Giorgia Manzo

Enhanced Amphiphilic Profile of a Short β-Stranded Peptide Improves Its Antimicrobial Activity

SB056 is a novel semi-synthetic antimicrobial peptide with a dimeric dendrimer scaffold. Active against both Gram-negative and -positive bacteria, its mechanism has been attributed to a disruption of bacterial membranes. The branched peptide was shown to assume a β-stranded conformation in a lipidic environment. Here, we report on a rational modification of the original, empirically derived linear peptide sequence [WKKIRVRLSA-NH2, SB056-lin]. We interchanged the first two residues [KWKIRVRLSA-NH2, β-SB056-lin] to enhance the amphipathic profile, in the hope that a more regular β-strand would lead to a better antimicrobial performance. MIC values confirmed that an enhanced amphiphilic profile indeed significantly increases activity against both Gram-positive and -negative strains. The membrane binding affinity of both peptides, measured by tryptophan fluorescence, increased with an increasing ratio of negatively charged/zwitterionic lipids. Remarkably, β-SB056-lin showed considerable binding even to purely zwitterionic membranes, unlike the original sequence, indicating that besides electrostatic attraction also the amphipathicity of the peptide structure plays a fundamental role in binding, by stabilizing the bound state. Synchrotron radiation circular dichroism and solid-state 19F-NMR were used to characterize and compare the conformation and mobility of the membrane bound peptides. Both SB056-lin and β-SB056-lin adopt a β-stranded conformation upon binding POPC vesicles, but the former maintains an intrinsic structural disorder that also affects its aggregation tendency. Upon introducing some anionic POPG into the POPC matrix, the sequence-optimized β-SB056-lin forms well-ordered β-strands once electro-neutrality is approached, and it aggregates into more extended β-sheets as the concentration of anionic lipids in the bilayer is raised. The enhanced antimicrobial activity of the analogue correlates with the formation of these extended β-sheets, which also leads to a dramatic alteration of membrane integrity as shown by 31P-NMR. These findings are generally relevant for the design and optimization of other membrane-active antimicrobial peptides that can fold into amphipathic β-strands.

Characterization of sodium dodecylsulphate and dodecylphosphocholine mixed micelles through NMR and dynamic light scattering.

The complexity of biological membranes leads to the use of extremely simplified models in biophysical investigations of membrane‐bound proteins and peptides. Liposomes are probably the most widely used membrane models due, especially, to their versatility in terms of electric charge and size. However, liquid‐state NMR suffers the lack of such a model, because even the smallest liposomes slowly tumble in solution, resulting in a dramatic signals broadening. Micelles are typically used as good substitutes, with sodium dodecylsulphate (SDS) and dodecylphosphocholine (DPC) being the most widely employed surfactants. However, they are always used separately to mimic prokaryotic and eukaryotic membranes, respectively, and accurate investigations as a function of surface charge cannot be performed. In this work, the critical micelle concentration (CMC) of binary mixtures with different SDS/DPC ratios has been determined by following the chemical shift variation of selected 1H and 31P NMR signals as a function of total surfactant concentration. The regular solution theory and the Motomuras formalism have been applied to characterize the micellization both in water and in phosphate buffer saline, and results were compared with those obtained directly from the experimental NMR chemical shift. The ζ‐potential and size distribution of the mixed micelles have been estimated with dynamic light scattering measurements. Results showed that SDS and DPC are synergic and can be used together to prepare mixed micelles with different negative/zwitterionic surfactants molar ratio. Copyright

Conformational Analysis of the Frog Skin Peptide, Plasticin-L1 and its Effects on the Production of Proinflammatory Cytokines by Macrophages

Plasticin-L1 (GLVNGLLSSVLGGGQGGGGLLGGIL) is a conformationally flexible glycine/leucine-rich peptide originally isolated from norepinephrine-stimulated skin secretions of the South-American Santa Fe frog Leptodactylus laticeps (Leptodactylidae). A nuclear magnetic resonance/molecular dynamics characterization of plasticin-L1 in the presence of dodecylphosphocholine (DPC) and DPC/sodium dodecylsulphate micelles as membrane-mimetic models showed that the peptide has affinity for both neutral and anionic membranes. The peptide adopts a stable helical conformation at the N-terminal region and a more disordered helix at the C-terminal region, separated by an unstructured loop wherein the highest number of glycines is localized. In both micelle environments, plasticin-L1 slowly inserts between the detergent head groups but always remains localized at the micelle/water interface. Plasticin-L1 lacks direct antimicrobial activity but stimulates cytokine production by macrophages. Incubation with plasticin-L1 (20 μg/mL) significantly (P < 0.05) increased the production of the proinflammatory cytokines IL-1β, IL-12, IL-23, and TNF-α from unstimulated peritoneal macrophages from both C57BL/6 and BALB/C mice. The peptide also increased IL-6 production by unstimulated (P < 0.01) and lipopolysaccharide-stimulated (P < 0.01) macrophages, whereas the effects on production of the anti-inflammatory cytokine IL-10 were not significant. These findings suggest that plasticin-L1 may play an immunomodulatory role in vivo by stimulating cytokine production from frog skin macrophages in response to microbial pathogens. This peptide may represent a template for the design of peptides with therapeutic applications as immunostimulatory agents.

Rational modification of a dendrimeric peptide with antimicrobial activity: consequences on membrane-binding and biological properties.

Peptide-based antibiotics might help containing the rising tide of antimicrobial resistance. We developed SB056, a semi-synthetic peptide with a dimeric dendrimer scaffold, active against both Gram-negative and Gram-positive bacteria. Being the mechanism of SB056 attributed to disruption of bacterial membranes, we enhanced the amphiphilic profile of the original, empirically derived sequence [WKKIRVRLSA-NH2] by interchanging the first two residues [KWKIRVRLSA-NH2], and explored the effects of this modification on the interaction of peptide, both in linear and dimeric forms, with model membranes and on antimicrobial activity. Results obtained against Escherichia coli and Staphylococcus aureus planktonic strains, with or without salts at physiological concentrations, confirmed the added value of dendrimeric structure over the linear one, especially at physiological ionic strength, and the impact of the higher amphipathicity obtained through sequence modification on enhancing peptide performances. SB056 peptides also displayed intriguing antibiofilm properties. Staphylococcus epidermidis was the most susceptible strain in sessile form, notably to optimized linear analog lin-SB056-1 and the wild-type dendrimer den-SB056. Membrane affinity of all peptides increased with the percentage of negatively charged lipids and was less influenced by the presence of salt in the case of dendrimeric peptides. The analog lin-SB056-1 displayed the highest overall affinity, even for zwitterionic PC bilayers. Thus, in addition to electrostatics, distribution of charged/polar and hydrophobic residues along the sequence might have a significant role in driving peptide–lipid interaction. Supporting this view, dendrimeric analog den-SB056-1 retained greater membrane affinity in the presence of salt than den-SB056, despite the fact that they bear exactly the same net positive charge.

Modifications of the 1H NMR metabolite profile of processed mullet (Mugil cephalus) roes under different storage conditions

1H NMR spectroscopy was employed to study the modifications over time of the water‐soluble low molecular weight metabolites extracted from samples of salted and dried mullet (Mugil cephalus) roes (mullet bottarga) stored at different conditions. Samples of grated mullet bottarga were stored for 7 months at −20 °C, at 3 °C, and at room temperature in the presence and in the absence of light and then timely extracted and analyzed by NMR. Principal component multivariate data analysis applied to the spectral data indicated that samples stored at −20 °C maintained similar features over time whereas, along PC1, samples stored at room temperature in the presence and in the absence of light showed, over time, marked metabolite modifications. The comparative analysis of the integrated areas of the selected regions of the 1H NMR spectra indicated that the major compositional changes due to storage conditions were (i) the increase of the derivatives of the breakdown of phosphatidylcholine (choline, phosphorylcholine, and glycerol), (ii) the breakdown of nucleosides, (iii) the decrease of methionine, tryptophan, and tyrosine, and (iv) the cyclization of creatine. These changes were observed at different storage conditions, with more pronounced trends in the samples stored at room temperature. The role of metabolites in food aging is discussed. Copyright

Toward an improved structural model of the frog-skin antimicrobial peptide esculentin-1b(1-18).

Antimicrobial peptides (AMPs) are found in various classes of organisms as part of the innate immune system. Despite high sequence variability, they share common features such as net positive charge and an amphipathic fold when interacting with biologic membranes. Esculentin-1b is a 46-mer frog-skin peptide, which shows an outstanding antimicrobial activity. Experimental studies revealed that the N-terminal fragment encompassing the first 18 residues, Esc(1-18), is responsible for the antimicrobial activity of the whole peptide, with a negligible toxicity toward eukaryotic cells, thus representing an excellent candidate for future pharmaceutical applications. Similarly to most of the known AMPs, Esc(1-18) is expected to act by destroying/permeating the bacterial plasma-membrane but, to date, its 3D structure and the detailed mode of action remains unexplored. Before an in-depth investigation on peptide/membranes interactions could be undertaken, it is necessary to characterize peptides folding propensity in solution, to understand what is intrinsically due to the peptide sequence, and what is actually driven by the membrane interaction. Circular dichroism and nuclear magnetic resonance spectroscopy were used to determine the structure adopted by the peptide, moving from water to increasing amounts of trifluoroethanol. The results showed that Esc(1-18) has a clear tendency to fold in a helical conformation as hydrophobicity of the environment increases, revealing an intriguing amphipathic structure. The helical folding is adopted only by the N-terminal portion of the peptide, while the rest is unstructured. The presence of a hydrophobic cluster of residues in the C-terminal portion suggests its possible membrane-anchoring role.

Folded structure and insertion depth of the frog-skin antimicrobial peptide esculentin-1b(1−18) in the presence of differently charged membrane-mimicking micelles

Antimicrobial peptides (AMPs) are effectors of the innate immunity of most organisms. Their role in the defense against pathogen attack and their high selectivity for bacterial cells make them attractive for the development of a new class of antimicrobial drugs. The N-terminal fragment of the frog-skin peptide esculentin-1b (Esc(1-18)) has shown broad-spectrum antimicrobial activity. Similarly to most cationic AMPs, it is supposed to act by binding to and damaging the negatively charged plasma membrane of bacteria. Differently from many other AMPs, Esc(1-18) activity is preserved in biological fluids such as serum. In this work, a structural investigation was performed through NMR spectroscopy. The 3D structure was obtained in the presence of either zwitterionic or negatively charged micelles as membrane models for eukaryotic and prokaryotic membranes, respectively. Esc(1-18) showed a higher affinity for and deeper insertion into the latter and adopted an amphipathic helical structure characterized by a kink at the residue G8. These findings were confirmed by measuring penetration into lipid monolayers. The presence of negatively charged lipids in the bilayer appears to be necessary for Esc(1-18) to bind, to fold in the right three-dimensional structure, and, ultimately, to exert its biological role as an AMP.

Antimicrobial Dendrimeric Peptides: Structure, Activity and New Therapeutic Applications

Microbial resistance to conventional antibiotics is one of the most outstanding medical and scientific challenges of our times. Despite the recognised need for new anti-infective agents, however, very few new drugs have been brought to the market and to the clinic in the last three decades. This review highlights the properties of a new class of antibiotics, namely dendrimeric peptides. These intriguing novel compounds, generally made of multiple peptidic sequences linked to an inner branched core, display an array of antibacterial, antiviral and antifungal activities, usually coupled to low haemolytic activity. In addition, several peptides synthesized in oligobranched form proved to be promising tools for the selective treatment of cancer cells.

Conformational analysis and cytotoxic activities of the frog skin host-defense peptide, hymenochirin-1Pa.

Hymenochirin-1Pa (LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2) is a host-defense peptide first isolated from skin secretions of the frog Pseudhymenochirus merlini (Pipidae). A nuclear magnetic resonance structural investigation demonstrates that the peptide has a random coil conformation in water but, in the membrane-mimetic solvent 50% (v/v) trifluoroethanol-water adopts a well-defined conformation characterized by two α-helical domains from residues K6 to G17 and from G21 to M28, with the N-terminal region unfolded. The presence of a GXXXG domain, the most common structural motif found at the interface between interacting trans-membrane helices, between residues 17 and 21, introduces a kink corresponding to a deviation from linearity of 93 ± 31°. Hymenochirin-1Pa shows broad spectrum anti-bacterial activity, including high potency against multidrug-resistant clinical isolates of Staphylococcus aureus, Acinetobacter baumannii, and Stenotrophomonas maltophilia. The peptide also shows high cytotoxic potency against human non-small lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells but its therapeutic potential as an anti-cancer agent is limited by moderate hemolytic activity against human erythrocytes and lack of selectivity for tumor cells. Increasing cationicity of the peptide by substituting the Asp(9) residue by either L-Lys (K) or D-Lys (k) has relatively minor effects on antimicrobial and anti-tumor potencies but the [D9k] analog is non-hemolytic LC50 > 400 μM. Thus, [D9k]hymenochirin-1Pa may serve as a template for the design of non-toxic antimicrobial agents for use against multidrug-resistant pathogenic bacteria.

Conformational Analysis of the Host-Defense Peptides Pseudhymenochirin-1Pb and -2Pa and Design of Analogues with Insulin-Releasing Activities and Reduced Toxicities.

Pseudhymenochirin-1Pb (Ps-1Pb; IKIPSFFRNILKKVGKEAVSLIAGALKQS) and pseudhymenochirin-2Pa (Ps-2Pa; GIFPIFAKLLGKVIKVASSLISKGRTE) are amphibian peptides with broad spectrum antimicrobial activities and cytotoxicity against mammalian cells. In the membrane-mimetic solvent 50% (v/v) trifluoroethanol-H2O, both peptides adopt a well-defined α-helical conformation that extends over almost all the sequence and incorporates a flexible bend. Both peptides significantly (p < 0.05) stimulate the rate of release of insulin from BRIN-BD11 clonal β-cells at concentrations ≥ 0.1 nM but produce loss of integrity of the plasma membrane at concentrations ≥ 1 μM. Increasing cationicity by the substitution Glu(17) → l-Lys in Ps-1Pb and Glu(27) → l-Lys in Ps-2Pa generates analogues with increased cytotoxicity and reduced insulin-releasing potency. In contrast, the analogues [R8r]Ps-1Pb and [K8k,K19k]Ps-2Pa, incorporating d-amino acid residues to destabilize the α-helical domains, retain potent insulin-releasing activity but are nontoxic to BRIN-BD11 cells at concentrations of 3 μM. [R8r]Ps-1Pb produces a significant increase in insulin release rate at 0.3 nM and [K8k,K19k]Ps-2Pa at 0.01 nM. Both analogues show low hemolytic activity (IC50 > 100 μM) but retain broad-spectrum antimicrobial activity and remain cytotoxic to a range of human tumor cell lines, albeit with lower potency than the naturally occurring peptides. These analogues show potential for development into agents for type 2 diabetes therapy.