Search Results (9)

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium responsible for severe nosocomial infections and is considered a critical pulmonary pathogen for both immunocompromised and cystic fibrosis patients. Planktonic cells of P. aeruginosa possess intrinsic and acquired resistances, inactivating several classes of conventional antibiotics. Additionally, this bacterium can grow, forming biofilms, and complex structures, further hampering the action of multiple antibiotics. Here, we report the biological properties of D-Q53 CecB, an all-D enantiomer of the silkworm natural peptide Q53 CecB. Compared to the L-variant, D-Q53 CecB was resistant to in vitro degradation by humans and P. aeruginosa elastases and showed an enhanced bactericidal activity against P. aeruginosa planktonic bacteria. D-Q53 CecB was thermostable and maintained its antimicrobial activity at high salt concentrations and in the presence of divalent cations or fetal-bovine serum, although at reduced levels. Against different types of human cells, D-Q53 CecB showed cytotoxic phenomena at concentrations several folds higher compared to those active against P. aeruginosa. When L- and D-Q53 CecB were compared for their antibiofilm properties, both peptides were active in inhibiting biofilm formation. However, the D-enantiomer was extremely effective in inducing biofilm degradation, suggesting this peptide as a favorable candidate in an anti-Pseudomonas therapy. Full article

Cell-penetrating peptides (CPPs), such as penetratin, are often investigated as drug delivery vectors and incorporating d-amino acids, rather than the natural l-forms, to enhance proteolytic stability could improve their delivery efficiency. The present study aimed to compare membrane association, cellular uptake, and delivery capacity for all-l and all-d enantiomers of penetratin (PEN) by using different cell models and cargos. The enantiomers displayed widely different distribution patterns in the examined cell models, and in Caco-2 cells, quenchable membrane binding was evident for d-PEN in addition to vesicular intracellular localization for both enantiomers. The uptake of insulin in Caco-2 cells was equally mediated by the two enantiomers, and while l-PEN did not increase the transepithelial permeation of any of the investigated cargo peptides, d-PEN increased the transepithelial delivery of vancomycin five-fold and approximately four-fold for insulin at an extracellular apical pH of 6.5. Overall, while d-PEN was associated with the plasma membrane to a larger extent and was superior in mediating the transepithelial delivery of hydrophilic peptide cargoes compared to l-PEN across Caco-2 epithelium, no enhanced delivery of the hydrophobic cyclosporin was observed, and intracellular insulin uptake was induced to a similar degree by the two enantiomers. Full article

The N-capping region of an α-helix is a short N-terminal amino acid stretch that contributes to nucleate and stabilize the helical structure. In the VEGF mimetic helical peptide QK, the N-capping region was previously demonstrated to be a key factor of QK helical folding. In this paper, we explored the effect of the chiral inversion of the N-capping sequence on QK folding, performing conformational analysis in solution by circular dichroism and NMR spectroscopy. The effect of such a modification on QK stability in serum and the proliferative effect were also evaluated. Full article

BP214 is an all-D antimicrobial peptide amide, kklfkkilryl, which shows an excellent activity against colistin-resistant Acinetobacter baumannii and a low hemolytic activity. The aim of the present work was to investigate how C-terminus-to-side chain macrocyclization and fatty acid modification affect the antimicrobial and hemolytic activity of this peptide. In total, 18 analogs of BP214 were synthesized using a combination of Fmoc-based solid-phase peptide synthesis and the submonomer approach. Cyclization was achieved by reacting the ε-amino group of a C-terminal lysine residue with a bromoacetylgroup attached to the N^α amino group of the N-terminal amino acid, generating a secondary amine at which the exocyclic lipopeptide tail was assembled. Three different ring sizes (i.e., 3–5 amino acid residues) of C-locked analogs combined with fatty acids of different lengths (i.e., C₁₀–C₁₄) were investigated. The antimicrobial activity of the analogs was tested against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. The most promising compound was analog 13 (MIC = 4 µg/mL (2.4 µM) against E. coli and 36% hemolysis of red blood cells at 150 µM). In a time-kill assay, this peptide showed a significant, concentration-dependent reduction in viable E. coli cells comparable to that seen for colistin. Full article

(1) Antimicrobial peptides (AMPs) are a promising alternative to conventional antibiotics. Among AMPs, the disulfide-rich β-defensin AvBD103b, whose antibacterial activities are not inhibited by salts contrary to most other β-defensins, is particularly appealing. Information about the mechanisms of action is mandatory for the development and approval of new drugs. However, data for non-membrane-disruptive AMPs such as β-defensins are scarce, thus they still remain poorly understood. (2) We used single-cell fluorescence imaging to monitor the effects of a β-defensin (namely AvBD103b) in real time, on living E. coli, and at the physiological concentration of salts. (3) We obtained key parameters to dissect the mechanism of action. The cascade of events, inferred from our precise timing of membrane permeabilization effects, associated with the timing of bacterial growth arrest, differs significantly from the other antimicrobial compounds that we previously studied in the same physiological conditions. Moreover, the AvBD103b mechanism does not involve significant stereo-selective interaction with any chiral partner, at any step of the process. (4) The results are consistent with the suggestion that after penetrating the outer membrane and the cytoplasmic membrane, AvBD103b interacts non-specifically with a variety of polyanionic targets, leading indirectly to cell death. Full article

Antimicrobial peptides (AMPs) are alternative therapeutics to traditional antibiotics against bacterial resistance. Our previous work identified an antimicrobial region at the N-terminus of the eosinophil cationic protein (ECP). Following structure-based analysis, a 30mer peptide (ECPep-L) was designed that combines antimicrobial action against Gram-negative species with lipopolysaccharides (LPS) binding and endotoxin-neutralization activities. Next, analogues that contain non-natural amino acids were designed to increase serum stability. Here, two analogues were selected for in vivo assays: the all-D version (ECPep-D) and the Arg to Orn version that incorporates a D-amino acid at position 2 (ECPep-2D-Orn). The peptide analogues retained high LPS-binding and anti-endotoxin activities. The peptides efficacy was tested in a murine acute infection model of Acinetobacter baumannii. Results highlighted a survival rate above 70% following a 3-day supervision with a single administration of ECPep-D. Moreover, in both ECPep-D and ECPep-2D-Orn peptide-treated groups, clinical symptoms improved significantly and the tissue infection was reduced to equivalent levels to mice treated with colistin, used as a last resort in the clinics. Moreover, treatment drastically reduced serum levels of TNF-α inflammation marker within the first 8 h. The present results support ECP-derived peptides as alternative candidates for the treatment of acute infections caused by Gram-negative bacteria. Full article

Gold nanoparticles (AuNPs) have been shown to be outstanding tools for drug delivery and biomedical applications, mainly owing to their colloidal stability, surface chemistry, and photothermal properties. The biocompatibility and stability of nanoparticles can be improved by capping the nanoparticles with endogenous proteins, such as albumin. Notably, protein coating of nanoparticles can interfere with and decrease their cell penetration. Therefore, in the present study, we functionalized albumin with the r₈ peptide (All-D, octaarginine) and used it for coating NIR-plasmonic anisotropic gold nanoparticles. Gold nanoprisms (AuNPrs) and gold nanorods (AuNRs) were coated with bovine serum albumin (BSA) previously functionalized using a cell penetrating peptide (CPP) with the r₈ sequence (BSA-r₈). The effect of the coated and r₈-functionalized AuNPs on HeLa cell viability was assessed by the MTS assay, showing a low effect on cell viability after BSA coating. Moreover, the internalization of the nanostructures into HeLa cells was assessed by confocal microscopy and transmission electron microscopy (TEM). As a result, both nanoconstructs showed an improved internalization level after being capped with BSA-r₈, in contrast to the BSA-functionalized control, suggesting the predominant role of CPP functionalization in cell internalization. Thus, our results validate both novel nanoconstructs as potential candidates to be coated by endogenous proteins and functionalized with a CPP to optimize cell internalization. In a further approach, coating AuNPs with CPP-functionalized BSA can broaden the possibilities for biomedical applications by combining their optical properties, biocompatibility, and cell-penetration abilities. Full article

The increasing emergence of multi-drug resistant bacteria is a serious threat to public health worldwide. Antimicrobial peptides have attracted attention as potential antibiotics since they are present in all multicellular organisms and act as a first line of defence against invading pathogens. We have previously identified a small all-d antimicrobial octapeptide amide kk(1-nal)fk(1-nal)k(nle)-NH₂ (D2D) with promising antimicrobial activity. In this work, we have performed a structure-activity relationship study of D2D based on 36 analogues aimed at discovering which elements are important for antimicrobial activity and toxicity. These modifications include an alanine scan, probing variation of hydrophobicity at lys⁵ and lys⁷, manipulation of amphipathicity, N-and C-termini deletions and lys-arg substitutions. We found that the hydrophobic residues in position 3 (1-nal), 4 (phe), 6 (1-nal) and 8 (nle) are important for antimicrobial activity and to a lesser extent cationic lysine residues in position 1, 2, 5 and 7. Our best analogue 5, showed MICs of 4 µg/mL against A. baumannii, E. coli, P. aeruginosa and S. aureus with a hemolytic activity of 47% against red blood cells. Furthermore, compound 5 kills bacteria in a concentration-dependent manner as shown by time-kill kinetics. Circular dichroism (CD) spectra of D2D and compounds 1–8 showed that they likely fold into α-helical secondary structure. Small angle x-ray scattering (SAXS) experiments showed that a random unstructured polymer-like chains model could explain D2D and compounds 1, 3, 4, 6 and 8. Solution structure of compound 5 can be described with a nanotube structure model, compound 7 can be described with a filament-like structure model, while compound 2 can be described with both models. Lipid interaction probed by small angle X-ray scattering (SAXS) showed that a higher amount of compound 5 (~50–60%) inserts into the bilayer compared to D2D (~30–50%). D2D still remains the lead compound, however compound 5 is an interesting antimicrobial peptide for further investigations due to its nanotube structure and minor improvement to antimicrobial activity compared to D2D. Full article

Initially characterized by its antimicrobial activities, LL-37 has also been shown to significantly contribute to tumor development. On breast cancer cell lines, LL-37 increases intracellular calcium via the TRPV2 channel and their migration via the activation of PI3K/AKT signaling. Its all-d enantiomer d-LL-37 induces similar effects, which excludes a protein-protein interaction of LL-37 in a classic ligand-receptor manner. Its net charge of +6 gave rise to the hypothesis that the peptide uses the negative charges of sulfoglycans or sialic acids to facilitate its attachment to the cell membrane and to induce its activities. Whereas several vegetal lectins, specifically attaching to sialylated or sulfated structures, blocked the activities of LL-37 on both calcium increase and cell migration, several sialidases had no effect. However, the competitive use of free sulfated glycoaminoglycans (GAGs) as chrondroitin and heparin, or treatment of the cell surface with chondroitinase and heparinase resulted in an activity loss of 50–100% for LL-37. Concordant results were obtained by blocking the synthesis of GAGs with 4-Methylumbelliferyl-β-d-xyloside, and by suppression of glycan sulfatation by sodium chlorate. Using a candidate approach by suppressing proteoglycan synthesis using RNA interference, syndecan-4 was shown to be required for the activities of LL-37 and its binding to the cell surface. This leads to the conclusion that syndecan-4, by means of sulfated GAGs, could act as a receptor for LL-37. Full article