Functional and structural characterization of recombinant dermcidin-1L, a human antimicrobial peptide

Antimicrobial peptides from human skin are an important component of the innate immune response and play a key role as a first line of defense against infections. One such peptide is the recently discovered dermcidin-1L. To better understand its mechanism and to further investigate its antimicrobial spectrum, recombinant dermcidin-1L was expressed in Escherichia coli as a fusion protein and purified by affinity chromatography. The fusion protein was cleaved by factor Xa protease to produce recombinant dermcidin-1L. Antimicrobial and hemolytic assays demonstrated that dermcidin-1L displayed microbicidal activity against several opportunistic nosocomial pathogens, but no hemolytic activity against human erythrocytes even at concentrations up to 100 microM. Structural studies performed by circular dichroism spectroscopy indicated that the secondary structure of dermcidin-1L was very flexible, and both alpha-helix and beta-sheet structures might be required for the antimicrobial activity. Our results confirmed previous findings indicating that dermcidin-1L could have promising therapeutic potentials and shed new light on the structure-function relationship of dermcidin-1L.     

Structure-activity relations of parasin I, a histone H2A-derived antimicrobial peptide

The structure-activity relations and mechanism of action of parasin I, a 19-amino acid histone H2A-derived antimicrobial peptide, were investigated. Parasin I formed an amphipathic alpha-helical structure (residues 9-17) flanked by two random coil regions (residues 1-8 and 18-19) in helix-promoting environments. Deletion of the lysine residue at the N-terminal [Pa(2-19)] resulted in loss of antimicrobial activity, but did not affect the alpha-helical content of the peptide. The antimicrobial activity was recovered when the lysine residue was substituted with another basic residue, arginine ([R(1)]Pa), but not with polar, neutral, or acidic residues. Progressive deletions from the C-terminal [Pa(1-17), Pa(1-15)] slightly increased the antimicrobial activity (1-4 microg/ml) without affecting the alpha-helical content of the peptide. However, further deletion [Pa(1-14)] resulted in nearly complete loss of antimicrobial activity and alpha-helical structure. Confocal microscopic analysis and membrane permeabilization assays showed that parasin I and its analogs with comparable antimicrobial activities localized to the cell membrane and subsequently permeabilized the outer and cytoplasmic membranes. Pa(1-14) also localized to the cell membrane, but lost membrane-permeabilizing activity, whereas Pa(2-19) showed poor membrane-binding and -permeabilizing activities. The results indicate that the basic residue at the N-terminal is essential for the membrane-binding activity of parasin I, and among the membrane-binding parasin I analogs, the alpha-helical structure is necessary for the membrane-permeabilizing activity.     

Structure and in vitro activities of a Copper II-chelating anionic peptide from the venom of the scorpion Tityus stigmurus

Anionic Peptides are molecules rich in aspartic acid (Asp) and/or glutamic acid (Glu) residues in the primary structure. This work presents, for the first time, structural characterization and biological activity assays of an anionic peptide from the venom of the scorpion Tityus stigmurus, named TanP. The three-dimensional structure of TanP was obtained by computational modeling and refined by molecular dynamic (MD) simulations. Furthermore, we have performed circular dichroism (CD) analysis to predict TanP secondary structure, and UV–vis spectroscopy to evaluate its chelating activity. CD indicated predominance of random coil conformation in aqueous medium, as well as changes in structure depending on pH and temperature. TanP has chelating activity on copper ions, which modified the peptide’s secondary structure. These results were corroborated by MD data. The molar ratio of binding (TanP:copper) depends on the concentration of peptide: at lower TanP concentration, the molar ratio was 1:5 (TanP:Cu²⁺), whereas in concentrated TanP solution, the molar ratio was 1:3 (TanP:Cu²⁺). TanP was not cytotoxic to non-neoplastic or cancer cell lines, and showed an ability to inhibit the in vitro release of nitric oxide by LPS-stimulated macrophages. Altogether, the results suggest TanP is a promising peptide for therapeutic application as a chelating agent.     

Structural and functional characterization of hBD-1(Ser35), a peptide deduced from a DEFB1 polymorphism

beta-Defensins are mammalian antimicrobial peptides that share a unique disulfide-bonding motif of six conserved cysteines. An intragenic polymorphism of the DEFB1 gene that changes a highly conserved Cys to Ser in the peptide coding region has recently been described. The deduced peptide cannot form three disulfide bonds, as one of the cysteines is unpaired. We have determined the cysteine connectivities of a corresponding synthetic hBD-1(Ser35) peptide, investigated the structure by circular dichroism spectroscopy, and assayed the in vitro antimicrobial activity. Despite a different arrangement of the disulfides, hBD-1(Ser35) proved as active as hBD-1 against the microorganisms tested. This activity likely depends on the ability of hBD-1(Ser35) to adopt an amphipathic conformation in hydrophobic environment, similar to the wild type peptide, as suggested by CD spectroscopy.     

Effect of a novel antifungal peptide P852 on cell morphology and membrane permeability of Fusarium oxysporum

P852, a novel cyclic peptide isolated from Bacillus amyloliquefaciens L-H15, showed potent antifungal activity against several major plant fungal pathogens including Fusarium oxysporum. To elucidate the antifungal mechanism, the impact of P852 on the cell morphology and membrane permeabilization of F. oxysporum was studied. By applying electron microscopy and fluorescent techniques, we showed that P852 treatment caused the morphological change of F. oxysporum cells and disrupted its cell structure, including formation of blebs, broken hyphae, deformation of membrane, intracellular organization disruption, pore formation, and cell lysis. Our findings provide insights into the mode of action of P852, which laying a foundation to develop P852 as a novel antifungal agent to control plant fungal pathogens.     

In vitro and in vivo antimicrobial activity of a synthetic peptide derived from the C-terminal region of human chemokine CCL13 against Pseudomonas aeruginosa

Chemokines are important mediators of immunological responses during inflammation and under steady-state conditions. In addition to regulating cell migration, some chemotactic cytokines have direct effects on bacteria. Here, we characterized the antibacterial ability of the synthetic oligopeptide CCL13_57-75, which corresponds to the carboxyl-terminal region of the human chemokine CCL13. In vitro measurements indicated that CCL13_57-75 disrupts the cell membrane of Pseudomonas aeruginosa through a mechanism coupled to an unordered-helicoidal conformational transition. In a murine pneumonic model, CCL13_57-75 improved mouse survival and bacterial clearance and decreased neutrophil recruitment, proinflammatory cytokines and lung pathology compared with that observed in untreated infected animals. Overall, our study supports the ability of chemokines and/or chemokine-derived oligopeptides to act as direct defense agents against pathogenic bacteria and suggests their potential use as alternative antibiotics.     

A strategic approach to the synthesis of ferrocene appended chalcone linked triazole allied organosilatranes: Antibacterial,antifungal, antiparasitic and antioxidant studies

A series of ferrocene appended chalcone allied triazole coupled organosilatranes (FCTSa 7–FCTSa 12) were synthesised with the aim of amalgamating the pharmacological action of the constituting moieties into a single molecular scaffold. All the synthesised silatranes were well characterized by various spectroscopic techniques like IR, ¹H NMR, ¹³C NMR and elemental analysis. Organosilatranes were then evaluated for their biological alacrity against bacterial and fungal strains compared with the standard drugs Rifampicin and Amphotericin B respectively. The ferrocene conjugates were found to be only moderately effective against the tested microbes. However, the organosilatranes conceded excellent efficacy against parasite G. lamblia with FCTSa 11 arraying the leading results. On the other hand against another parasite T. vaginalis, FCTSa 8 has emerged as an outstanding composite. Further, Total Antioxidant Assay (TAA) with 2,2′-azino-bis-3-(ethylbenzothiazoline-6-sulphonic acid) revealed FCTSa 10 to be the best claimant for radical scavenging activity. Along these lines, introducing some different substituents in the synthesised hybrids may act as a useful strategy for increasing the biological profile of the drugs.     

Structure-activity relationships of the intramolecular disulfide bonds in coprisin,a defensin from the dung beetle

Defensins, which are small cationic molecules produced by organisms as part of their innate immune response, share a common structural scaffold that is stabilized by three disulfide bridges. Coprisin is a 43-amino acid defensin-like peptide from Copris tripartitus. Here, we report the intramolecular disulfide connectivity of cysteine-rich coprisin, and show that it is the same as in other insect defensins. The disulfide bond pairings of coprisin were determined by combining the enzymatic cleavage and mass analysis. We found that the loss of any single disulfide bond in coprisin eliminated all antibacterial, but not antifungal, activity. Circular dichroism (CD) analysis showed that two disulfide bonds, Cys20-Cys39 and Cys24-Cys41, stabilize coprisin’s α-helical region. Moreover, a BLAST search against UniProtKB database revealed that coprisin’s α-helical region is highly homologous to those of other insect defensins. [BMB Reports 2014; 47(11): 625-630]     

Spectroscopic investigations of the binding mechanisms between antimicrobial peptides and membrane models of Pseudomonas aeruginosa and Klebsiella pneumoniae

CD spectroscopy was used to investigate the interactions of a series of synthetic AMPs with LPS isolated from Pseudomonas aeruginosa and Klebsiella pneumoniae, as well as with various phospholipids to better approximate the chemical composition of the membranes of these two strains of Gram-negative bacteria. This investigation was conducted in order to probe how the contributions of key physicochemical properties of an AMP vary in different regions of the membranes of these two bacteria. The conclusions from this study are as follows. (1) The binding interactions between the AMP and the membranes are defined by the complementarity of delocalization of positive charge density of the basic amino side chains (i.e., electrostatics), molecular flexibility of the peptide backbone, and overall hydrophobicity. (2) The binding interactions of these AMPs to LPS seem to be predominantly with the lipid A region of the LPS. (3) Incorporation of phospholipids into the LPS containing SUVs resulted in dramatic changes in the conformational equilibrium of the bound AMPs. (4) For the LPS-phospholipid models of Pseudomonas aeruginosa, delocalization of the side chain positive charge plays a major role in determining the number of conformers that contribute to the binding conformational equilibrium. This relationship was not observed for the models of the outer and inner membranes of Klebsiella pneumoniae.     

Membrane-induced peptide structural changes monitored by infrared and circular dichroism spectroscopy

As more peptide secondary structures deduced by infrared spectroscopy (IR) have been reported in the literature, there have been overlaps in assignments of elements of secondary structure to carbonyl vibrational frequencies. We have investigated this phenomenon with regards to the use of IR for monitoring membrane-induced structural changes using conformationally diverse peptides. These IR studies, complemented by circular dichroism (CD) experiments, revealed that peptide–solvent interactions can mask membrane-induced conformational changes monitored by IR. A structural transition from random coil to α-helix upon the binding of mastoparan X to a membrane was clearly observed by CD but obscured in the amide I region of the IR spectrum. In addition, unlike the buried helical peptides gramicidin D and P16 in micelles, the amide II peak for mastoparan X was absent, likely due to H–D exchange. This suggests information on the peptide's membrane-bound solvent accessibility could be obtained from this region of the spectrum.     

Conformations of a synthetic peptide which facilitates the cellular delivery of nucleic acids

We describe the synthesis of an amphipathic vectorpeptide which is able to form complexes with nucleicacids. Based on circular dichroism investigations, the nature of thestructure obtained in water is questioned. Thepeptide adopts an -helical structure in TFEand is partially in a -sheet conformation inphosphate buffer at low peptide concentrations.     

Conformations of a synthetic peptide which facilitates the cellular delivery of nucleic acids

Summary We describe the synthesis of an amphipathic vector peptide which is able to form complexes with nucleic acids. Based on circular dichroism investigations, the nature of the structure obtained in water is questioned. The peptide adopts an α-helical structure in TFE and is partially in a β-sheet conformation in phosphate buffer at low peptide concentrations.     

Cell penetrating peptide TAT can kill cancer cells via membrane disruption after attachment of camptothecin

Attachment of traditional anticancer drugs to cell penetrating peptides is an effective strategy to improve their application in cancer treatment. In this study, we designed and synthesized the conjugates TAT-CPT and TAT-2CPT by attaching camptothecin (CPT) to the N-terminus of the cell penetrating peptide TAT. Interestingly, we found that TAT-CPT and especially TAT-2CPT could kill cancer cells via membrane disruption, which is similar to antimicrobial peptides. This might be because that CPT could perform as a hydrophobic residue to increase the extent of membrane insertion of TAT and the stability of the pores. In addition, TAT-CPT and TAT-2CPT could also kill cancer cells by the released CPT after they entered cells. Taken together, attachment of CPT could turn cell penetrating peptide TAT into an antimicrobial peptide with a dual mechanism of anticancer action, which presents a new strategy to develop anticancer peptides based on cell penetrating peptides.     

Bombinins, antimicrobial peptides from Bombina species

The skin secretions of Bombina species contain peptides and small proteins with interesting biological properties. These include bombesin, thyrotropin releasing hormone, BSTI and Bv8. In this review, the biosynthesis and antimicrobial activity of two groups of peptides, bombinins and bombinins H, are described. To date, these have only been found in Bombina skin. They are derived from common precursors containing one or two bombinin copies at the amino and a single bombinin H at the carboxyl end. Bombinins are active against Gram-positive and Gram-negative bacteria and fungi but virtually inactive in haemolysis assays. Conversely, bombinins H have lower bactericidal activities but lyse erythrocytes. In the skin secretions, bombinins H are present in two sizes with either 20 or 17 amino acids. Moreover, they occur as epimers with either an l- or a d-amino acid at position 2. An enzyme catalyzing this inversion of chirality of an amino acid in peptide linkage has been isolated from Bombina skin secretions. In different tests, also with different stages of the life cycle of Leishmania parasites, the d-forms were found to be more active. Biophysical studies have yielded some insight into the different behaviours of the epimers in model membranes.     

Biological activity and structural aspects of PGLa interaction with membrane mimetic systems

Peptidyl-glycine-leucine-carboxyamide (PGLa), isolated from granular skin glands of Xenopus laevis, is practically devoid of secondary structure in aqueous solution and in the presence of zwitterionic phospholipids, when added exogenously, but adopts an α-helix in the presence of anionic lipids. The peptide was shown to exhibit antifungal activity and to have antimicrobial activity towards both Gram-negative and Gram-positive bacteria. As a broad variety of peptides is found in the secretions of amphibian skin combinatorial treatment of PGLa and magainin 2 was studied showing enhanced activity by a heterodimer formation. Thus production of mutually recognizing peptides seems to be an effective way in nature to increase selective membrane activity. Biophysical studies on membrane mimics demonstrated that PGLa can discriminate between different lipid species, preferentially interacting with negatively charged lipids, which are major components of bacterial but not mammalian cell membranes. This emphasizes the role of electrostatic interactions as a major determinant to trigger the affinity of antimicrobial peptides towards bacterial membranes. PGLa induced the formation of a quasi-interdigitated phase in phosphatidylglycerol bilayers below their chain melting transition, which is due to the creation of voids below the peptide being aligned parallel to the membrane surface. In the fluid phase of phosphatidylglycerol the peptide inserts perpendicularly into the bilayer above a threshold concentration, which results in a hydrophobic mismatch of the peptide length and bilayer core for lipids ≤ C16. This mismatch is compensated by stretching of the acyl chains and in turn thickening of the bilayer demonstrating that membrane thinning cannot be taken generally as the hallmark of pore formation by antimicrobial peptides. Furthermore, PGLa was shown to affect membrane curvature strain of phosphatidylethanolamine, another main lipid component of bacterial membranes, where a cubic phase coexists with the fluid bilayer phase. Investigations on living Escherichia coli showed distinct changes in cell envelope morphology, when treated with the peptide. In a first stage loss of surface stiffness and consequently of topographic features was observed, followed in a second stage by permeabilization of the outer membrane and rupture of the inner (cytoplasmic) membrane supposedly by the mechanism(s) derived from model studies.     

Synthesis and CD studies of an 88-residue peptide containing the main receptor binding site of HTLV-I SU-glycoprotein

Essential HTLV-I biological functions, like host-cell receptor recognition, depend on the structural motives on the surface glycoprotein gp46. We defined a peptide of 88 amino acids [Arg¹⁴⁷-Leu²³⁴] corresponding to the central part of the protein sequence, where major neutralizing epitopes are localized. After evaluating the feasibility of its chemical synthesis, the chosen sequence was realized using the stepwise solid-phase methodology. Multiple chromatographic purification steps were required to obtain a sample suitable for structural analysis. Correct folding was supported by strong binding of monooclonal antibodies, recognizing known exposed immunodominant regions. Circular dichroism studies confirmed a non-random conformation of at least 70–80% of the synthetic peptide. Investigation of the 3D-structure of the synthetic peptide will provide useful information for future vaccine and drug-design strategies. © 1997 European Peptide Society and John Wiley & Sons, Ltd. J. Pep. Sci.3: 347–353 No. of Figures: 5. No. of Tables: 0. No. of References: 23     

Indolicidin,a 13-residue basic antimicrobial peptide rich in tryptophan and proline,interacts with Ca(2+)-calmodulin

Indolicidin, ILPWKWPWWPWRR-NH(2), a short 13-residue antimicrobial and cytolytic peptide characterized from bovine neutrophils, has the calmodulin-recognition 1-5-10 hydrophobic pattern (indicated by amino acids in bold), is cationic, and thereby fulfills the requirements to interact with calmodulin. Hence, we have investigated the calmodulin-binding properties of indolicidin. Indolicidin interacted with calmodulin with fairly high affinity in a Ca(2+)-dependent manner. However, when bound, the peptide did not adopt helical conformation. Indolicidin also inhibited calmodulin-stimulated phosphodiesterase activity with IC(50) values in the nanomolar range. Replacement of either the proline residues of indolicidin with alanines or tryptophan residues with phenylalanines did not affect binding to calmodulin. However, these replacements had distinctive effects on the conformations of the bound peptides. While the alanine analog of indolicidin adopted predominantly alpha-helical conformation, the phenylalanine analog remained largely unordered. Differences in the ability of these analogs to inhibit the calmodulin-stimulated phosphodiesterase activity were observed. While the alanine analog was capable of inhibiting the activity with IC(50) values comparable to that of indolicidin, the phenylalanine analog did not inhibit the activity. Our results indicate that ability to adopt amphiphilic alpha-helical structure is not a prerequisite for binding to calmodulin and also binding does not necessarily result in inhibition of calmodulin-stimulated enzyme activities.     

Antimicrobial activity and conformation of gaegurin-6 amide and its analogs

A function of the intra-disulfide bridge located at the C-terminal of Rana peptides has not been extensively studied. To investigate the function of the disulfide bridge related to the activity and the structure, we chose Gaegurin-6, isolated from Rana rugosa as a model peptide and synthesized linear analogs. The reduction of the disulfide bridge resulted in the complete loss of antimicrobial activity while replacements of cysteines by serines retained antimicrobial activity. Circular dichroism spectra from a titration of the peptides in sodium dodecyl sulfate indicated that the disulfide bridge of Gaegurin-6 might stabilize the induction of an helical structure in lipid membranes and the helical forming propensity of the peptides correlated with antimicrobial activity.     

Structure-activity relationship of an antibacterial peptide, maculatin 1.1, from the skin glands of the tree frog, Litoria genimaculata.

Maculatin 1.1 (Mac) is a cationic antibacterial peptide isolated from the dorsal glands of the tree frog, Litoria genimaculata, and has a sequence of GLFGVLAKVAAHVVPAIAEHF-NH2. A short peptide lacking the N-terminal two residues of Mac was reported to have no activity. To investigate the structure-activity relationship in detail, several analogs and related short peptides of Mac were synthesized. CD measurement showed that all the peptides took more or less an alpha-helical structure in the presence of anionic lipid vesicles. Analogs which are more basic than Mac had strong antibacterial and hemolytic activities, while short peptides lacking one or two terminal residues exhibited weak or no activity. Outer and inner membrane permeabilization activities of the peptides were also reduced with shortening of the peptide chain. These results indicate that the entire chain length of Mac is necessary for full activity, and the basicity of the peptides greatly affects the activity.