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Structural conversion of cellular prion protein (PrPC) into scrapie PrP (PrPSc) and subsequent aggregation are key events associated with the onset of transmissible spongiform encephalopathies (TSEs). Experimental evidence supports the role of nucleic acids (NAs) in assisting this conversion. Here, we asked whether PrP undergoes liquid-liquid phase separation (LLPS) and if this process is modulated by NAs. To this end, two 25-mer DNA aptamers, A1 and A2, were selected against the globular domain of recombinant murine PrP (rPrP90-231) using SELEX methodology. Multiparametric structural analysis of these aptamers revealed that A1 adopts a hairpin conformation. Aptamer binding caused partial unfolding of rPrP90-231 and modulated its ability to undergo LLPS and fibrillate. In fact, although free rPrP90-231 phase separated into large droplets, aptamer binding increased the number of droplets but noticeably reduced their size. Strikingly, a modified A1 aptamer that does not adopt a hairpin structure induced formation of amyloid fibrils on the surface of the droplets. We show here that PrP undergoes LLPS, and that the PrP interaction with NAs modulates phase separation and promotes PrP fibrillation in a NA structure and concentration-dependent manner. These results shed new light on the roles of NAs in PrP misfolding and TSEs.

Antimicrobial peptides are recognized candidates with pharmaceutical potential against epidemic emerging multi-drug resistant bacteria. In this study, we use nuclear magnetic resonance spectroscopy and molecular dynamics simulations to determine the unknown structure and evaluate the interaction with dodecylphosphatidylcholine (DPC) and sodium dodecylsulphate (SDS) micelles with three W6 -Hylin-a1 analogs antimicrobial peptides (HyAc, HyK, and HyD). The HyAc, HyK, and HyD bound to DPC micelles are all formed by a unique α-helix structure. Moreover, all peptides reach the DPC micelles' core, which thus suggests that the N-terminal modifications do not influence the interaction with zwiterionic surfaces. On the other hand, only HyAc and HyK peptides are able to penetrate the SDS micelle core while HyD remains always at its surface. The stability of the α-helical structure, after peptide-membrane interaction, can also be important to the second step of peptide insertion into the membrane hydrophobic core during permeabilization. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Studies have suggested that antimicrobial peptides act by different mechanisms, such as micellisation, self-assembly of nanostructures and pore formation on the membrane surface. This work presents an extensive investigation of the membrane interactions of the 14 amino-acid antimicrobial peptide hylaseptin P1-NH2 (HSP1-NH2), derived from the tree-frog Hyla punctata, which has stronger antifungal than antibacterial potential. Biophysical and structural analyses were performed and the correlated results were used to describe in detail the interactions of HSP1-NH2 with zwitterionic and anionic detergent micelles and phospholipid vesicles. HSP1-NH2 presents similar well-defined helical conformations in both zwitterionic and anionic micelles, although NMR spectroscopy revealed important structural differences in the peptide N-terminus. 2H exchange experiments of HSP1-NH2 indicated the insertion of the most N-terminal residues (1–3) in the DPC-d38 micelles. A higher enthalpic contribution was verified for the interaction of the peptide with anionic vesicles in comparison with zwitterionic vesicles. The pore formation ability of HSP1-NH2 (examined by dye release assays) and its effect on the size and surface charge as well as on the lipid acyl chain ordering (evaluated by Fourier-transform infrared spectroscopy) of anionic phospholipid vesicles showed membrane disruption even at low peptide-to-phospholipid ratios, and the effect increases proportionately to the peptide concentration. On the other hand, these biophysical investigations showed that a critical peptide-to-phospholipid ratio around 0.6 is essential for promoting disruption of zwitterionic membranes. In conclusion, this study demonstrates that the binding process of the antimicrobial HSP1-NH2 peptide depends on the membrane composition and peptide concentration.

© 2018 Elsevier B.V. All rights reserved., Antimicrobial peptides (AMPs) are promising candidates for the development of future antibiotics. In an attempt to increase the efficacy of therapeutic AMPs, computer-based design methods appear as a reliable strategy. In this study, we evaluated the antimicrobial efficiency and mechanism of action of a novel designed AMP named PaDBS1R1, previously designed by means of the Joker algorithm, using a fragment of the ribosomal protein L39E from the archaeon Pyrobaculum aerophilum as a template. PaDBS1R1 displayed low micromolar broad-spectrum antimicrobial activity against Gram-negative (MIC of 1.5 μM) and Gram-positive (MIC of 3 μM) bacteria, including carbapenem-resistant Klebsiella pneumoniae (MIC of 6.25 μM) and methicillin-resistant Staphylococcus aureus (MIC of 12.5 μM), without cytotoxicity towards HEK-293 cells. In addition, membrane permeabilization and depolarization assays, combined with time-kill studies and FEG-SEM imaging, indicated a fast membrane permeation and further leakage of intracellular content. Biophysical studies with lipid vesicles show a preference of PaDBS1R1 for Gram-negative bacteria-like membranes. We investigated the three-dimensional structure of PaDBS1R1 by CD and NMR analyses. Our results suggest that PaDBS1R1 adopts an amphipathic α-helix upon interacting with hydrophobic environments, after an initial electrostatic interaction with negative charges, suggesting a membrane lytic effect. This study reveals that PaDBS1R1 has potential application in antibiotic therapy., The authors acknowledge Fundação para a Ciência e a Tecnologia – Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal) for funding, including fellowship SFRH/BD/100517/2014 to M.R.F., Marie Sklodowska-Curie Research and Innovation Staff Exchange (MSCA-RISE, European Union) project INPACT (call H2020-MSCA-RISE-2014, grant agreement 644167), IMPC (Institut des Matériaux de Paris Centre, FR 2482) and the C'Nano projects of the Region Ile-de-France for SEM-FEG funding. Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq for financial support and researcher fellowship (Processes: 141327/2012-0; 407181/2013-0; 303181/20150), Coordenação de Aperfeiçoamento de Pessoal do Nível Superior – CAPES, for scholarship (Process: BEX 2758/15-6), and Fundação de Amparo à Pesquisa do Estado de Goiás – FAPEG (Process: 201710267000062). This work was also supported by Fundação de Amparo a Pesquisa do Mato Grosso do Sul (FUNDECT) and Fundação de Amapro a Pesquisa do Distrito Federal (FAPDF).

Infections caused by Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa foremost among them, constitute a major worldwide health problem. Bioinformatics methodologies are being used to rationally design new antimicrobial peptides, a potential alternative for treating these infections. One of the algorithms used to develop antimicrobial peptides is the Joker, which was used to design the peptide PaDBS1R6. This study evaluates the antibacterial activities of PaDBS1R6 in vitro and in vivo, characterizes the peptide interaction to target membranes, and investigates the PaDBS1R6 structure in contact with mimetic vesicles. Moreover, we demonstrate that PaDBS1R6 exhibits selective antimicrobial activity against Gram-negative bacteria. In the presence of negatively charged and zwitterionic lipids the structural arrangement of PaDBS1R6 transits from random coil to α-helix, as characterized by circular dichroism. The tertiary structure of PaDBS1R6 was determined by NMR in zwitterionic dodecylphosphocholine (DPC) micelles. In conclusion, PaDBS1R6 is a candidate for the treatment of nosocomial infections caused by Gram-negative bacteria, as template for producing other antimicrobial agents.

Plants are extensively used in traditional medicine, and several plant antimicrobial peptides have been described as potential alternatives to conventional antibiotics. However, after more than four decades of research no plant antimicrobial peptide is currently used for treating bacterial infections, due to their length, post-translational modifications or high dose requirement for a therapeutic effect . Here we report the design of antimicrobial peptides derived from a guava glycine-rich peptide using a genetic algorithm. This approach yields guavanin peptides, arginine-rich α-helical peptides that possess an unusual hydrophobic counterpart mainly composed of tyrosine residues. Guavanin 2 is characterized as a prototype peptide in terms of structure and activity. Nuclear magnetic resonance analysis indicates that the peptide adopts an α-helical structure in hydrophobic environments. Guavanin 2 is bactericidal at low concentrations, causing membrane disruption and triggering hyperpolarization. This computational approach for the exploration of natural products could be used to design effective peptide antibiotics., Antimicrobial peptides are considered promising alternatives to antibiotics. Here the authors developed a computational algorithm that starts with peptides naturally occurring in plants and optimizes this starting material to yield new variants which are highly distinct from the parent peptide.

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4, Due to the growing concern about antibiotic-resistant microbial infections, increasing support has been given to new drug discovery programs. A promising alternative to counter bacterial infections includes the antimicrobial peptides (AMPs), which have emerged as model molecules for rational design strategies. Here we focused on the study of Pa-MAP 1.9, a rationally designed AMP derived from the polar fish Pleuronectes americanus. Pa-MAP 1.9 was active against Gram-negative planktonic bacteria and biofilms, without being cytotoxic to mammalian cells. By using AFM, leakage assays, CD spectroscopy and in silico tools, we found that Pa-MAP 1.9 may be acting both on intracellular targets and on the bacterial surface, also being more efficient at interacting with anionic LUVs mimicking Gram-negative bacterial surface, where this peptide adopts α-helical conformations, than cholesterolenriched LUVs mimicking mammalian cells. Thus, as bacteria present varied physiological features that favor antibiotic-resistance, Pa-MAP 1.9 could be a promising candidate in the development of tools against infections caused by pathogenic bacteria., Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R21AI098701, and by a grant from the Canadian Institutes. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. R.E.W.H. holds a Canada Research Chair in Health and Genomics. C.D.L.F.-N. received a scholarship from the Fundación “la Caixa” and Fundación Canadá (Spain), and currently holds a postdoctoral scholarship from Fundación Ramón Areces (Spain). This work was also supported by Fundaçao para a Ciencia e a Tecnologia–Ministério da Educaçao e Ciencia (FCT-MEC, Portugal), including the fellowship SPRH/BD/100517/2014 to M.R.F., and by Marie Skłodowska-Curie Research and Innovation Staff Exchange (MSCA-RISE, European Union) project INPACT (call H2020-MSCA-RISE-2014, grant agreement 644167). M.H.C., L.M. and O.L.F. acknowledge funding from CNPq, CAPES, FUNDECT and FAPDF.