dicroísmo Circular , DNA/química , Modelos Moleculares , Conformação Molecular , Morfolinos/química , Peptídeos/químicaRESUMO
Nanofiber films produced by electrospinning currently provide a promising platform for different applications. Although nonfunctionalized nanofiber films from natural or synthetic polymers are extensively used, electrospun materials combined with peptides are gaining more interest. In fact, the selection of specific peptides improves the performance of the material for biological applications and mainly for tissue engineering, mostly by maintaining similar mechanical properties with respect to the simple polymer. The main drawback in using peptides blended with a polymer is the quick release of the peptides. To avoid this problem, covalent linking of the peptide is more beneficial. Here, we reviewed synthetic protocols that enable covalent grafting of peptides to polymers before or after the electrospinning procedures to obtain more robust electrospun materials. Applications and the performance of the new material compared to that of the starting polymer are discussed.
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Nanofibras , Materiais Biocompatíveis , Peptídeos , Polímeros , Engenharia Tecidual , Alicerces TeciduaisRESUMO
Biofilms consist of a complex microbial community adhering to biotic or abiotic surfaces and enclosed within a protein/polysaccharide self-produced matrix. The formation of this structure represents the most important adaptive mechanism that leads to antibacterial resistance, and therefore, closely connected to pathogenicity. Antimicrobial peptides (AMPs) could represent attractive candidates for the design of new antibiotics because of their specific characteristics. AMPs show a broad activity spectrum, a relative selectivity towards their targets (microbial membranes), the ability to act on both proliferative and quiescent cells, a rapid mechanism of action, and above all, a low propensity for developing resistance. This article investigates the effect at subMIC concentrations of Temporin-L (TL) on biofilm formation in Pseudomonas fluorescens (P. fluorescens) both in static and dynamic conditions, showing that TL displays antibiofilm properties. Biofilm formation in static conditions was analyzed by the Crystal Violet assay. Investigation of biofilms in dynamic conditions was performed in a commercial microfluidic device consisting of a microflow chamber to simulate real flow conditions in the human body. Biofilm morphology was examined using Confocal Laser Scanning Microscopy and quantified via image analysis. The investigation of TL effects on P. fluorescens showed that when subMIC concentrations of this peptide were added during bacterial growth, TL exerted antibiofilm activity, impairing biofilm formation both in static and dynamic conditions. Moreover, TL also affects mature biofilm as confocal microscopy analyses showed that a large portion of preformed biofilm architecture was clearly perturbed by the peptide addition with a significative decrease of all the biofilm surface properties and the overall biomass. Finally, in these conditions, TL did not affect bacterial cells as the live/dead cell ratio remained unchanged without any increase in damaged cells, confirming an actual antibiofilm activity of the peptide.
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Peptídeos Catiônicos Antimicrobianos/farmacologia , Biofilmes/efeitos dos fármacos , Polissacarídeos Bacterianos/química , Pseudomonas fluorescens/efeitos dos fármacos , Antibacterianos/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Biomassa , Farmacorresistência Bacteriana/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Microfluídica , Microscopia Confocal , Polímeros/química , Resistência ao Cisalhamento , Estresse Mecânico , Propriedades de SuperfícieRESUMO
The outer membrane (OM) of Gram-negative bacteria is a highly selective permeability barrier due to its asymmetric structure with lipopolysaccharide (LPS) in the outer leaflet. In Escherichia coli, LPS is transported to the cell surface by the LPS transport (Lpt) system composed of seven essential proteins forming a transenvelope bridge. Transport is powered by the ABC transporter LptB2FGC, which extracts LPS from the inner membrane (IM) and transfers it, through LptC protein, to the periplasmic protein LptA. Then, LptA delivers LPS to the OM LptDE translocon for final assembly at the cell surface. The Lpt protein machinery operates as a single device, since depletion of any component leads to the accumulation of a modified LPS decorated with repeating units of colanic acid at the IM outer leaflet. Moreover, correct machine assembly is essential for LPS transit and disruption of the Lpt complex results in LptA degradation. Due to its vital role in cell physiology, the Lpt system represents a good target for antimicrobial drugs. Thanatin is a naturally occurring antimicrobial peptide reported to cause defects in membrane assembly and demonstrated in vitro to bind to the N-terminal ß-strand of LptA. Since this region is involved in both LptA dimerization and interaction with LptC, we wanted to elucidate the mechanism of inhibition of thanatin and discriminate whether its antibacterial effect is exerted by the disruption of the interaction of LptA with itself or with LptC. For this purpose, we here implemented the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system to probe in vivo the Lpt interactome in the periplasm. With this system, we found that thanatin targets both LptC-LptA and LptA-LptA interactions, with a greater inhibitory effect on the former. We confirmed in vitro the disruption of LptC-LptA interaction using two different biophysical techniques. Finally, we observed that in cells treated with thanatin, LptA undergoes degradation and LPS decorated with colanic acid accumulates. These data further support inhibition or disruption of Lpt complex assembly as the main killing mechanism of thanatin against Gram-negative bacteria.
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BACKGROUND: The comprehension of the mechanism of action of antimicrobial peptides is fundamental for the design of new antibiotics. Studies performed looking at the interaction of peptides with bacterial cells offer a faithful picture of what really happens in nature. METHODS: In this work we focused on the interaction of the peptide Temporin L with E. coli cells, using a variety of biochemical and biophysical techniques that include: functional proteomics, docking, optical microscopy, TEM, DLS, SANS, fluorescence. RESULTS: We identified bacterial proteins specifically interacting with the peptides that belong to the divisome machinery; our data suggest that the GTPase FtsZ is the specific peptide target. Docking experiments supported the FtsZ-TL interaction; binding and enzymatic assays using recombinant FtsZ confirmed this hypothesis and revealed a competitive inhibition mechanism. Optical microscopy and TEM measurements demonstrated that, upon incubation with the peptide, bacterial cells are unable to divide forming long necklace-like cell filaments. Dynamic light scattering studies and Small Angle Neutron Scattering experiments performed on treated and untreated bacterial cells, indicated a change at the nanoscale level of the bacterial membrane. CONCLUSIONS: The peptide temporin L acts by a non-membrane-lytic mechanism of action, inhibiting the divisome machinery. GENERAL SIGNIFICANCE: Identification of targets of antimicrobial peptides is pivotal to the tailored design of new antimicrobials.
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Peptídeos Antimicrobianos , Escherichia coli , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Divisão Celular , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/metabolismoRESUMO
The purpose of this work is to set up a general protocol for the production of antimicrobial materials based on cellulose and peptides. We exploited the chemical ligation reaction to achieve the conjugation of peptides to cellulose; to this aim, we produced thioester peptides and cysteine-modified cellulose. As the thioester handle can be inserted at any position of the peptide, the peptide can be immobilized onto the cellulose through its N- or C-terminal end or through any other position within the sequence. Our experiments performed on Escherichia coli cultures show that the cellulose conjugated to the peptides lasioglossin-III and TBKKG6A causes a significant reduction in the concentration of viable cells as compared to unmodified cellulose. In conclusion, antimicrobial peptides bound to cellulose through a covalent bond retain their activity and therefore have the potential to be used as active ingredients in antimicrobial materials.
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Gaining new understanding on the mechanism of action of antimicrobial peptides is the basis for the design of new and more efficient antibiotics. To this aim, it is important to detect modifications occurring to both the peptide and the bacterial cell upon interaction; this will help to understand the peptide structural requirement, if any, at the base of the interaction as well as the pathways triggered by peptides ending in cell death. A limited number of papers have described the interaction of peptides with bacterial cells, although most of the studies published so far have been focused on model membrane-peptides interactions. Investigations carried out with bacterial cells highlighted the limitations connected to the use of oversimplified model membranes and, more importantly, helped to identify molecular targets of antimicrobial peptides and changes occurring to the bacterial membrane. In this review, details on the mechanism of action of antimicrobial peptides, as determined by the application of spectroscopic techniques, as well as scattering, microscopy, and calorimetry techniques, to complex systems such as peptide/bacteria mixtures are discussed.