Inhibition of MRSA and of Clostridium difficile by durancin 61A: synergy with bacteriocins and antibiotics.

AIM: The aim of this study was to evaluate the efficacy of durancin 61A alone or in combination with nisin, pediocin PA-1, reuterin, microcin J25, vancomycin or tetracycline as an inhibitor of resistant clinical pathogens and to shed light on its mode of action. RESULTS: Durancin and reuterin were effective inhibitors of Clostridium difficile, vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus. The combination of durancin and reuterin was highly synergistic against C. difficile (fractional inhibitory concentration index = 0.2). Durancin/vancomycin combination was synergistic against S. aureus ATCC® 700699 (fractional inhibitory concentration index = 0.3). Conclusion & future perspective: Durancin 61A alone or combined with other bacteriocins or antibiotics may therefore provide a possible therapeutic option for the treatment of infections by these pathogens.

ANTISTAPHYBASE: database of antimicrobial peptides (AMPs) and essential oils (EOs) against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus.

Staphylococcus aureus and methicillin-resistant S. aureus are major pathogens. The antimicrobial peptides and essential oils (EOs) display narrow- or broad-spectrum activity against bacteria including these strains. A centralized resource, such as a database, designed specifically for anti-S. aureus/anti-methicillin-resistant S. aureus antimicrobial peptides and EOs is therefore needed to facilitate the comprehensive investigation of their structure/activity associations and combinations. The database ANTISTAPHYBASE is created to facilitate access to important information on antimicrobial peptides and essential peptides against methicillin-resistant S. aureus and S. aureus. At the moment, the database contains 596 sequences of antimicrobial peptides produced by diverse organisms and 287 essential oil records. It permits a quick and easy search of peptides based on their activity as well as their general, physicochemical properties and literature data. These data are very useful to perform further bioinformatic or chemometric analysis and would certainly be useful for the development of new drugs for medical use. The ANTISTAPHYBASE database is freely available at: https://www.antistaphybase.com/ .

Inhibition of methicillin-resistant Staphylococcus aureus (MRSA) by antimicrobial peptides (AMPs) and plant essential oils.

CONTEXT: Drug-resistant bacterial infections cause considerable patient mortality and morbidity. The annual frequency of deaths from methicillin-resistant Staphylococcus aureus (MRSA) has surpassed those caused by human immunodeficiency virus/acquired immune deficiency syndrome. The antimicrobial peptides (AMPs), plant essential oils (EOs) and their combinations have proven to be quite effective in killing a wide selection of bacterial pathogens including MRSA. OBJECTIVES: This review summarizes the studies in the use of AMPs, plant EOs and their combinations for coping with MRSA bacteria, and to formulate new prospects for future studies on this topic. METHODS: The sources of scientific literature such as PubMed, library search, Google Scholar, Science Direct and electronic databases such as 'The Antimicrobial Peptide Database', 'Collection of Anti-Microbial Peptides' and 'YADAMP'. Physicochemical data of anti-MRSA peptides were determined by Scientific DataBase Maker software. RESULTS: Of the 118 peptides, 88 exhibited an activity against MRSA with the highest activity of minimum inhibitory concentration values. Various plant EOs have been effective against MRSA. Remarkably, lemongrass EOs completely inhibited all MRSA growth on the plate. Lemon myrtle, Mountain savory, Cinnamon bark and Melissa EOs showed a significant inhibition. CONCLUSION: Several of these AMPs, EOs and their combinations were effective against MRSA. Their activities have implications for the development of new drugs for medical use.

Simultaneous Production of Formylated and Nonformylated Enterocins L50A and L50B as well as 61A, a New Glycosylated Durancin, by Enterococcus durans 61A, a Strain Isolated from Artisanal Fermented Milk in Tunisia.

Enterococcus durans 61A, a broad-spectrum strain, was isolated from artisanal fermented dairy products. The strain is a multibacteriocin producer, free from virulence genes, and could be considered a good candidate for application in food preservation. In the present study, E. durans 61A was shown to produce simultaneously formylated and nonformylated forms of leaderless enterocins L50A and L50B as well as 61A, a new glycosylated durancin. Bacteriocins were characterized using mass spectrometry. Formylation was found to increase enterocin antimicrobial activity of enterocin L50A (8×) and, to a lesser extent, the activity of L50B (2×). Durancin 61A was found glycosylated by two hexoses (glucose and arabinose) and exhibited broad-spectrum inhibition against Gram-positive and Gram-negative bacteria and fungal spores. Durancin 61A was highly bactericidal at 15.6 µg/mL (10× the MIC) on Listeria innocua HPB13 and seems to target bacterial membrane as shown by ion efflux and transmission electron microscopy.

Correspondences between the binding characteristics of a non-natural peptide, Lei-Dab7, and the distribution of SK subunits in the rat central nervous system.

Small-conductance calcium-activated potassium channels (SK1-SK3 channels) are responsible for long-lasting hyperpolarization following action potential and contribute to the neuronal firing and integration signal. Two peptide toxins: apamin and Leiurotoxin 1, block this SK channels with high affinities. We generated a modified Leiurotoxin 1 (Lei-Dab7) that inhibits SK2 channels with a high selectivity. Competitive binding of radio-iodinated apamin to different rat brain structures, in the presence of native apamin and Lei-Dab7, has shown that dissociation constants differ by a factor of 1000 and thus demonstrated that ligand affinity is as important as ligand selectivity for a specific receptor. However, the lack of ligands discriminating between SK channel subunits is impeding the understanding of the role of each heteromeric SK channel type in different tissues. Our study aims to better understand the molecular combinations of SK channels and their association with specific functional implications. On this purpose, a clustering technique allows us to identify five groups of brain structures reflecting singular profiles of affinity and selectivity of Lei-Dab7 in comparison with apamin. The analysis of correspondences between Lei-Dab7 binding and distribution of SK subunits in these groups of brain structures suggests that functional heteromeric SK channels are involved in specific information processes.

Polymorphisms in oxidative stress-related genes are associated with nasopharyngeal carcinoma susceptibility.

Nasopharyngeal carcinoma (NPC) is a complex multifactorial disorder involving both genetic and environmental factors. Polymorphisms of genes encoding nitric oxide synthase (NOS) and antioxidant glutathione-S transferases (GSTs) have been associated with various tumors. We examined the combined role of NOS3, NOS2 and GST polymorphisms in NPC risk in Tunisians. We found that NOS3−786C allele and −786 CC genotype, NOS3+894T allele and +894 GT+TT genotypes, NOS2−277 G allele and −277 GG genotype, and GSTT1 del/del genotype, are more prevalent in NPC patients as compared to healthy controls. Our results suggest that genetically driven dysfunction in red­ox stress pathway could augment the risk in NPC-susceptible individuals.

SKCa Channels Blockage Increases the Expression of Adenosine A2A Receptor in Jurkat Human T Cells.

Adenosine is a nucleoside displaying various biological effects via stimulation of four G-protein-coupled receptors, A1, A2A, A2B, and A3. Adenosine also modulates voltage-gated (Kv) and small conductance calcium-activated (SKCa) potassium channels. The effect of these potassium channels on the expression of adenosine receptors is poorly understood. We evaluated the action of BgK (a natural Kv channel blocker) and Lei-Dab7 (a synthetic SKCa channel blocker) on the expression of adenosine A2A receptors (A2AR) in Jurkat human T cells. We found that Lei-Dab7, but not BgK, increased the maximal binding value of the tritiated ligand ZM241385 to A2AR in a dose-dependent manner (+45% at 5 nM; +70% at 50 nM as compared to control). These results were further confirmed by Western blotting using a specific monoclonal antibody to human A2AR. The ligand affinity-related dissociation constant and A2AR mRNA amount were not significantly modified by either drug. We suggest that modulation of SKCa channels can influence membrane expression of A2AR and thus has a therapeutic potential.

Homology modeling and virtual screening approaches to identify potent inhibitors of VEB-1 ß-lactamase.

BACKGROUND: blaVEB-1 is an integron-located extended-spectrum ß-lactamase gene initially detected in Escherichia coli and Pseudomonas aeruginosa strains from south-east Asia. Several recent studies have reported that VEB-1-positive strains are highly resistant to ceftazidime, cefotaxime and aztreonam antibiotics. One strategy to overcome resistance involves administering antibiotics together with ß-lactamase inhibitors during the treatment of infectious diseases. During this study, four VEB-1 ß-lactamase inhibitors were identified using computer-aided drug design. METHODS: The SWISS-MODEL tool was utilized to generate three dimensional structures of VEB-1 ß-lactamase, and the 3D model VEB-1 was verified using PROCHECK, ERRAT and VERIFY 3D programs. Virtual screening was performed by docking inhibitors obtained from the ZINC Database to the active site of the VEB-1 protein using AutoDock Vina software. RESULTS AND CONCLUSION: Homology modeling studies were performed to obtain a three-dimensional structure of VEB-1 ß-lactamase. The generated model was validated, and virtual screening of a large chemical ligand library with docking simulations was performed using AutoDock software with the ZINC database. On the basis of the dock-score, four molecules were subjected to ADME/TOX analysis, with ZINC4085364 emerging as the most potent inhibitor of the VEB-1 ß-lactamase.

Capture of sunflower seedlings lipase using polyclonal antibodies.

True lipolytic activity is observed in different subcellular fractions of germinating sunflower seedlings (Helianthus annuus L.) in delipidated oleosomes and microsomes. Triacylglycerol lipase (EC. 3.1.1.3) catalyses the first catabolic step of lipolysis. To our knowledge, this plant lipase has not yet been identified. Our aim was to develop a method to collect the lipase for further studies. An immunological method was used to capture sunflower seedling lipase from oleosomes and microsomes. This method uses an immunoaffinity column prepared with polyclonal antibodies (anti-P-61) directed against oleosomal activity. Our results verify that we have successfully adapted a purification procedure of plant lipase using anti-P-61. Since the eluted lipolytic activity is distributed among diverse proteic peaks, we changed the elution procedure: the introduction of CHAPS, a zwitterionic detergent, allowed us to recover all the lipolytic activity in a single proteic peak. This may help us to characterise the studied lipase.

Three-Dimensional Structure of Arabidopsis thaliana Lipase Predicted by Homology Modeling Method.

Triacylglycerol lipases have been thoroughly characterized in mammals and microorganisms. By contrast, very little is known about plant lipases. In this investigation, a homology model of Arabidopsis thaliana lipase (NP_179126) was constructed using a human gastric lipase (PDB ID: 1HLG), as a template for model building. This model was then assessed for stereochemical quality and side chain environment. Natural substrates: tributyrin, trioctanoin and triolen were docked into the model to investigate ligand-substrate interaction.

BACTIBASE second release: a database and tool platform for bacteriocin characterization.

BACKGROUND: BACTIBASE is an integrated open-access database designed for the characterization of bacterial antimicrobial peptides, commonly known as bacteriocins. DESCRIPTION: For its second release, BACTIBASE has been expanded and equipped with additional functions aimed at both casual and power users. The number of entries has been increased by 44% and includes data collected from published literature as well as high-throughput datasets. The database provides a manually curated annotation of bacteriocin sequences. Improvements brought to BACTIBASE include incorporation of various tools for bacteriocin analysis, such as homology search, multiple sequence alignments, Hidden Markov Models, molecular modelling and retrieval through our taxonomy Browser. CONCLUSION: The provided features should make BACTIBASE a useful tool in food preservation or food safety applications and could have implications for the development of new drugs for medical use. BACTIBASE is available at http://bactibase.pfba-lab-tun.org.

Purification and characterization of alpha-amylase from safflower (Carthamus tinctorius L.) germinating seeds.

alpha-Amylase (alpha-1-4 D-glucan glucanohydrolase EC 3.2.1.1) crude extract was obtained from safflower (Carthamus tinctorius L.) cotyledons excised from 5-day-old dark grown seedlings. The enzyme was purified by precipitating the crude extract with ammonium sulphate at 20-60% saturation, and then by subjecting this fraction to affinity chromatography on a beta-cyclodextrin-Sepharose 6B column. The active fraction was dialysed and concentrated. An overall purification of about 131 folds with an activity yield of 81.25% was achieved. The molecular mass of purified enzyme determined by SDS-PAGE was 35 kD. When the purified alpha-amylase was subjected to gel electrophoresis followed by negative staining, only one band of active protein was detected. Its maximal activity was in the pH 6.0 and at a temperature of 55 degrees C. This enzyme was activated by Ca(2+) and inhibited by Fe(2+).

Physiological behaviour of four rapeseed cultivar (Brassica napus L.) submitted to metal stress.

Eliminating heavy metals in the environment by phytoremediation is a method that uses, generally, plants with a low biomass yielded and feeble depth of root system. For the purpose of improving this technique, we have tested four varieties of productive specie with high yields, the rapeseed (Brassica napus L.). In particular, we have studied metal stress effect on biomass, growth, and endogenous Zn and Cd content. Metal treatment caused significant dry weight differences between metal-treated and control plants. A significant genotypic difference has been noticed between the four cv. For two varieties, Jumbo and Drakkar, the accumulation is more important in the stems and petioles, whereas, this accumulation is at a maximum level in the root system for the two varieties, Cossair and Pactol. Chlorophyll and carotenoïd content, as well as lipid peroxidation, known as stress markers, were also evaluated. Metal treatment led to an increase in the amount of malondialdehyde (MDA) in the leaves. However, the increase of Zn and Cd levels in the tissue culture was followed by a decrease in the photosynthetic pigments.

PhytAMP: a database dedicated to antimicrobial plant peptides.

Plants produce small cysteine-rich antimicrobial peptides as an innate defense against pathogens. Based on amino acid sequence homology, these peptides were classified mostly as alpha-defensins, thionins, lipid transfer proteins, cyclotides, snakins and hevein-like. Although many antimicrobial plant peptides are now well characterized, much information is still missing or is unavailable to potential users. The compilation of such information in one centralized resource, such as a database would therefore facilitate the study of the potential these peptide structures represent, for example, as alternatives in response to increasing antibiotic resistance or for increasing plant resistance to pathogens by genetic engineering. To achieve this goal, we developed a new database, PhytAMP, which contains valuable information on antimicrobial plant peptides, including taxonomic, microbiological and physicochemical data. Information is very easy to extract from this database and allows rapid prediction of structure/function relationships and target organisms and hence better exploitation of plant peptide biological activities in both the pharmaceutical and agricultural sectors. PhytAMP may be accessed free of charge at http://phytamp.pfba-lab.org.

SciDBMaker: new software for computer-aided design of specialized biological databases.

BACKGROUND: The exponential growth of research in molecular biology has brought concomitant proliferation of databases for stocking its findings. A variety of protein sequence databases exist. While all of these strive for completeness, the range of user interests is often beyond their scope. Large databases covering a broad range of domains tend to offer less detailed information than smaller, more specialized resources, often creating a need to combine data from many sources in order to obtain a complete picture. Scientific researchers are continually developing new specific databases to enhance their understanding of biological processes. DESCRIPTION: In this article, we present the implementation of a new tool for protein data analysis. With its easy-to-use user interface, this software provides the opportunity to build more specialized protein databases from a universal protein sequence database such as Swiss-Prot. A family of proteins known as bacteriocins is analyzed as 'proof of concept'. CONCLUSION: SciDBMaker is stand-alone software that allows the extraction of protein data from the Swiss-Prot database, sequence analysis comprising physicochemical profile calculations, homologous sequences search, multiple sequence alignments and the building of new and more specialized databases. It compiles information with relative ease, updates and compares various data relevant to a given protein family and could solve the problem of dispersed biological search results.

BACTIBASE: a new web-accessible database for bacteriocin characterization.

BACKGROUND: Bacteriocins are very diverse group of antimicrobial peptides produced by a wide range of bacteria and known for their inhibitory activity against various human and animal pathogens. Although many bacteriocins are now well characterized, much information is still missing or is unavailable to potential users. The assembly of such information in one central resource such as a database would therefore be of great benefit to the exploitation of these bioactive molecules in the present context of increasing antibiotic resistance and natural bio-preservation need. DESCRIPTION: In the present paper, we present the development of a new and original database BACTIBASE that contains calculated or predicted physicochemical properties of 123 bacteriocins produced by both Gram-positive and Gram-negative bacteria. The information in this database is very easy to extract and allows rapid prediction of relationships structure/function and target organisms of these peptides and therefore better exploitation of their biological activity in both the medical and food sectors. CONCLUSION: The BACTIBASE database is freely available at http://bactibase.pfba-lab.org, web-based platform enabling easy retrieval, via various filters, of sets of bacteriocins that will enable detailed analysis of a number of microbiological and physicochemical data.

Morphological and physiological characteristics of rapeseed plants regenerated in vitro from thin cell layers in the presence of zinc.

Phytoremediation offers owners and managers of metal-contaminated sites an innovative and cost-effective option to address recalcitrant environmental contamination. The use of plants to restore or stabilize contaminated sites, known as phytoremediation, takes advantage of the natural abilities of plants to take up, accumulate or store metals. This includes the use of plants that tolerate and accumulate metals at high levels for phytoextraction and the use of plants growing under conditions that are toxic to other plants, for preventing, for example, soil erosion (phytostabilisation). Rapeseed (Brassica napus L.) was shown to be able to accumulate substantial amounts of metals combined with high biomass. Brassica napus was therefore selected for heavy metal (HM) tolerance and accumulation through in vitro selection. A selective pressure applied during the neoformation process from transversal thin cell layers (tTCLs) allowed us to select tolerant cells and tissues. Toxic metals (such as Zn) were added to the culture media in order to select zinc-tolerant plants. Exerting a selective pressure during tTCLs regeneration aimed at selecting plants with exceptional zinc tolerance and/or accumulating capacity. The morphological and physicochemical characteristics of regenerated plants cultivated in greenhouse appeared to depend very significantly on the concentration of ZnSO(4) applied during the neoformation process. Plants regenerated in the presence of ZnSO(4) at 100 microM exhibited a greater size and a higher biomass together with flowering precocity. The contents of zinc, chlorophyll, and proline were modified in the regenerated plants. Pre-treatment with an excess of ZnSO(4) (>500 microM) was responsible for a percentage of tTCLs intolerance above 96%. With lower Zn concentrations (100-250 microM), the survival rates (33-15%) were higher.

Proteomic analysis of the effect of heat stress on hexaploid wheat grain: characterization of heat-responsive proteins from non-prolamins fraction.

The effect of heat stress on hexaploid wheat grain proteome was recently analyzed in our previous works. Proteomic tools allowed the characterization of heat-responsive proteins of total endosperm, composed mainly of prolamins. The present work completes this study; our aim was to analyze the effect of heat stress on the water-soluble fraction, composed essentially of albumins and globulins. These proteins were separated by two-dimensional electrophoresis (2-DE), visualized by Coomassie Brilliant Blue (CBB) staining and analyzed by Melanie-3 software. Of the 43 heat-changed proteins, 24 were found to be up-regulated whereas 19 spot proteins were down-regulated. All of these proteins were subjected to matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by database searching which allowed the identification of 42 spots. Of these, some were enzymes involved in different metabolic pathways of plants, such as granule-bound starch synthase and glucose-1-phosphate adenyltransferase, involved in the starch synthesis pathway; beta-amylase, involved in carbohydrate metabolism, and the ATP synthase beta-chain that was related to four heat-decreased proteins. Moreover, five heat up-regulated proteins showed similarities with small heat shock proteins while three other spots were related to elongation factors or eucaryotic translation initiation factors. Proteins involved in abiotic stresses or in plant defense mechanism were also identified and are discussed.

Proteomic analysis of the effect of heat stress on hexaploid wheat grain: Characterization of heat-responsive proteins from total endosperm.

High temperatures during grain filling have been reported to be one of the factors that can affect the dough properties and quality characteristics of wheat. Responses to high temperature have been related to changes in protein composition at both quantitative and qualitative levels. The present study was conducted to determine the influence of high temperature during grain filling on the protein composition of bread wheat evaluated by proteomic tools. Plants were grown in the field and transferred to cabinets soon after flowering. They were subjected to two thermal regimes 18 degrees C/10 degrees C (day/night) and 34 degrees C/10 degrees C. Total proteins were extracted from control grains and treated plants at three different post-anthesis stages. The proteins were separated by two-dimensional gel electrophoresis and analysed by Melanie 3 software. Of the total number of mature wheat grain proteins, 37 were identified as significantly changed by heat treatment. Analysis by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry coupled with database searching allowed the characterization of 25 heat-induced proteins and only one heat-decreased protein spot. To learn more about the function of the identified proteins, we examined their expression during treatment.