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      Antimicrobial activity, improved cell selectivity and mode of action of short PMAP-36-derived peptides against bacteria and Candida

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          Abstract

          Antimicrobial peptides (AMPs) have recently attracted a great deal of attention as promising antibiotic candidates, but some obstacles such as toxicity and high synthesis cost must be addressed before developing them further. For developing short peptides with improved cell selectivity, we designed a series of modified PMAP-36 analogues. Antimicrobial assays showed that decreasing chain length in a certain range retained the high antimicrobial activity of the parental peptide and reduced hemolysis. The 18-mer peptide RI18 exhibited excellent antimicrobial activity against both bacteria and fungi, and its hemolytic activity was observably lower than PMAP-36 and melittin. The selectivity indexes of RI18 against bacteria and fungi were improved approximately 19-fold and 108-fold, respectively, compared to PMAP-36. In addition, serum did not affect the antibacterial activity of RI18 against E. coli but inhibited the antifungal efficiency against C. albicans. Flow cytometry and electron microscopy observation revealed that RI18 killed microbial cells primarily by damaging membrane integrity, leading to whole cell lysis. Taken together, these results suggest that RI18 has potential for further therapeutic research against frequently-encountered bacteria and fungi. Meanwhile, modification of AMPs is a promising strategy for developing novel antimicrobials to overcome drug-resistance.

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          Most cited references 46

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          Rational design of alpha-helical antimicrobial peptides with enhanced activities and specificity/therapeutic index.

          In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAVKTVLHTALKAISS-amide (V681) was utilized as the framework to study the effects of peptide hydrophobicity/hydrophilicity, amphipathicity, and helicity (induced by single amino acid substitutions in the center of the polar and nonpolar faces of the amphipathic helix) on biological activities. The peptide analogs were also studied by temperature profiling in reversed-phase high performance liquid chromatography, from 5 to 80 degrees C, to evaluate the self-associating ability of the molecules in solution, another important parameter in understanding peptide antimicrobial and hemolytic activities. A higher ability to self-associate in solution was correlated with weaker antimicrobial activity and stronger hemolytic activity of the peptides. Biological studies showed that strong hemolytic activity of the peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. In most cases, the D-amino acid substituted peptides possessed an enhanced average antimicrobial activity compared with L-diastereomers. The therapeutic index of V681 was improved 90- and 23-fold against Gram-negative and Gram-positive bacteria, respectively. By simply replacing the central hydrophobic or hydrophilic amino acid residue on the nonpolar or the polar face of these amphipathic derivatives of V681 with a series of selected D-/L-amino acids, we demonstrated that this method has excellent potential for the rational design of antimicrobial peptides with enhanced activities.
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            Optimization of the antimicrobial activity of magainin peptides by modification of charge.

            Investigation of magainin II amide analogs with cationic charges ranging between +3 and +7 showed that enhancement of the peptide charge up to a threshold value of +5 and conservation of appropriate hydrophobic properties optimized the antimicrobial activity and selectivity. High selectivity was the result of both enhanced antimicrobial and reduced hemolytic activity. Charge increase beyond +5 with retention of other structural motifs led to a dramatic increase of hemolytic activity and loss of antimicrobial selectivity. Selectivity could be restored by reduction of the hydrophobicity of the hydrophobic helix surface (H(hd)), a structural parameter not previously considered to modulate activity. Dye release experiments with lipid vesicles revealed that the potential of peptide charge to modulate membrane activity is limited: on highly negatively charged 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol bilayers, reinforcement of electrostatic interactions had an activity-reducing effect. On neutral 1-palmitoyl-2-oleoylphosphatidylcholine bilayers, the high activity was determined by H(hd). H(hd) values above a certain threshold led to effective permeabilization of all lipid systems and even compensated for the activity-reducing effect of charge increase on highly negatively charged membranes.
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              Investigation of the cytotoxicity of eukaryotic and prokaryotic antimicrobial peptides in intestinal epithelial cells in vitro.

              Antimicrobial peptides (AMPs) are a diverse group of proteinaceous compounds ranging in size, complexity and antimicrobial spectrum. The activity of AMPs against gut pathogens warrants the study of the interaction of AMPs with the mammalian gastrointestinal tract. In particular, the investigation of the in vitro cytotoxicity of these peptides is critical before they can be considered in clinical infections. The cytotoxicity of gallidermin, nisin A, natural magainin peptides, and melittin was investigated in two gastrointestinal cell models (HT29 and Caco-2) with the MTT conversion assay, neutral red dye uptake assay and compared with that of vancomycin. The hemolytic activities were also investigated in sheep erythrocytes and the effect of AMPs on paracellular permeability was examined by transepithelial resistance (TEER) and TEM. Gallidermin was the least cytotoxic AMP followed by nisin A, magainin I, magainin II and melittin. Melittin and nisin were the only peptides to result in significant hemolysis. However, while nisin caused hemolysis at concentrations which were 1000-fold higher than those required for antimicrobial activity, melittin was hemolytic at concentrations in the same order of magnitude as its antimicrobial activity. Melittin was the only AMP to affect paracellular permeability. Long term melittin treatment also resulted in loss of microvilli, an increase in cell debris and destruction of intestinal tight junctions and cell-cell adhesion. Gallidermin shows most promise as a therapeutic agent, with relatively low cytotoxicity and potent antimicrobial activities. Melittin, while showing little potential as an antimicrobial agent, may have potential in delivery of poorly bioavailable drugs.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                02 June 2016
                2016
                : 6
                Affiliations
                [1 ]Laboratory of Molecular Nutrition and Immunity, Institute of Animal Nutrition, Northeast Agricultural University , Harbin, Heilongjiang, P.R. China
                Author notes
                Article
                srep27258
                10.1038/srep27258
                4890124
                27251456
                a2e7778a-647f-4f34-8cba-599bb283ad91
                Copyright © 2016, Macmillan Publishers Limited

                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/4.0/

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