Characterization of an antagonistic peptide produced by a Bacteroides Fragilis isolate obtained from a patient with intra-abdominal infection / Caracterização de um peptido antagonístico produzido por Bacteroides Fragilis isolado obtido em um paciente com infecção intra-abdominal

The indigenous microbiota of humans is extremely rich and diverse, with special emphasis on the intestinal microbiota. As a constituent of this microbiota, it is mentioned the genus Bacteroides, whose members are Gram negative rods, obligate anaerobes, amphibionts, associated to the etiopathogenesis of important infectious diseases, such as intra-abdominal infections. Bacteroides strains have the ability to synthesize antagonistic substances that play an ecological role, especially in densely colonized habitats, giving a competitive advantage to the producing samples. The objective of this study was to evaluate the synthesis capacity of antagonistic substances by 40 samples of Bacteroides and Parabacteroides isolated from patients with intra-abdominal infections. The expression of antagonism was evaluated by the overlay diffusion method, using, as well as the test samples, 36 reference samples of Gram negative and Gram-positive bacteria. Subsequently, a production strain (Bacteroides fragilis) was used for extraction, purification and partial characterization of the detected antagonistic substance. As indicator strains, Bacteroides ovatus and Bacteroides caccae were used. The production strain was submitted to protein extraction, and the activity of the precipitated intracellular extract was detected with (NH4) 2SO4 in concentrations of 30% (C30) and 50% (C50). C30 and C50 were inactivated by proteases and high temperatures and remained active after exposure to organic solvents and a wide pH range. Both fractions presented antagonistic activity of bacteriostatic nature. The C50 extract was subjected to ion exchange chromatography, and 50 fractions were recovered. Among them, fractions 1 to 4, referring to a single peak, that were not able to bind to the column, presented antagonistic activity. Fractions from the ion exchange chromatography were applied in gel filtration chromatography. Among them, fractions 2 and 3 were able to inhibit the developing sample. These fractions were submitted to reverse phase chromatography, and 50 fractions were collected. One of them, fraction 2C, remained active against the revealing sample. Mass spectrometry, from fraction 2C obtained from reverse phase chromatography, presented ions of approximately 1300.00 Da, which generated a more intense signal. The search performed by similarity between the sequenced peptides and proteins described in the BLASTP database, from fragmentation obtained in reverse phase chromatography, resulted in 100% identity between two peptides. One of the sequenced peptides showed 100% identity to a type VII secretion protein. The search performed by similarity between the sequenced peptides and proteins described in the ANTIMICROBIAL PEPTIDE DATABASE database, from fragmentation obtained with trypsin digestion, resulted in 42% identity with a Streptomyces microcine. Together, the results indicate the production of antagonistic substances by the B. fragilis sample under study. It is plausible to assume that they play a relevant role in interbacterial relationships, as a virulence factor, in a complex environment such as intra-abdominal infection

Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies

The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires a-helical conformation, the helix spanning residues 3-11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide: lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential () of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. the mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism. (C) 2014 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Institut Nacional de Ciencia e Tecnologia de fluidos complexos (INCTFCx)Nude de Apoio Pesquisa de Fluidos Complexos (NAPFCx)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ São Paulo, Inst Chem, Dept Biochem, BR-05513970 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Med Biochem, Nucl Magnet Resonance Natl Ctr, Rio de Janeiro, BrazilEmbrapa Recursos Genet & Biotecnol, BR-70770917 Brasilia, DF, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilFAPESP: 2007/50970-5FAPESP: 2013/08166-5Web of Scienc

Novel ocellatin peptides mitigate LPS-induced ROS formation and NF-kB activation in microglia and hippocampal neurons

© The Author(s) 2020. Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per-mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.Cutaneous secretions of amphibians have bioactive compounds, such as peptides, with potential for biotechnological applications. Therefore, this study aimed to determine the primary structure and investigate peptides obtained from the cutaneous secretions of the amphibian, Leptodactylus vastus, as a source of bioactive molecules. The peptides obtained possessed the amino acid sequences, GVVDILKGAAKDLAGH and GVVDILKGAAKDLAGHLASKV, with monoisotopic masses of [M + H]± = 1563.8 Da and [M + H]± = 2062.4 Da, respectively. The molecules were characterized as peptides of the class of ocellatins and were named as Ocellatin-K1(1-16) and Ocellatin-K1(1-21). Functional analysis revealed that Ocellatin-K1(1-16) and Ocellatin-K1(1-21) showed weak antibacterial activity. However, treatment of mice with these ocellatins reduced the nitrite and malondialdehyde content. Moreover, superoxide dismutase enzymatic activity and glutathione concentration were increased in the hippocampus of mice. In addition, Ocellatin-K1(1-16) and Ocellatin-K1(1-21) were effective in impairing lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) formation and NF-kB activation in living microglia. We incubated hippocampal neurons with microglial conditioned media treated with LPS and LPS in the presence of Ocellatin-K1(1-16) and Ocellatin-K1(1-21) and observed that both peptides reduced the oxidative stress in hippocampal neurons. Furthermore, these ocellatins demonstrated low cytotoxicity towards erythrocytes. These functional properties suggest possible to neuromodulatory therapeutic applications.Alexandra Plácido is a recipient of a post-doctoral grant from the project FCT (PTDC/BII-BIO/31158/2017). Renato Socodato and Camila Cabral Portugal hold postdoctoral fellowships from FCT (Refs: SFRH/BPD/91833/2012 and FRH/BPD/91962/2012, respectively). This work was funded through project UID/QUI/50006/2013-POCI/01/0145/FEDER/007265 (LAQV/REQUIMTE) with financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT 2020info:eu-repo/semantics/publishedVersio

Structure and membrane interactions of the homodimeric antibiotic peptide homotarsinin

Antimicrobial peptides (AMPs) from amphibian skin are valuable template structures to find new treatments against bacterial infections. This work describes for the first time the structure and membrane interactions of a homodimeric AMP. Homotarsinin, which was found in Phyllomedusa tarsius anurans, consists of two identical cystine-linked polypeptide chains each of 24 amino acid residues. The high-resolution structures of the monomeric and dimeric peptides were determined in aqueous buffers. The dimer exhibits a tightly packed coiled coil three-dimensional structure, keeping the hydrophobic residues screened from the aqueous environment. An overall cationic surface of the dimer assures enhanced interactions with negatively charged membranes. An extensive set of biophysical data allowed us to establish structure-function correlations with antimicrobial assays against Gram-positive and Gram-negative bacteria. Although both peptides present considerable antimicrobial activity, the dimer is significantly more effective in both antibacterial and membrane biophysical assays.PMC524437

In Silico, In Vitro and In Vivo Toxicological Assessment of BPP-BrachyNH2, A Vasoactive Proline-Rich Oligopeptide from Brachycephalus ephippium

BPP-BrachyNH2 is a proline-rich oligopeptide (PRO) firstly identified in skin secretion of the frog Brachycephalus ephippium, which possess in vitro inhibitory activity of angiotensin-I converting enzyme (ACE) and endothelium-dependent vasorelaxant activity. Considering its potential application in the treatment of cardiovascular diseases, the present work assessed the toxicological profile of the BPP-BrachyNH2. The in silico toxicity prediction was performed from the best model obtained through the optimization of the FASTA query peptide. This prediction study revealed that BPP-BrachyNH2 induced high predicted LD50 values for both humans and rats, and then is well-tolerated in the recommended range. The MTT assay was applied for the in vitro cytotoxic evaluation in murine macrophages. In this assay, a decrease of cell viability was not observed. The in vivo acute toxicological study was performed after the intraperitoneal administration of BPP-BrachyNH2 at doses of 5 and 50 mg/kg. After intraperitoneal administration, no death, alterations in behavioral parameters or weight gain curve was observed, as well as none in the serum biochemical parameters, and gross pathological and histopathological analyses. These observations demonstrates an acceptable safety profile for BPP-BrachyNH2, leading towards further studies focused on investigation of pharmacological and therapeutical applications for this peptide.info:eu-repo/semantics/publishedVersio

Pepsin‐catalyzed peptide synthesis in organic media : studies with free and immobilized enzyme

Pepsin‐catalyzed synthesis of protected peptides was studied in two‐phase systems containing up to 50% (by volume) of aqueous phase. A methodological study was carried out to determine the optimum conditions for the synthesis of the model protected peptide Z‐Phe‐Phe‐OMe. Several parameters such as concentrations of carboxylic and amino components, pH of the aqueous phase, ratio of organic to aqueous phase volumes and nature of the organic solvent were investigated. It was observed that the most hydrophobic solvents produced the best yields, despite the low solubility of substrates in these media. The log P of the solvent could be used to predict the solvent effect over the reaction yields. Pepsin immobilized by adsorption onto the solid supports Celite and Chromosorb was employed to perform a study of secondary specificity of the enzyme in organic media through the coupling between Z‐X‐Phe‐OH (X = Ala, Asp, Glu, Gly, Phe, Ile, Val, Trp and Tyr) and Phe‐OMe. This investigation was performed in two solvent systems: (A) ethyl acetate:citrate buffer pH 4.5 (98:02, v:v) and (B) acetonitrile:citrate buffer pH 4.5 (96:04, v:v). Reaction rate data showed that pepsin had a preference for more hydrophilic substituents in the P2 position. These data are in contrast to the literature for a similar reaction performed in predominantly aqueous media. Thus, for mainly organic media, partition phenomena are very important and may cause an apparent modification of enzyme specificity

Enzymatic Activity in the Gastrointestinal Tract of Pimelodus maculatus (Teleostei, Siluriformes) in Two Neotropical Reservoirs with Different Trophic Conditions

Enzymatic activities for digestion of proteins and carbohydrates were compared among three organs of the digestive system of Pimelodus maculatus in two reservoirs with different trophic conditions during the winter of 2006. The aim was to test the hypothesis that enzymatic activity for the digestion of proteins and carbohydrates differed among organs and that such activities differ between the trophic state of the environment. Enzymatic activities were determined through the assays of specificity for trypsin, chymotrypsin and β-glucosidase enzymes. The intestine had higher trypsin-like enzymatic activities compared to the stomach and liver. The highest β-glucosidase activity was found in the liver compared to the stomach and intestine in the oligotrophic reservoir only. Overall, enzymatic activity did not differ between the eutrophic and oligotrophic reservoirs, although the intestinal chymotrypsin was comparatively higher in the eutrophic reservoir and the hepatic β-glucosidase was higher in the oligotrophic reservoir. These findings indicated that most digestive activity occurred in the intestine for P. maculatus, which was probably related to its omnivorous/carnivorous feeding habits. The highest proteolytic activity in the intestine was expected for most fishes, but the high hepatic β-glucosidase in the oligotrophic reservoir was unexpected. The hepatic β-glucosidase as well as the intestinal chymotrypsin-like activity could be considered as the candidates for biomarkers of environmental quality