Studies on the membrane interactions of the cyclotides kalata B1 and kalata B6 on model membrane systems by surface plasmon resonance

In this study we have demonstrated the interactions of kalata B1 and its naturally occurring analogue kalata B6 with five model lipid membranes and have analyzed the binding kinetics using surface plasmon resonance. Two kalata peptides showed a higher affinity for the phosphatidylethanolamine-containing membranes, indicating that the peptides would bind selectively to bacterial membranes. Also we have optimized the procedure for the immobilization of five liposome mixtures and have shown that the procedure provides reproducible levels of immobilized liposomes and could be used to screen the selective binding of putative antimicrobial peptides to model mammalian or microbial phospholipid membranes. (C) 2004 Elsevier Inc. All rights reserved.

Altered fungal sensitivity to a plant antimicrobial peptide through over-expression of yeast cDNAs

A yeast cDNA expression library was screened to identify genes and cellular processes that influence fungal sensitivity to a plant antimicrobial peptide. A plasmid-based, GAL1 promoter-driven yeast cDNA expression library was introduced into a yeast genotype susceptible to the antimicrobial peptide MiAMP1 purified from Macadamia integrifolia. Following a screen of 20,000 cDNAs, three yeast cDNAs were identified that reproducibly provided transformants with galactose-dependent resistance to MiAMP1. These cDNAs encoded a protein of unknown function, a component (VMA11) of the vacuolar H+-ATPase and a component (cytochrome c oxidase subunit VIa) of the mitochondrial electron transport chain, respectively. To identify genes that increased sensitivity to MiAMP1, the yeast cDNA expression library was introduced into a yeast mutant with increased resistance to MiAMP1. From 11,000 cDNAs screened, two cDNA clones corresponding to a ser/thr kinase and a ser/thr phosphatase reproducibly increased MiAMP1 susceptibility in the mutant in a galactose-dependent manner. Deletion mutants were available for three of the five genes identified but showed no change in their sensitivity to MiAMP1, indicating that these genes could not be detected by screening of yeast deletion mutant libraries. Yeast cDNA expression library screening therefore provides an alternative approach to gene deletion libraries to identify genes that can influence the sensitivity of fungi to plant antimicrobial peptides.

The mode of action of the plant antimicrobial peptide MiAMP1 differs from that of its structural homologue, the yeast killer toxin WmKT

The plant antimicrobial peptide MiAMP1 from Macadamia integrifolia and the yeast killer toxin peptide WmKT from Williopsis mrakii are structural homologues. Comparative studies of yeast mutants were performed to test their sensitivity to these two antimicrobial peptides. No differences in susceptibility to MiAMP1 were detected between wild-type and several WmKT-resistant mutant yeast strains. A yeast mutant MT1, resistant to MiAMP1 but unaffected in its susceptibility to plant defensins and hydrogen peroxide, also did not show enhanced tolerance towards WmKT. It is therefore probable that the Greek key beta-barrel structure shared by MiAMP1 and WmKT provides a robust structural framework ensuring stability for the two proteins but that the specific action of the peptides depends on other motifs. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Structural and functional characterization of the conserved salt bridge in mammalian Paneth cell alpha-defensins - Solution structures of mouse cryptdin-4 AND (E15D)-cryptdin-4

Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell α-defensin cryptdin-4 (Crp4) and a mutant (E15D)-Crp4 peptide, in which a conserved Glu15 residue was replaced by Asp. Structural analysis of the two peptides confirms the involvement of this Glu in a conserved salt bridge that is removed in the mutant because of the shortened side chain. Despite disruption of this structural feature, the peptide variant retains a well defined native fold because of a rearrangement of side chains, which result in compensating favorable interactions. Furthermore, salt bridge-deficient Crp4 mutants were tested for bactericidal effects and resistance to proteolytic degradation, and all of the variants had similar bactericidal activities and stability to proteolysis. These findings support the conclusion that the function of the conserved salt bridge in Crp4 is not linked to bactericidal activity or proteolytic stability of the mature peptide.

Synthesis of 'difficult' peptides free of aspartimide and related products, using peptoid methodology

We developed an efficient, cost effective strategy for Fmoc-based solid phase synthesis of 'difficult' peptides and/or peptides containing Asp/Asn-Gly sequences, free of aspartimide and related products, using a peptoid methodology for the preparation of N-substituted glycines.

Protein and peptides in pictures:imaging with MALDI mass spectrometry

Imaging using MS has the potential to deliver highly parallel, multiplexed data on the specific localization of molecular ions in tissue samples directly, and to measure and map the variations of these ions during development and disease progression or treatment. There is an intrinsic potential to be able to identify the biomarkers in the same experiment, or by relatively simple extension of the technique. Unlike many other imaging techniques, no a priori knowledge of the markers being sought is necessary. This review concentrates on the use of MALDI-MS for MS imaging (MSI) of proteins and peptides, with an emphasis on mammalian tissue. We discuss the methodologies used, their potential limitations, overall experimental considerations and progress that has been made towards establishing MALDI-MSI as a routine technique for the spatially resolved measurement of peptides and proteins. As well as determining the local abundance of individual molecular ions, there is the potential to determine their identity within the same experiment using relatively simple extensions of the basic techniques. In this way MSI offers an important opportunity for biomarker discovery and identification.

Individual monitoring of immune responses in rainbow trout after cohabitation and intraperitoneal injection challenge with Yersinia ruckeri

Acknowledgements This work was funded by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs, grant G1100675). The authors are grateful to the aquarium staff at the University of Aberdeen (Karen Massie) and Dr David Smail at Marine Scotland for valuable discussion during the establishment of the experimental design.

Chitosan gel film bandages: correlating structure, composition, and antimicrobial properties

Chitosan gel films were successfully obtained by evaporation cast from chitosan solutions in aqueous acidic solutions of organic acids (lactic and acetic acid) as gel film bandages, with a range of additives that directly influence film morphology and porosity. We show that the structure and composition of a wide range of 128 thin gel films, is correlated to the antimicrobial properties, their biocompatibility and resistance to biodegradation. Infrared spectroscopy and solid-state 13C nuclear magnetic resonance spectroscopy was used to correlate film molecular structure and composition to good antimicrobial properties against 10 of the most prevalent Gram positive and Gram negative bacteria. Chitosan gel films reduce the number of colonies after 24 h of incubation by factors of ∼105–107 CFU/mL, compared with controls. For each of these films, the structure and preparation condition has a direct relationship to antimicrobial activity and effectiveness. These gel film bandages also show excellent stability against biodegradation with lysozyme under physiological conditions (5% weight loss over a period of 1 month, 2% in the first week), allowing use during the entire healing process. These chitosan thin films and subsequent derivatives hold potential as low-cost, dissolvable bandages, or second skin, with antimicrobial properties that prohibit the most relevant intrahospital bacteria that infest burn injuries.

Identification and bioactivity evaluation of two novel temporins from the skin secretion of the European edible frog, Pelophylax kl. Esculentus

The amphibian temporins, amongst the smallest antimicrobial peptides (AMPs), are α-helical, amphipathic, hydrophobic and cationic and are active mainly against Gram-positive bacteria but inactive or weakly active against Gram-negative bacteria. Here, we report two novel members of the temporin family, named temporin-1Ee (FLPVIAGVLSKLFamide) and temporin-1Re (FLPGLLAGLLamide), whose biosynthetic precursor structures were deduced from clones obtained from skin secretion-derived cDNA libraries of the European edible frog, Pelophylax kl. esculentus, by ‘shotgun’ cloning. Deduction of the molecular masses of each mature processed peptide from respective cloned cDNAs was used to locate respective molecules in reverse-phase HPLC fractions of secretion. Temporin-1Ee (MIC = 10 μM) and temporin-1Re (MIC = 60 μM) were both found to be active against Gram-positive Staphylococcus aureus, but retaining a weak haemolytic activity. To our knowledge, Single-site substitutions can dramatically change the spectrum of activity of a given temporin. Compared with temporine-1Ec, just one chemically-conservative substitution (Val8 instead of Leu8), temporin-1Ee bearing a net charge of +2 displays broad-spectrum activity with particularly high potency on the clinically relevant Gram-negative strains, Escherichia coli (MIC = 40 μM). These factors bode well for translating temporins to be potential drug candidates for the design of new and valuable anti-infective agents.

Molecular Characterization of Three Novel Phospholipase A2 Proteins from the Venom of Atheris chlorechis, Atheris nitschei and Atheris squamigera

Secretory phospholipase A2 (sPLA2) is known as a major component of snake venoms and displays higher-order catalytic hydrolysis functions as well as a wide range of pathological effects. Atheris is not a notoriously dangerous genus of snakes although there are some reports of fatal cases after envenomation due to the effects of coagulation disturbances and hemorrhaging. Molecular characterization of Atheris venom enzymes is incomplete and there are only a few reports in the literature. Here, we report, for the first time, the cloning and characterization of three novel cDNAs encoding phospholipase A2 precursors (one each) from the venoms of the Western bush viper (Atheris chlorechis), the Great Lakes bush viper (Atheris nitschei) and the Variable bush viper (Atheris squamigera), using a “shotgun cloning” strategy. Open-reading frames of respective cloned cDNAs contained putative 16 residue signal peptides and mature proteins composed of 121 to 123 amino acid residues. Alignment of mature protein sequences revealed high degrees of structural conservation and identity with Group II venom PLA2 proteins from other taxa within the Viperidae. Reverse-phase High Performance Liquid Chromatography (HPLC) profiles of these three snake venoms were obtained separately and chromatographic fractions were assessed for phospholipase activity using an egg yolk suspension assay. The molecular masses of mature proteins were all identified as approximately 14 kDa. Mass spectrometric analyses of the fractionated oligopeptides arising from tryptic digestion of intact venom proteins, was performed for further structural characterization.

Kunitzins: Prototypes of a new class of protease inhibitor from the skin secretions of European and Asian frogs

Amphibian skin secretions contain biologically-active compounds, such as anti-microbial peptides and trypsin inhibitors, which are used by biomedical researchers as a source of potential novel drug leads or pharmacological agents. Here, we report the application of a recently developed technique within our laboratory to “shotgun” clone the cDNAs encoding two novel but structurally-related peptides from the lyophilized skin secretions of one species of European frog, Rana esculenta and one species of Chinese frog, Odorrana schmackeri. Bioanalysis of the peptides established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17, which is a canonical Kunitz-type protease inhibitor motif (-CKAAFC-). Due to the presence of this structural attribute, these peptides were named kunitzin-RE (AAKIILNPKFRCKAAFC) and kunitzin-OS (AVNIPFKVHLRCKAAFC). Synthetic replicates of these two novel peptides were found to display a potent inhibitory activity against Escherichia coli but were ineffective at inhibiting the growth of Staphylococcus aureus and Candida albicans at concentrations up to 160 μM, and both showed little haemolytic activity at concentrations up to 120 μM. Subsequently, kunitzin-RE and kunitzin-OS were found to be a potent inhibitor of trypsin with a Ki of 5.56 μM and 7.56 μM that represent prototypes of a novel class of highly-attenuated amphibian skin protease inhibitor. Substitution of Lys-13, the predicted residue occupying the P1 position within the inhibitory loop, with Phe (F) resulted in decrease in trypsin inhibitor effectiveness and antimicrobial activity against Esherichia coli, but exhibits a potential inhibition activity against chymotrypsin.

Gene therapy tools: oligonucleotides and peptides

Genetic mutations can cause a wide range of diseases, e.g. cancer. Gene therapy has the potential to alleviate or even cure these diseases. One of the many gene therapies developed so far is RNA-cleaving deoxyribozymes, short DNA oligonucleotides that specifically bind to and cleave RNA. Since the development of these synthetic catalytic oligonucleotides, the main way of determining their cleavage kinetics has been through the use of a laborious and error prone gel assay to quantify substrate and product at different time-points. We have developed two new methods for this purpose. The first one includes a fluorescent intercalating dye, PicoGreen, which has an increased fluorescence upon binding double-stranded oligonucleotides; during the course of the reaction the fluorescence intensity will decrease as the RNA is cleaved and dissociates from the deoxyribozyme. A second method was developed based on the common denominator of all nucleases, each cleavage event exposes a single phosphate of the oligonucleotide phosphate backbone; the exposed phosphate can simultaneously be released by a phosphatase and directly quantified by a fluorescent phosphate sensor. This method allows for multiple turnover kinetics of diverse types of nucleases, including deoxyribozymes and protein nucleases. The main challenge of gene therapy is often the delivery into the cell. To bypass cellular defenses researchers have used a vast number of methods; one of these are cell-penetrating peptides which can be either covalently coupled to or non-covalently complexed with a cargo to deliver it into a cell. To further evolve cell-penetrating peptides and understand how they work we developed an assay to be able to quickly screen different conditions in a high-throughput manner. A luciferase up- and downregulation experiment was used together with a reduction of the experimental time by 1 day, upscaling from 24- to 96-well plates and the cost was reduced by 95% compared to commercially available assays. In the last paper we evaluated if cell-penetrating peptides could be used to improve the uptake of an LNA oligonucleotide mimic of GRN163L, a telomerase-inhibiting oligonucleotide. The combination of cell-penetrating peptides and our mimic oligonucleotide lead to an IC50 more than 20 times lower than that of GRN163L.

Genetic Modification of Disease Resistance: Fungal and Oomycete Pathogens

Development of recombinant DNA technology allowed scientists to manipulate plant genomes, making it possible to study genes and exploit them to modify novel agronomic traits. Here, we review the current and future potential of genetic modification (GM) strategies used to increase the resistance of plants to oomycete and fungal pathogens. Numerous resistance genes (R-genes) have been cloned, and under laboratory conditions, transgenic plants have given promising results against some important plant pathogens. However, only a few have so far been deployed as commercial crop plants.GMof plants to disrupt pathogenicity, such as by inhibiting or degrading pathogenicity factors, especially by necrotrophic pathogens, has also been exploited. The potential to engineer plants for the production of antimicrobial peptides or to modify defense-signaling pathways have been successfully demonstrated under laboratory conditions. The most promising current technology is genome editing, which allows researchers to edit DNA sequences directly in their endogenous environment. The potential of this approach is discussed in detail and examples where broad-spectrum resistance has been achieved are given.

Channel-forming activity of syringopeptin 25 A in mercury-supported phospholipid monolayers and negatively charged bilayers

Interactions of the cationic lipodepsipeptide syringopeptin 25 A (SP25A) with mercury-supported dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS) and dioeleoylphosphatidic acid (DOPA) self-assembled monolayers (SAMs) were investigated by AC voltammetry in 0.1 M KCl at pH 3, 5.4 and 6.8. SP25A targets and penetrates the DOPS SAM much more effectively than the other SAMs not only at pH 6.8, where the DOPS SAM is negatively charged, but also at pH 3, where it is positively charged just as SP25A. Similar investigations at tethered bilayer lipid membranes (tBLMs) consisting of a thiolipid called DPTL anchored to mercury, with a DOPS, DOPA or DOPC distal monolayer on top of it, showed that, at physiological transmembrane potentials, SP25A forms ion channels spanning the tBLM only if DOPS is the distal monolayer. The distinguishing chemical feature of the DOPS SAM is the ionic interaction between the protonated amino group of a DOPS molecule and the carboxylate group of an adjacent phospholipid molecule. Under the reasonable assumption that SP25A preferentially interacts with this ion pair, the selective lipodepsipeptide antimicrobial activity against Gram-positive bacteria may be tentatively explained by its affinity for similar protonated amino-carboxylate pairs, which are expected to be present in the peptide moieties of peptidoglycan strands.

Phenolic profile of Cynara scolymus L. and Silybum marianum(L.) Gaertn and related antimicrobial properties

Cynara scolymus L. (artichoke) and Silybum marianum (L.) Gaertn (milk thistle), belonging to the Asteraceae family, are medicinal plants vo.ith well-reported antioxidant and hepatoprotective effects. Widely consumed as infusions, these plants can also be found in several formulations to allow an easier consumption. The bioactivity of infusions, pills, and syrups based on artichoke and milk thistle was previously reported by our research group [1 ,2] and among the various phytochemicals present in these dietary supplements, phenolic compounds are pointed out as the most responsible for their beneficial properties. With the aim of studying the antimicrobial activity and possible relation vo.ith the phenolic composition, two different formulations of each plant were assessed (pills and syrups). The phenolic profiles were obtained by HPLC-DAD-ESIIMS, and the antimicrobial activity was performed with clinical isolates from hospitalized patients, namely Escherichia coli, Escherichia coli spectrum extended producer of P-lactarnases (ESBL), Proteus mirabilis, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA). Vanillic acid (5.58 J.tg/g) and luteolin-7-0-glucoside (2.2 J.tg/g) were the most abundant compounds in artichoke syrup, that did not reveal antimicrobial activity against the studied strains, which could be due to their low concentrations. On the other hand, artichoke pills presented a prevalence of 5-0-caffeoylquinic (28.2 J.tg/g), 1,3-dicaffeoylquinic (24 J.tg/g), and 4-0-Caffeoylquinic acids (13.3 J.tg/g); revealing the capacity to inhibit MRSA vo.ith a MIC value of 1.9 mg!g. Regarding milk thistle, isorhamnetin-0-deoxyhexoside-0-hexoside, isorhamnetin-3-0-rutinoside, and isorhamnetin-0-deoxyhexoside-0-dihexoside were the major compounds detected in the syrup, in concentrations of 7.26, 5. 75, and 3.64 J.tg/g, respectively. This formulation proved to be able to inhibit the growth of E. coli, ESBL, MRSA and P. aeruginosa, with MIC values ranging from 0.2 to 1.3 mg!mL. Hydroxylated silibinin (1.565 J.!g/g) was the major flavonoid found in the pills, that revealed antimicrobial activity against ESBL, with a MIC value of 15 mg!mL, but did not inhibit the growth of the remaining bacteria None of the studied samples was able to inhibit P. mirabilis at the studied concentrations (1000 and 26.4 mg!mL for the syrups of artichoke and milk thistle, respectively; 150 mg/mL for both pills). Overall, the studied syrups and pills of artichoke and milk thistle revealed to be a good source of phenolic compounds, with some of these formulations revealing antimicrobial activity.