Expression and Purification of the VpDef Defensin in Escherichia coli using the Small Metal-Binding Proteins CusF3H+ and SmbP.

BACKGROUND: The heterologous production of antimicrobial peptides in bacterial models can produce insoluble proteins due to the lack of proper folding. Fusion proteins have been used to increase the expression and solubility of these types of proteins with varying degrees of success. OBJECTIVES: Here, we demonstrate the use of the small metal-binding proteins CusF3H+ (9.9kDa) and SmbP (9.9kDa) as fusion partners for the soluble expression of the bioactive antimicrobial peptide VpDef(6.9 kDa) in Escherichia coli. METHODS: The recombinant VpDef (rVpDef) peptide was expressed as a translational fusion with CusF3H+ and SmbP in Escherichia coli SHuffle under different small-scale culture conditions. The best conditions were applied to 1-liter cultures, with subsequent purification of the recombinant protein through IMAC chromatography. The recombinant protein was digested using enterokinase to liberate the peptide from the fusion protein, and a second IMAC chromatography step removed the fusion protein. The purified peptide was tested against two Gram-positive and two Gram-negative bacteria. RESULTS: The use either of CusF3H+ or of SmbP results in recombinant proteins that are found in the soluble fraction of the bacterial lysate; these recombinant proteins are easily purified through IMAC chromatography, and rVpDef is readily separated following enterokinase treatment. The purified rVpDef peptide exhibits antimicrobial properties against both Gram-positive and Gram-negative. CONCLUSION: Use of the fusion proteins CusF3H+ and SmbP results in production of a soluble recombinant protein containing the antimicrobial peptide rVpDef that is correctly folded and that retains its antimicrobial properties once purified.

Expression and Functional Characterization of a Novel Antimicrobial Peptide: Human Beta-Defensin 118.

PURPOSE: ß-Defensin 118 (DEFB118) is a novel host defense peptide (HDP) identified in humans. To evaluate its potentials for future utilization, the DEFB118 gene was expressed in Escherichia coli (E. coli) and the recombinant protein was fully characterized. METHODS: The DEFB118 protein was obtained by heterologous expression using E. coli Rosetta (DE3). Antibacterial activity of DEFB118 was determined by using various bacterial strains. IPEC-J cells challenged by E. coli K88 were used to determine its influences on inflammatory responses. RESULTS: The E. coli transformants yielded more than 250 µg/mL DEFB118 protein after 4 h induction by 1.0 mM IPTG. The DEFB118 was estimated by SDS-PAGE to be 30 kDa, and MALDI-TOF analysis verified that it is a human ß-defensin 118. Importantly, the DEFB118 showed antimicrobial activities against both Gram-negative bacteria (E. coli K88 and E. coli DH5α) and Gram-positive bacteria (S. aureus and B. subtilis), with a minimum inhibitory concentration (MIC) of 4 µg/mL. Hemolytic assays showed that DEFB118 had no detrimental impact on cell viability. Additionally, DEFB118 was found to elevate the viability of IPEC-J2 cells upon E. coli K88 challenge. Moreover, DEFB118 significantly decreased cell apoptosis in the late apoptosis phase and downregulated the expression of inflammatory cytokines such as IL-1ß and TNF-α in IPEC-J2 cell exposure to E. coli K88. CONCLUSIONS: These results suggested a novel function of the mammalian defensins, and the antibacterial and anti-inflammatory properties of DEFB118 may allow it as a potential substitute for conventionally used antibiotics or drugs.

Identification and Characterization of Two Defensins from Capsicum annuum Fruits that Exhibit Antimicrobial Activity.

Scientific advances have not been enough to combat the growing resistance to antimicrobial medicines. Antimicrobial peptides (AMPs) are effector molecules of the innate immune defense system in plants and could provide an important source of new antimicrobial drugs. The aim of this work was to extract, purify, characterize, and evaluate the antifungal activities present in fractions obtained from Capsicum annum fruits through reversed-phase chromatography. The fractions named F2 and F3 presented the highest inhibitory activity against Candida and Mycobacterium tuberculosis species. In addition, we identified two sequences of AMPs in the F2 and F3 fractions through mass spectrometry that showed similarity to an already well-characterized family of plant defensins. A plasma membrane permeabilization assay demonstrated that the peptides present in F2, F3, and F4 fractions induced changes in the membrane of some yeast strains, culminating in permeabilization. The production of reactive oxygen species was induced by the fractions in some yeast strains. Fractions F2, F3, and F4 also did not show toxicity in macrophage or monocyte cultures. In conclusion, the obtained data demonstrate that the AMPs, especially those present in the fractions F2 and F3, are promising antimicrobial agents that may be useful to enhance the development of new therapeutic agents for the treatment of diseases.

Class I defensins (BraDef) from broccoli (Brassica oleracea var. italica) seeds and their antimicrobial activity.

The objective of this study was to determine whether seeds of Brassica oleracea var. italica (i.e. broccoli, an edible plant) produce defensins that inhibit phytopathogenic fungi and pathogenic bacteria of clinical significance. Crude extracts obtained from broccoli seeds were fractioned by molecular exclusion techniques and analyzed by liquid chromatography-high-resolution mass spectrometry. Two peptides were identified, BraDef1 (10.68 kDa) and BraDef2 (9.9 kDa), which were categorized as Class I defensins based on (a) their primary structure, (b) the presence of four putative cysteine disulfide bridges, and (c) molecular modeling predictions. BraDef1 and BraDef2 show identities of, respectively, 98 and 71%, and 67 and 85%, with defensins from Brassica napus and Arabidopsis thaliana. BraDef (BraDef1 + BraDef2) disrupted membranes of Colletotrichum gloeosporioides and Alternaria alternata and also reduced hyphal growth of C. gloeosporioides by ~ 56% after 120 h of incubation. Pathogenic bacteria (Bacillus cereus 183, Listeria monocytogenes, Salmonella typhimurium, Pseudomonas aeruginosa, and Vibrio parahaemolitycus) were susceptible to BraDef, but probiotic bacteria such as Bifidobacterium animalis, Lactobacillus acidophilus, and Lactobacillus casei were not inhibited. To our knowledge, this is the first report of defensins present in seeds of B. oleracea var. italica (i.e. edible broccoli). Our findings suggest an applied value for BraDef1/BraDef2 in controlling phytopathogenic fungi and pathogenic bacteria of clinical significance.

A novel recombinant javanicin with dual antifungal and anti-proliferative activities.

Resistance to common drugs by microorganisms and cancers has become a major issue in modern healthcare, increasing the number of deaths worldwide. Novel therapeutic agents with a higher efficiency and less side effects for the treatment of certain diseases are urgently needed. Plant defensins have an integral role in a hosts' immune system and are attractive candidates for combatting drug-resistant microorganisms. Interestingly, some of these defensins also showed great potential due to their cytotoxic activity toward cancer cells. In this study, a defensin encoding gene was isolated from five legume seeds using 3' rapid amplification of cDNA ends (3' RACE) with degenerate primers and cDNA cloning strategies. Bioinformatic tools were used for in silico identification and the characterization of new sequences. To study the functional characteristics of these unique defensins, the gene encoded for Sesbania javanica defensin, designated as javanicin, was cloned into pTXB-1 plasmid and expressed in the Escherichia coli Origami 2 (DE3) strain. Under optimized conditions, a 34-kDa javanicin-intein fusion protein was expressed and approximately 2.5-3.5 mg/L of soluble recombinant javanicin was successfully extracted with over 90% purity. Recombinant javanicin displayed antifungal properties against human pathogenic fungi, including resistant strains, as well as cytotoxic activities toward the human breast cancer cell lines, MCF-7 & MDA-MB-231. Recombinant javanicin holds great promise as a novel therapeutic agent for further medical applications.

Isolation and characterisation of the antifungal activity of the cowpea defensin Cp-thionin II.

As a result of the rapidly growing human population, reducing post-harvest crop losses of cereals due to microbial pests has major importance. Plant defensins have the potential to fulfil these demands, being highly specific and efficient antimicrobial agents. Hence, this study aimed to extract and characterise a peptide from cowpea seeds and investigate its antifungal performance. After extraction and partial purification, N-terminal sequencing was used to identify the primary peptide in the extract as cowpea-thionin II. Antifungal activity in vitro was found against Fusarium culmorum (MIC = 50 µg/mL), but Aspergillus niger and Penecillium expansum showed an MIC > 500 µg/mL. The extract was resistant against heat treatment (100 °C, 15 min) but lost its antifungal activity in presence of cations (Na+, K+, Ca2+ and Mg2+, respectively). Membrane permeabilization of fungal hyphae was evident at 25 µg/mL, while induction of oxidative stress only had minor contribution to the antifungal performance. The extract did not induce haemolysis at all concentrations tested (up to 200 µg/mL). Finally, it was successfully used to protect stored wheat grains from fungal spoilage (determined via ergosterol content) when applied at 100 µg/mL. In conclusion, the defensin Cp-thionin II showed the potential for future application as food bio-preservative.

Expression and production of recombinant scorpine as a potassium channel blocker protein in Escherichia coli.

Scorpine is a cationic protein from the venom of Pandinus imperator, belonging to potassium channel blocker family, which has been shown to have antibacterial, antiviral, and antiplasmodia activities. In the present study, a pET-44a vector containing scorpine synthetic gene with T7 Promoter (pET 44a-His6-Nus-His6-tev-scorpine) was transferred into Escherichia coli Rosetta-gami B (DE3) for soluble expression of the protein in the cytoplasm and its overproduction. After confirming recombinant scorpine peptide expression using SDS-PAGE and Western blot, augmentation of production was performed during two stages. At first, effects of three parameters including carbon source concentration of medium, temperature, and induction time were investigated in terrific broth (TB) medium. Afterward, the overexpression was performed by response surface methodology in TB + glucose. Under the optimized conditions, the highest production of 3.5 g/L in the TB + glucose medium (7.5 g/L glucose, induction at OD600 = 3.5 and 25 °C) was increased to 4.1 g/L in TB medium (2.5 g/L glycerol, induction at OD600 = 0.7 and 25 °C). Then, in order to increase the amount of protein production, effects of carbon concentration in the fermenter under the primary optimized condition was investigated. The amount of produced recombinant protein increased from 0.12 to 2.1 g/L.H. The results were similar to previous studies on optimizing and increasing the production of recombinant protein and in particular recombinant scorpine.

Characterization of the mature form of a ß-defensin-like peptide, Hoa-D1, in the lobster Homarus americanus.

We report on the characterization of the native form of an American lobster, Homarus americanus, ß-defensin-like putative antimicrobial peptide, H. americanus defensin 1 (Hoa-D1), sequenced employing top-down and bottom-up peptidomic strategies using a sensitive, chip-based nanoLC-QTOF-MS/MS instrument. The sequence of Hoa-D1 was determined by mass spectrometry; it was found to contain three disulfide bonds and an amidated C-terminus. The sequence was further validated by searching publicly-accessible H. americanus expressed sequence tag (EST) and transcriptome shotgun assembly (TSA) datasets. Hoa-D1, SYVRScSSNGGDcVYRcYGNIINGAcSGSRVccRSGGGYamide (with c representing a cysteine participating in a disulfide bond), was shown to be related to ß-defensin-like peptides previously reported from Panulirus japonicas and Panulirus argus. We found Hoa-D1 in H. americanus hemolymph, hemocytes, the supraoesophageal ganglion (brain), eyestalk ganglia, and pericardial organ extracts, as well as in the plasma of some hemolymph samples. Using discontinuous density gradient separations, we fractionatated hemocytes and localized Hoa-D1 to hemocyte sub-populations. While Hoa-D1 was detected in semigranulocytes and granulocytes using conventional proteomic strategies for analysis, the direct analysis of cell lysates exposed evidence of Hoa-D1 processing, including truncation of the C-terminal tyrosine residue, in the granulocytes, but not semigranulocytes. These measurements demonstrate the insights regarding post-translational modifications and peptide processing that can be revealed through the MS analysis of intact peptides. The identification of Hoa-D1 as a widely-distributed peptide in the lobster suggests the possibility that it may be pleiotropic, with functions in addition to its proposed role as an antimicrobial molecule in the innate immune system.

Expression and activity analysis of ß Gallinacin-3 in Arabidopsis.

ß Gallinacin-3 (ß Gal-3) is an antimicrobial peptide with strong antibacterial activity against Escherichia coli, Staphylococcus aureus and Salmonella typhimurium. In this study, the ß Gal-3 gene was transferred into a plant genome by genetic engineering techniques. These transgenic plants can be used as feed additives to prevent poultry diseases and them might replace the antibiotics used in poultry industry. To ensure the ß Gal-3 expresses effectively in Arabidopsis seeds, the expression was driven by promoter Ppha cloned from the ß-phaseolin storage protein gene. A total of 294 transgenic lines were obtained by Agrobacterium-mediated transformation into Arabidopsis, and five transgenic lines were selected in which the expression levels of ß Gal-3 were more than 0.10% of the total soluble proteins. The transgenic lines with single locus were identified by Southern blotting. The expression of ß Gal-3 and the highest protein accumulation level (about 4.76 mg/g fresh weight with a maximum of 0.27% of total soluble proteins) was measured by Western blotting and ELISA, respectively. After ultrafiltration by centrifugation, the purity of recombinant ß Gal-3 was up to 73%. Taken together, our data showed that expression of ß Gal-3 with antimicrobial activity is possible and effective in Arabidopsis seeds.

Molecular characterization and protein localization of the antimicrobial peptide big defensin from the scallop Argopecten purpuratus after Vibrio splendidus challenge.

Big defensins are antimicrobial peptides (AMPs) that are proposed as important effectors of the immune response in mollusks, chelicerates and chordates. At present, only two members of the big defensin family have been identified in scallop. In the present work, a cDNA sequence encoding a new big defensin homologue was characterized from the scallop Argopecten purpuratus, namely ApBD1. ApBD1 cDNA sequence comprised 585 nucleotides, with an open reading frame of 375 bp and 5'- and 3'-UTRs of 41 and 167 bp, respectively. The deduced protein sequence contains 124 amino acids with a molecular weight of 13.5 kDa, showing characteristic motifs of the big defensin family and presenting 76% identity with the big defensin from the scallop A. irradians. Phylogenetic analysis revealed that ApBD1 is included into the cluster of big defensins from mollusks. Tissue-specific transcript expression analysis by RT-qPCR showed that ApBD1 was present in all tissues tested from non-immune challenged scallops but it was most strongly expressed in the mantle. The transcript levels of ApBD1 were significantly up-regulated in gills at 24 and 48 h post-injection with the heat-attenuated bacteria Vibrio splendidus. Additionally, immunofluorescence analysis using a polyclonal anti-ApBD1 antibody showed that this protein was abundantly located in epithelial linings of gills and mantle; and also in digestive gland showing ApBD1-infiltrating hemocytes from immune challenged scallops. This is the first time that a big defensin is detected and located at the protein level in a mollusk. These results suggest an important role of ApBD1 in the mucosal immune response of A. purpuratus.

Antibacterial activity and phospholipid recognition of the recombinant defensin J1-1 from Capsicum genus.

The gene of the four disulfide-bridged defensin J1-1 from Capsicum was cloned into the expression vector pQE30 containing a 6His-tag as fusion protein. This construct was transfected into Origami strain of Escherichia coli and expressed after induction with isopropyl thiogalactoside (IPTG). The level of expression was 4 mg/L of culture medium, and the His-tagged recombinant defensin (HisXarJ1-1) was expressed exclusively into inclusion bodies. After solubilization, HisXarJ1-1 was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisXarJ1-1 product obtained from the affinity chromatography step showed single main peptide fraction of molecular masses of 7050.6 Da and after treatment with DTT a single fraction of 7, 042.6 Da corresponding to the reduced peptide was observed. An in vitro folding step of the HisXarJ1-1 generated a distinct profile of oxidized forms of the peptide this oxidized peptide was capable of binding phosphatidic acid in vitro. Possible dimer and oligomer of HisXarJ1-1 were visible in gel electrophoresis and immunodetected with anti-His antibodies. Pure recombinant defensin HisXarJ1-1 exhibited antibacterial activity against Pseudomonas aeruginosa.

Hydrolysate from a mixture of legume flours with antifungal activity as an ingredient for prolonging the shelf-life of wheat bread.

Aiming at identifying antifungal compounds from plant matrices to be used as ingredients in the bakery industry, a water/salt-soluble extract (WSE) was produced from a legume enzyme hydrolysate, consisting of a mixture of pea, lentil, and faba bean flours, and assayed towards Penicillium roqueforti DPPMAF1. Agar diffusion assays allowed the selection of the optimal processing conditions for hydrolysis. As shown by hyphal radial growth rate, the inhibition was observed towards several fungi, including Aspergillus parasiticus CBS971.97, Penicillium carneum CBS 112297, Penicillium paneum CBS 101032, Penicillium polonicum 112490. A multi-step purification was carried out to identify the active compounds. The antifungal activity was attributed to native proteins (nsLTP, ubiquitin, lectin alpha-1 chain, wound-induced basic protein, defensin-1, defensin-2) and a mixture of peptides, which were released during hydrolysis. Nine peptides were purified and identified as sequences encrypted in legume vicilins, lectins and chitinases. WSE was used as ingredient for making bread under pilot plant conditions. Chemical, structural and sensory characterization of bread showed the lack of significant changes compared to control. The bread made with the legume hydrolysate had a longer shelf-life than that of the control.

Two novel antimicrobial defensins from rice identified by gene coexpression network analyses.

Defensins form an antimicrobial peptides (AMP) family, and have been widely studied in various plants because of their considerable inhibitory functions. However, their roles in rice (Oryza sativa L.) have not been characterized, even though rice is one of the most important staple crops that is susceptible to damaging infections. Additionally, a previous study identified 598 rice genes encoding cysteine-rich peptides, suggesting there are several uncharacterized AMPs in rice. We performed in silico gene expression and coexpression network analyses of all genes encoding defensin and defensin-like peptides, and determined that OsDEF7 and OsDEF8 are coexpressed with pathogen-responsive genes. Recombinant OsDEF7 and OsDEF8 could form homodimers. They inhibited the growth of the bacteria Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, and Erwinia carotovora subsp. atroseptica with minimum inhibitory concentration (MIC) ranging from 0.6 to 63µg/mL. However, these OsDEFs are weakly active against the phytopathogenic fungi Helminthosporium oryzae and Fusarium oxysporum f.sp. cubense. This study describes a useful method for identifying potential plant AMPs with biological activities.

An anionic defensin from Plutella xylostella with potential activity against Bacillus thuringiensis.

Insect defensins, are cationic peptides that play an important role in immunity against microbial infection. In the present study, an anionic defensin from Plutella xylostella, (designated as PxDef) was first cloned and characterized. Amino acid sequence analysis showed that the mature peptide owned characteristic six-cysteine motifs with predicted isoelectric point of 5.57, indicating an anionic defensin. Quantitative real-time polymerase chain reaction analysis showed that PxDef was significantly induced in epidermis, fat body, midgut and hemocytes after injection of heat-inactivated Bacillus thuringiensis, while such an induction was delayed by the injection of live B. thuringiensis in the 4th instar larvae of P. xylostella. Knocking down the expression of nuclear transcription factor Dorsal in P. xylostella by RNA interference significantly decreased the mRNA level of PxDef, and increased the sensitivity of P. xylostella larvae to the infection by live B. thuringiensis. The purified recombinant mature peptide (PxDef) showed higher activity against Gram-positive bacteria, with the minimum inhibition concentrations of 1.6 and 2.6 µM against B. thuringiensis and Bacillus subtilis, respectively. To our knowledge, this is the first report about an anionic PxDef, which may play an important role in the immune system of P. xylostella against B. thuringiensis.

Comparative Analysis of the Antimicrobial Activities of Plant Defensin-Like and Ultrashort Peptides against Food-Spoiling Bacteria.

UNLABELLED: Antimicrobial peptides offer potential as novel therapeutics to combat food spoilage and poisoning caused by pathogenic and nonpathogenic bacteria. Our previous studies identified the peptide human beta-defensin 3 (HBD3) as a potent antimicrobial agent against a wide range of beer-spoiling bacteria. Thus, HBD3 is an excellent candidate for development as an additive to prevent food and beverage spoilage. To expand the repertoire of peptides with antimicrobial activity against bacteria associated with food spoilage and/or food poisoning, we carried out an in silico discovery pipeline to identify peptides with structure and activity similar to those of HBD3, focusing on peptides of plant origin. Using a standardized assay, we compared the antimicrobial activities of nine defensin-like plant peptides to the activity of HBD3. Only two of the peptides, fabatin-2 and Cp-thionin-2, displayed antimicrobial activity; however, the peptides differed from HBD3 in being sensitive to salt and were thermostable. We also compared the activities of several ultrashort peptides to that of HBD3. One of the peptides, the synthetic tetrapeptide O3TR, displayed biphasic antimicrobial activity but had a narrower host range than HBD3. Finally, to determine if the peptides might act in concert to improve antimicrobial activity, we compared the activities of the peptides in pairwise combinations. The plant defensin-like peptides fabatin-2 and Cp-thionin-2 displayed a synergistic effect with HBD3, while O3TR was antagonistic. Thus, some plant defensin-like peptides are effective antimicrobials and may act in concert with HBD3 to control bacteria associated with food spoilage and food poisoning. IMPORTANCE: Food spoilage and food poisoning caused by bacteria can have major health and economic implications for human society. With the rise in resistance to conventional antibiotics, there is a need to identify new antimicrobials to combat these outbreaks in our food supply. Here we screened plant peptide databases to identify peptides that share structural similarity with the human defensin peptide HBD3, which has known antimicrobial activity against food-spoiling bacteria. We show that two of the plant peptides display antimicrobial activity against bacteria associated with food spoilage. When combined with HBD3, the peptides are highly effective. We also analyzed the activity of an easily made ultrashort synthetic peptide, O3TR. We show that this small peptide also displays antimicrobial activity against food-spoiling bacteria but is not as effective as HBD3 or the plant peptides. The plant peptides identified are good candidates for development as natural additives to prevent food spoilage.

cDNA cloning and molecular characterization of a defensin-like antimicrobial peptide from larvae of Protaetia brevitarsis seulensis (Kolbe).

We identified new defensin-like cDNA (called Psdefensin) by searching data set of high-throughput RNA sequencing (RNA-seq) expression profiling of immunized larva of white-spotted flower chafers, Protaetia brevitarsis seulensis. The length of the analyzed new defensin-like sequences were 240 base pair (bp) and encoded the deduced polypeptide of 79 amino acid residues with signal peptides (amino acids 1-20), pro-peptide region (amino acids 21-36), and mature peptide region (amino acids 37-79). The Psdefensin transcript levels were slightly up-regulated at 4 h post-infection and were highly expressed at 8 h post-infection compared to control larvae injected with sterile water. In addition, the Psdefensin did have antimicrobial activity against both Gram-negative bacteria, E. coli and Gram-positive bacteria, B. subtilis suggesting potentially pharmacologic agent.

Hit 'em where it hurts: The growing and structurally diverse family of peptides that target lipid-II.

Understanding the mode of action of antibiotics is becoming more and more important in the time that microorganisms start to develop resistance. One very well validated target of several classes of antibiotics is the peptidoglycan precursor lipid II. In this review different classes of lipid II targeting antibiotics will be discussed in detail, including the lantibiotics, human invertebrate defensins and the recently discovered teixobactin. By hitting bacteria where it hurts, at the level of lipid II, we expect to be able to develop efficient antibacterial agents in the future. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

An intimate link between antimicrobial peptide sequence diversity and binding to essential components of bacterial membranes.

Antimicrobial peptides and proteins (AMPs) are widespread in the living kingdom. They are key effectors of defense reactions and mediators of competitions between organisms. They are often cationic and amphiphilic, which favors their interactions with the anionic membranes of microorganisms. Several AMP families do not directly alter membrane integrity but rather target conserved components of the bacterial membranes in a process that provides them with potent and specific antimicrobial activities. Thus, lipopolysaccharides (LPS), lipoteichoic acids (LTA) and the peptidoglycan precursor Lipid II are targeted by a broad series of AMPs. Studying the functional diversity of immune effectors tells us about the essential residues involved in AMP mechanism of action. Marine invertebrates have been found to produce a remarkable diversity of AMPs. Molluscan defensins and crustacean anti-LPS factors (ALF) are diverse in terms of amino acid sequence and show contrasted phenotypes in terms of antimicrobial activity. Their activity is directed essentially against Gram-positive or Gram-negative bacteria due to their specific interactions with Lipid II or Lipid A, respectively. Through those interesting examples, we discuss here how sequence diversity generated throughout evolution informs us on residues required for essential molecular interaction at the bacterial membranes and subsequent antibacterial activity. Through the analysis of molecular variants having lost antibacterial activity or shaped novel functions, we also discuss the molecular bases of functional divergence in AMPs. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Anti-inflammatory and anti-endotoxin properties of peptides derived from the carboxy-terminal region of a defensin from the tick Ornithodoros savignyi.

Antimicrobial peptides are small cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti-inflammatory and antioxidant activities. Several antimicrobial peptides are known to inhibit lipopolysaccharide (LPS)-induced inflammation in vitro and to protect animals from sepsis. In this study, the cellular anti-inflammatory and anti-endotoxin activities of Os and Os-C, peptides derived from the carboxy-terminal of a tick defensin, were investigated. Both Os and Os-C were found to bind LPS in vitro, albeit to a lesser extent than polymyxin B and melittin, known endotoxin-binding peptides. Binding to LPS was found to reduce the bactericidal activity of Os and Os-C against Escherichia coli confirming the affinity of both peptides for LPS. At a concentration of 25 µM, the nitric oxide (NO) scavenging activity of Os was higher than glutathione, a known NO scavenger. In contrast, Os-C showed no scavenging activity. Os and Os-C inhibited LPS/IFN-γ induced NO and TNF-α production in RAW 264.7 cells in a concentration-dependent manner, with no cellular toxicity even at a concentration of 100 µM. Although inhibition of NO and TNF-α secretion was more pronounced for melittin and polymyxin B, significant cytotoxicity was observed at concentrations of 1.56 µM and 25 µM for melittin and polymyxin B, respectively. In addition, Os, Os-C and glutathione protected RAW 264.7 cells from oxidative damage at concentrations as low as 25 µM. This study identified that besides previously reported antibacterial activity of Os and Os-C, both peptides have in addition anti-inflammatory and anti-endotoxin properties.

Peanut defensins: Novel allergens isolated from lipophilic peanut extract.

BACKGROUND: Peanut is one of the most hazardous sources of food allergens. Unknown allergens are still hidden in the complex lipophilic matrix. These allergens need to be discovered to allow estimation of the allergenic risk for patients with peanut allergy and to further improve diagnostic measures. OBJECTIVE: We performed detection, isolation, and characterization of novel peanut allergens from lipophilic peanut extract. METHODS: Extraction of roasted peanuts were performed under defined extraction conditions and examined by means of 2-dimensional PAGE. Subsequently, chromatographic methods were adapted to isolate low-molecular-weight components. Proteins were studied by using SDS-PAGE and immunoblotting with sera from patients with peanut allergy. For allergen identification protein sequencing, homology search and mass spectrometry were applied. Functional characterization for allergenicity was performed by using the basophil activation assay and for antimicrobial activity by using inhibition assays of different bacteria and fungi. RESULTS: IgE-reactive proteins of 12, 11, and 10 kDa were first detected after chloroform/methanol extraction in the flow through of hydrophobic interaction chromatography. The proteins were able to activate basophils of patients with peanut allergy. N-terminal sequencing and homology search in the expressed sequence tag database identified the allergens as peanut defensins, which was confirmed by using mass spectrometry. On microbial cell cultures, the peanut defensins showed inhibitory effects on the mold strains of the genera Cladosporium and Alternaria but none on bacteria. CONCLUSIONS: We identified defensins as novel peanut allergens (Ara h 12 and Ara h 13) that react in particular with IgE of patients with severe peanut allergy. Their antimicrobial activity is solely antifungal.