Recombinant Defensin Research Articles

Scots pine defensins inhibit Ips acuminatus α-amylase activity

Background. Pine bark beetle Ips acuminatus (Gyllenhal, 1827) is one of the most harmful pests of pine trees as it affects the phloem of the upper part of the stem and branches, disrupting the flow of nutrients and water to the crown. I. acuminatus feeds by plant tissues rich in starch, so α-amylases must play a pivotal role in the carbohydrate metabolism of these insects. However, in conifer bark beetles, α-amylases remain poorly understood. Materials and Methods. To detect the α-amylase activity in the digestive system of I. acuminatus, we obtained extracts from larvae, pupae, and adults that were collected from naturally infested Scots pine. The α-amylase activity of crude extracts from different stages and parts of the bark beetle’s body was assessed using 1% starch agar plates. The quantitative evaluation of the α-amylase inhibitory activity of recombinant defensins PsDef1, PsDef2, and PsDef5.1 was performed using the Bernfeld method. The docking models of Scots pine defensins and Ips typographus L. α-amylase (AmyIp) complexes were predicted using the ClusPro 2.0 web server. Results and Discussion. As a result, we found the presence of α-amylase activity in the digestive systems of both larvae and adults of I. acuminatus, but not in pupae. All tested defensins, PsDef1, PsDef2, and PsDef5.1, exhibited inhibitory activity against insect α-amylase at micromolar concentrations. The IC50 values for these peptides were 4.9±0.6 μM, 4.6±0.8 μM, and 2.8±0.5 μM, respectively. In the PsDefs-AmyIt complexes, a network of hydrogen bonds, ionic bridges, and nonbonded contacts are formed between the enzyme and its inhibitor, which prevents the substrate from reaching the catalytic site. The PsDef5.1-AmyIt complex has the largest interfacial contact area, 2328 Å2, in comparison with two other defensins, which correlates well with the inhibitory activity of defensins in this study. Conclusion. Thus, we have identified α-amylase activity in I. acuminatus and demonstrated the ability of Scots pine defensins to inhibit it, sugges­ting that they play a role in pine defenses against this pest.

Exploring the potential role of defensins in differential vector competence of body and head lice for Bartonella quintana

BackgroundThe body and head lice of humans are conspecific, but only the body louse functions as a vector to transmit bacterial pathogens such as Bartonella quintana. Both louse subspecies have only two antimicrobial peptides, defensin 1 and defensin 2. Consequently, any differences in the molecular and functional properties of these two louse subspecies may be responsible for the differential vector competence between them.MethodsTo elucidate the molecular basis of vector competence, we compared differences in the structural properties and transcription factor/microRNA binding sites of the two defensins in body and head lice. Antimicrobial activity spectra were also investigated using recombinant louse defensins expressed via baculovirus.ResultsThe full-length amino acid sequences of defensin 1 were identical in both subspecies, whereas the two amino acid residues in defensin 2 were different between the two subspecies. Recombinant louse defensins showed antimicrobial activities only against the representative Gram-positive Staphylococcus aureus but not against either Gram-negative Escherichia coli or the yeast Candida albicans. However, they did show considerable activity against B. quintana, with body louse defensin 2 being significantly less potent than head louse defensin 2. Regulatory sequence analysis revealed that the gene units of both defensin 1 and defensin 2 in body lice possess decreased numbers of transcription factor-binding sites but increased numbers of microRNA binding sites, suggesting relatively lower transcription activities of body louse defensins.ConclusionsThe significantly lower antibacterial activities of defensin 2 along with the reduced probability of defensin expression in body lice likely contribute to the relaxed immune response to B. quintana proliferation and viability, resulting in higher vector competence of body lice compared to head lice.Graphical

Exploring the impact of the recombinant Escherichia coli strain on defensins antimicrobial activity: BL21 versus Origami strain

The growing emergence of microorganisms resistant to antibiotics has prompted the development of alternative antimicrobial therapies. Among them, the antimicrobial peptides produced by innate immunity, which are also known as host defense peptides (HDPs), hold great potential. They have been shown to exert activity against both Gram-positive and Gram-negative bacteria, including those resistant to antibiotics. These HDPs are classified into three categories: defensins, cathelicidins, and histatins. Traditionally, HDPs have been chemically synthesized, but this strategy often limits their application due to the high associated production costs. Alternatively, some HDPs have been recombinantly produced, but little is known about the impact of the bacterial strain in the recombinant product. This work aimed to assess the influence of the Escherichia coli strain used as cell factory to determine the activity and stability of recombinant defensins, which have 3 disulfide bonds. For that, an α-defensin [human α-defensin 5 (HD5)] and a β-defensin [bovine lingual antimicrobial peptide (LAP)] were produced in two recombinant backgrounds. The first one was an E. coli BL21 strain, which has a reducing cytoplasm, whereas the second was an E. coli Origami B, that is a strain with a more oxidizing cytoplasm. The results showed that both HD5 and LAP, fused to Green Fluorescent Protein (GFP), were successfully produced in both BL21 and Origami B strains. However, differences were observed in the HDP production yield and bactericidal activity, especially for the HD5-based protein. The HD5 protein fused to GFP was not only produced at higher yields in the E. coli BL21 strain, but it also showed a higher quality and stability than that produced in the Origami B strain. Hence, this data showed that the strain had a clear impact on both HDPs quantity and quality.

Differential activity on trypanosomatid parasites of a novel recombinant defensin type 1 from the insect Triatoma (Meccus) pallidipennis

Defensins are one of the major families of antimicrobial peptides (AMPs) that are widely distributed in insects. In Triatomines (Hemiptera: Reduviidae) vectors of Trypanosoma cruzi the causative agent of Chagas disease, two large groups of defensin isoforms have been described: type 1 and type 4. The aim of this study was to analyze the trypanocidal activity of a type 1 recombinant defensin (rDef1.3) identified in Triatoma (Meccus) pallidipennis, an endemic specie from México. The trypanocidal activity of this defensin was evaluated in vitro, against the parasites T. cruzi, T. rangeli, and two species of Leishmania (L. mexicana and L. major) both causative agents of cutaneous leishmaniasis. Our data demonstrated that the defensin was active against all the parasites although in different degrees. The defensin altered the morphology, reduced the viability and inhibited the growth of T.cruzi. When tested against T. rangeli (a parasite that infects a variety of mammalian species), stronger morphological effects where observed. Surprisingly the greatest effects were observed against the two Leishmania species, of which L. major was the parasite most affected with 50% of dead cells or with damaged membranes, in addition of a reduction in its proliferative capacity in culture. These results suggest that rDef1.3 has an important antimicrobial effect against trypanosomatids which cause some of the more important neglected tropical diseases transmitted by insect vectors.

Utilization of a recombinant defensin from Maize (Zea mays L.) as a potential antimicrobial peptide

The search for effective and bioactive antimicrobial molecules to encounter the medical need for new antibiotics is an encouraging area of research. Plant defensins are small cationic, cysteine-rich peptides with a stabilized tertiary structure by disulfide-bridges and characterized by a wide range of biological functions. The heterologous expression of Egyptian maize defensin (MzDef) in Escherichia coli and subsequent purification by glutathione affinity chromatography yielded 2 mg/L of recombinant defensin peptide. The glutathione-S-transferase (GST)-tagged MzDef of approximately 30 kDa in size (26 KDa GST + ~ 4 KDa MzDef peptide) was immunodetected with anti-GST antibodies. The GST-tag was successfully cleaved from the MzDef peptide by thrombin, and the removal was validated by the Tris-Tricine gel electrophoresis. The MzDef induced strong growth inhibition of Rhizoctonia solani, Fusarium verticillioides, and Aspergillus niger by 94.23%, 93.34%, and 86.25%, respectively, whereas relatively weak growth inhibitory activity of 35.42% against Fusarium solani was recorded. Moreover, strong antibacterial activities were demonstrated against E. coli and Bacillus cereus and the moderate activities against Salmonella enterica and Staphylococcus aureus at all tested concentrations (0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 µM). Furthermore, the in vitro MTT assay exhibited promising anticancer activity against all tested cell lines (hepatocellular carcinoma, mammary gland breast cancer, and colorectal carcinoma colon cancer) with IC50 values ranging from 14.85 to 29.85 µg/mL. These results suggest that the recombinant peptide MzDef may serve as a potential alternative antimicrobial and anticancer agent to be used in medicinal application.

Multi-functionPlantDefensin,AntimicrobialandHeavyMetal Adsorbent Peptide.

Background: Defensin peptide isolated from plants are often heterogeneous in length, sequence and structure, but they are mostly small, cationic and amphipathic. Plant defensins exhibit broad-spectrum antibacterial and antifungal activities against Gram-positive and Gram-negative bacteria, fungi and etc. Plant defensins also play an important role in innate immunity, such as heavy metal and some abiotic stresses tolerance.Objectives: In this paper, in vitro broad-spectrum activities, antimicrobial and heavy metal absorption, of a recombinant plant defensin were studied.Material and Methods: SDmod gene, a modified plant defensin gene, was cloned in pBISN1-IN (EU886197) plasmid, recombinant protein was produced by transient expression via Agroinfiltration method in common bean. The recombinant protein was tested for antibacterial activity against Gram-negative, Gram-positive bacteria and Fusarium sp. the effects of different treatments on heavy metal zinc absorption by this peptide were tested.Results: We confirmed the antibacterial activities of this peptide against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Bacillus cereus) bacteria, and antifungal activities of this peptide against Fusarium spp. (Fusarium oxysporum and Fusarium solani). High metal absorption coefficient for this peptide was also observed. Results:Out of six actinobacterial isolates, VITVAMB 1 possessed the most efficient RO-16 decolorization property. It decolorized 85.6% of RO-16 (250 mg L-1) within 24hrs. Isolate VITVAMB 1 was identified to be Nocardiopsis sp. Maximum dye decolorization occurred at pH 8, temperature 35oC, 3% salt concentration and a dye concentration of 50 mg L-1.Conclusions: Results suggesting that modified defensin peptide facilitates a broader range of defense activities. dedefensins are an important part of the innate immune system in eukaryotes. These molecules have multidimensional properties that making them promising agents for therapeutic drugs.

Gene isolation and structural characterization of a legume tree defensin with a broad spectrum of antimicrobial activity.

The recombinant EcgDf1 defensin has an antimicrobial effect against both plant and human pathogens. In silico analyses predict that EcgDf1 is prone to form dimers capable of interacting with the membranes of microorganisms. Plant defensins comprise a large family of antimicrobial peptides (AMP) with a wide range of biological functions. They are cysteine-rich molecules, highly sequence diverse but with a conserved and stable structure. In this work, a defensin gene (EcgDf1) was isolated from Erythrina crista-galli, a legume tree native from South America. The predicted peptide presents eight cysteines, with a γ-core motif GXCX3-9C and six cysteines distributed like the typical defensin αβ motif. The mature EcgDf1 coding sequence was heterologously expressed in Escherichia coli strains and purified by affinity chromatography. Possible dimer and oligomers of EcgDf1 were visible in SDS electrophoresis. Moreover, its 3D structure, determined by homology modeling, docking, and molecular dynamics simulations, was found to be compatible with the formation of homodimers between the β3 and β1-loop-α1, leaving the β2-loop-β3 free to interact with lipid membranes. The purified recombinant peptide inhibited the growth of several critical plant and human pathogens, like the opportunistic fungi Candida albicans and Aspergillus niger and the plant pathogens Clavibacter michiganensis ssp. michiganensis, Penicillium expansum, Botrytis cinerea, and Alternaria alternata. EcgDf1 is a promising candidate for the development of antimicrobial products for use in agriculture and medicine.

Prokaryotic expression and purification of bioactive defension 2 from Pinus sylvestris L.

Plant defensins are highly stable cysteine-rich peptides consisting of 45–54 amino acid residues with a characteristic conservative βαββ structure stabilized by 4–5 disulfide bridges. These peptides are key molecules of innate immune system in plants. They inhibit growth of many phytopathogenic fungi, and some of them exhibit antibacterial acti­vity. Defensins also possess other biological functions. The multifunctional properties of the defensin peptides make them attractive candidates for creation of new remedies with antimicrobial properties. Elucidation of nature of the structural and functional relationships in the antimicrobial peptides is an essential step in the development of drugs with acti­vity against pathogens. Previously, we have purified endogenous and recombinant Scots pine defensin 1 ( Ps Def1) demonstrating high activity against fungi and bacteria. Importantly, Ps Def1 is the first defensin from coniferous plants whose NMR structure and pro­perties have been thoroughly investigated, also by the authors of this work. In this study, we presented the expression and affinity purification of recombinant defensin 2 from Pinus sylvestris L. ( Ps Def2), whose sequence has 90 % identity to Ps Def1. We used pET32/BL21-CodonPlus (DE3)-RIL Escherichia coli expression system to produce large quantities of the recombinant Ps Def2 peptide conjugated to thioredoxin (TRX). We found that the highest yield of recombinant protein in its soluble form was obtained at 0.5 mM of isopropyl-β-D-thiogalactoside (IPTG) concentration for 3 h of induction at 25 °С. After isolation of TRX- Ps Def2 on HisPurNi-NTA resin, the fusion protein was subjected to proteolytic cleavage by enterokinase. Ps Def2 was separated from the proteolytic fragments using the ion exchange on the SP-Sepharose Fast Flow column and a step gradient of 0.05–1 M NaCl. The purity of obtained recombinant Ps Def2 was higher than 95 %, as verified by 16.5 % SDS-PAGE. The recombinant peptide Ps Def2 showed activity against phytopathogenic Fusarium sporotrichiela fungus and Phythophtora gonapodyides oomycete at 5 µM concentration. The availability of recombinant Ps Def2 gives an option not only to examine its antimicrobial properties but to study its structure by spectroscopic methods (circular dichroism, NMR) in order to esta­blish relationships between the structure and function of pine defensins. Keywords : defensins, Scots pine, heterologous expression, protein purification, antimicrobial activity

ATF4 Deficiency Promotes Intestinal Inflammation in Mice by Reducing Uptake of Glutamine and Expression of Antimicrobial Peptides

Activating transcription factor 4 (ATF4) regulates genes involved in the inflammatory response, amino acid metabolism, autophagy, and endoplasmic reticulum stress. We investigated whether its activity is altered in patients with inflammatory bowel diseases (IBDs) and mice with enterocolitis. We obtained biopsy samples during endoscopy from inflamed and/or uninflamed regions of the colon from 21 patients with active Crohn's disease (CD), 22 patients with active ulcerative colitis (UC), and 38 control individuals without IBD and of the ileum from 19 patients with active CD and 8 individuals without IBD in China. Mice with disruption of Atf4 specifically in intestinal epithelial cells (Atf4ΔIEC mice) and Atf4-floxed mice (controls) were given dextran sodium sulfate (DSS) to induce colitis. Some mice were given injections of recombinant defensin α1 (DEFA1) and supplementation of l-alanyl-glutamine or glutamine in drinking water. Human and mouse ileal and colon tissues were analyzed by quantitative real-time polymerase chain reaction, immunoblots, and immunohistochemistry. Serum and intestinal epithelial cell (IEC) amino acids were measured by high-performance liquid chromatography-tandem mass spectrometry. Levels of ATF4 were knocked down in IEC-18 cells with small interfering RNAs. Microbiomes were analyzed in ileal feces from mice by using 16S ribosomal DNA sequencing. Levels of ATF4 were significantly decreased in inflamed intestinal mucosa from patients with active CD or active UC compared with those from uninflamed regions or intestinal mucosa from control individuals. ATF4 was also decreased in colonic epithelia from mice with colitis vs mice without colitis. Atf4ΔIEC mice developed spontaneous enterocolitis and colitis of greater severity than control mice after administration of DSS. Atf4ΔIEC mice had decreased serum levels of glutamine and reduced levels of antimicrobial peptides, such as Defa1, Defa4, Defa5, Camp, and Lyz1, in ileal Paneth cells. Atf4ΔIEC mice had alterations in ileal microbiomes compared with control mice; these changes were reversed by administration of glutamine. Injections of DEFA1 reduced the severity of spontaneous enteritis and DSS-induced colitis in Atf4ΔIEC mice. We found that expression of solute carrier family 1 member 5 (SLC1A5), a glutamine transporter, was directly regulated by ATF4 in cell lines. Overexpression of SLC1A5 in IEC-18 or primary IEC cells increased glutamine uptake and expression of antimicrobial peptides. Knockdown of ATF4 in IEC-18 cells increased expression of inflammatory cytokines, whereas overexpression of SLC1A5 in the knockdown cells reduced cytokine expression. Levels of SLC1A5 were decreased in inflamed intestinal mucosa of patients with CD and UC and correlated with levels of ATF4. Levels of ATF4 are decreased in inflamed intestinal mucosa from patients with active CD or UC. In mice, ATF4 deficiency reduces glutamine uptake by intestinal epithelial cells and expression of antimicrobial peptides by decreasing transcription of Slc1a5. ATF4 might therefore be a target for the treatment of IBD.

Limen, Non-Toxic Recombinant Plant Defensin and Its Effect against Pathogenic Yeast and Fungi

Limen is a small cysteine-rich peptide that was originally isolated from lima beans, and its activity against Micromycetes was described. Recombinant expression of limen in both a prokaryotic and plant system was performed to demonstrate a wide selection of its activities with potential for further use. At first limen was expressed in E. coli cells and after purification steps, its antimicrobial activity was tested on pathogenic fungi. This showed that limen can significantly eliminate the growth of several pathogenic yeasts such as Candida albicans, while its hemolytic activity was not recognizable. To confirm limen’s activity in vivo, transformation of the barley Hordeum vulgare L. was chosen. Transgenic barley was obtained using Agrobacterium-mediated transformation and the production of limen peptide in plant tissues was verified by molecular techniques. Young barley plants were repeatedly exposed to Fusarium oxysporum spores. Increased resistance to the most important pathogen was followed using chlorophyll measurements as indicators of fusariosis. Focusing on the food safety aspect of the use of GM plants, cytotoxicity was followed in extracts from transgenic stressed plants. No or a lower cytotoxic effect was found on human keratinocytes and human embryonic kidney cells than extracts from the non-transformed plants.

Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity.

In order to develop a secure and competent technique to express the human immune gene for fighting infections, we cloned and expressed the BD2/3 using VR1020 (a eukaryotic expression plasmid). BD2/3 contains human β-defensin 2 (BD2) and human BD3. To explore safe and effective DNA delivery molecules in vitro and in vivo, the fusion genes of BD2/3 were used as an immune-labelled gene to verify transfection effectivness of modified chitosan (CS). Plasmid of VR1020-BD2/3 was packed with biomaterials: CS, average molecular weight: 25000D; polyethylene glycol-O-chitosan-polyethylenimine (PEG-O-CS-PEI); liposomes (LP); polyamine cationic liposomes (PCL); polyamine cationic liposomes of protamine (PCL-protamine) by ionotropic gelation. We observed that BD2/3 fusion gene showed high bioactivity in vitro and in vivo. The BD2/3 fusion protein inhibited the proliferation of bacteria (S. aureus, S. pneumoniae, P. aeruginosa and E. coli). The Kunming mice were immune to these nanoparticles and we analyzed their delivery efficiency and gene expression effect. BD2/3 results in multiple changes of innate and required immune system of mice. BD2/3 increases expression of IgG, IgG1, IgG2a, IL-2, IL-6, IFN-γ, as well as of lymphocytes and monocytes. Following challenge with virulent E. coli, CD4+ and CD8+ positive T-cell counts were highly elevated in the BD2/3 immunized mice, resulting in higher survival rates of mice. These results indicate that nanoparticles containing modified CS and BD2/3 are potentially safe and effective drugs in vivo to improve the immunity against bacterial infection and enhance innate immunity and adaptive immunity against infectious diseases.

Use of antimicrobial peptides as a feed additive for juvenile goats

Although antimicrobial peptides (AMPs) have been used as feed additives, only a few studies have examined their use in ruminants. In this study, we evaluated the use of AMPs(recombinant swine defensin and a fly antibacterial peptide were mixed by 1:1) as a medicated feed additive for juvenile goats. Dietary treatments included control groups (group I: 300 g concentrate; group III: 600 g concentrate), and AMP-supplemented groups (group II: 300 g concentrate + 3.0 g AMPs; group IV: 600 g concentrate + 3.0 g AMPs). AMP-treated groups exhibited an increase in bacterial genera, including Fibrobacter, Anaerovibrio, and Succiniclasticum, and the ciliate genus Ophryoscolex; as well a reduction in bacterial genera, such as Selenomonas, Succinivibrio, and Treponema, and the ciliate genera Polyplastron, Entodinium, and Isotricha. The changes in Fibrobacter, Anaerovibrio, Ophryoscolex, Polyplastron, Entodinium, and Isotricha were related to the concentrate. AMP treatment led to increased body weight, average daily weight gain, enzymatic activity (pectinase, xylanase, and lipase), especially in the normal concentrate group, and influence on ruminal fermentation function. In addition, goats treated with AMPs had higher rumen microorganism diversity indices than the control groups. Our results demonstrate that AMPs can be utilized as feed additives for juvenile goats.

The Effects of Human Beta-Defensins on Skin Cells in vitro

Background: Defensins are antimicrobial peptides that exert immunomodulatory and chemotactic functions. Based on these properties and their high expression levels in the skin, they are likely to affect skin inflammation, infection, and wound healing. This may lead to therapeutic applications in (burn) wound healing. Objective: We aimed to investigate the effects of human β-defensins (hBDs) on keratinocytes and fibroblasts, 2 major skin cell types involved in skin regeneration. Methods: Monolayer keratinocyte and fibroblast cultures were exposed to recombinant hBDs, and we overexpressed hBD2 and hBD3 in keratinocytes of reconstructed epidermal equivalents by lentiviral transduction. The effects were measured by immunohistochemistry, quantitative real-time PCR, and migration assays. Kinome analyses were performed on cultured keratinocytes to investigate the signal transduction events elicited by hBD stimulation. Results: We found that hBD3 induced the expression of cytokines and chemokines in keratinocytes, which was not observed in fibroblasts. hBD2, however, stimulated cell migration only in fibroblasts, which was not found for hBD3. Both defensins are likely to exert receptor-mediated effects in keratinocytes, as witnessed by changes in protein kinase activation following stimulation by hBD2 and hBD3. Kinome analysis suggested that protein kinase C activation was a common event for both defensins. We observed, however, considerable differences in keratinocyte responses between stimulation by exogenous recombinant defensins and endogenous defensins expressed following lentiviral transduction. Conclusion: Defensins exert modest biological effects on skin cells that are potentially beneficial in wound healing, but many questions regarding the biological mechanisms of action and relevance for the in vivo situation are still remaining.

Experimental and theoretical studies of cadmium ions absorption by a new reduced recombinant defensin

Heavy metal pollutants such as Cd, Hg, Pb, As, and Se are considered as both a global problem and a growing threat to the humanity. Being strongly poisonous to the metal-sensitive enzymes and leading to the growth inhibition and death of organisms, these metals have a toxic impact on the plants and animals. Inducing the metal-binding cysteine-rich peptides such as metallothioneins, phytochelatins, and defensins, higher organisms like plants and animals usually react to the heavy metal stress. In this study, a recombinant defensin protein was expressed in bean and its ability in the cadmium absorption was determined. Experimental studies revealed that this protein was able to absorb cadmium ions in reduced form more than oxide one. Molecular dynamics simulations were carried out in order to evaluation of experimental studies, using a model of Cd2+ or Na+ and Cl– ions enclosed in a fully hydrated simulation box with the recombinant defensin. The theoretical results also suggested that the reduced recombinant defensin was more powerful in the absorption of Cd2+ than its oxide form. The present study is the first report of Cd2+ absorption potential of this new reduced recombinant defensin. The results unraveled that this recombinant defensin can be adopted as a molecular switch in the cadmium pollution of the environment and also the important role of sulfur groups in the absorption of cadmium ions.

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.

Expression of the ZmDEF1 gene and α-amylase inhibitory activityof recombinant defensin against maize weevils

Plant defensins are multifunctional small cysteine-rich proteins. They are active against fungi, bacteria, and many viruses, and they inhibit trypsin and α-amylase activities. In this study, we expressed the maize defensin gene (ZmDEF1) in tobacco seeds in order to establish the basis for generating transgenic maize plants resistant to weevils. The ZmDEF1 gene was isolated from Maison, a Vietnamese local maize cultivar, which is well known for having the highest resistance to weevils among other local cultivars. The ZmDEF1 gene was cloned into a binary vector, pBetaPhaso-dest, which carries phaseolin, a seed-specific promoter, to direct defensin expression in tobacco seeds. We obtained 13 transgenic tobacco lines from Agrobacterium-mediated transformation and regeneration, called the T0 generation. T0's seeds (called the T1 generation) were collected and analyzed for ZmDEF1 gene expression. Reverse-transcription polymerase chain reaction (RT-PCR) results showed that 4 out of 13 lines (T1-1, T1-3, T1-10, and T1-17) expressed the ZmDEF1 gene at the transcriptional level. These lines were further analyzed by real-time RT-PCR until their transcript expression could be identified. The results showed that the line T1-17 expressed it at the highest expression level. The western blot method also showed that ZmDEF1 was expressed in all four lines above. These transgenic seeds can inhibit maize weevils' α-amylase activity. Extracted protein from transgenic lines reduced weevil α-amylase activity by 67.9%-71.4% in comparison with protein extracted from nontransgenic plants. These results will hopefully provide a useful background to create transgenic maize plants with high resistance to weevils.

Phylogenetic characterization and antifungal activity of recombinant defensin protein from Triticum aestivum

Defensins protein plays an important role in innate immune defense against infectious diseases in animals and plants. In our study and for the first time, common wheat (Triticum aestivum) defensin gene was fully characterized. The protein encodes from a signal peptide region of 25 amino acids. Homology searches showed that T. aestivum defensin have a highest identity (72-64 %) with other defensin selected sequences. A multiple sequence alignment indicates very well highly conserved regions include eight cystiene residues, α-helix, loop, and β-sheet. A phylogenetic analysis of the T. aestivum defensin gene sequence among other plant defensin sequences further confirmed that the T. aestivum sequence is very closely related to Triticum durum defensin sequences, and thus, is likely to have the same expressed structure and function. Moreover, the recombinant defensin protein was expressed in vitro and it show a strong antifungal activity against pathogenic strain Puccinia striiformis. Our study indicate that recombinant defensin protein may be a powerful tool for common wheat treatment.

The recombinant pea defensin Drr230a is active against impacting soybean and cotton pathogenic fungi from the genera Fusarium, Colletotrichum and Phakopsora.

Plant defensins are antifungal peptides produced by the innate immune system plants developed to circumvent fungal infection. The defensin Drr230a, originally isolated from pea, has been previously shown to be active against various entomopathogenic and phytopathogenic fungi. In the present study, the activity of a yeast-expressed recombinant Drr230a protein (rDrr230a) was tested against impacting soybean and cotton fungi. First, the gene was subcloned into the yeast expression vector pPICZαA and expressed in Pichia pastoris. Resulting rDrr230a exhibited in vitro activity against fungal growth and spore germination of Fusarium tucumaniae, which causes soybean sudden death syndrome, and against Colletotrichum gossypii var. cephalosporioides, which causes cotton ramulosis. The rDrr230a IC50 corresponding to inhibition of fungal growth of F. tucumaniae and C. gossypii var. cephalosporioides was 7.67 and 0.84 µM, respectively, demonstrating moderate activity against F. tucumaniae and high potency against C. gossypii var. cephalosporioides. Additionally, rDrr230a at 25 ng/µl (3.83 µM) resulted in 100 % inhibition of spore germination of both fungi, demonstrating that rDrr230a affects fungal development since spore germination. Moreover, rDrr230a at 3 µg/µl (460.12 µM) inhibited 100 % of in vitro spore germination of the obligatory biotrophic fungus Phakopsora pachyrhizi, which causes Asian soybean rust. Interestingly, rDrr230a substantially decreased the severity of Asian rust, as demonstrated by in planta assay. To our knowledge, this is the first report of a plant defensin active against an obligatory biotrophic phytopathogenic fungus. Results revealed the potential of rDrr230a as a candidate to be used in plant genetic engineering to control relevant cotton and soybean fungal diseases.

Tribolium castaneum defensins are primarily active against Gram-positive bacteria

The red flour beetle Tribolium castaneum is a destructive insect pest of stored food and feed products, and a model organism for development, evolutionary biology and immunity. The insect innate immune system includes antimicrobial peptides (AMPs) with a wide spectrum of targets including viruses, bacteria, fungi and parasites. Defensins are an evolutionarily-conserved class of AMPs and a potential new source of antimicrobial agents. In this context, we report the antimicrobial activity, phylogenetic and structural properties of three T. castaneum defensins (Def1, Def2 and Def3) and their relevance in the immunity of T. castaneum against bacterial pathogens. All three recombinant defensins showed bactericidal activity against Micrococcus luteus and Bacillus thuringiensis serovar tolworthi, but only Def1 and Def2 showed a bacteriostatic effect against Staphylococcus epidermidis. None of the defensins showed activity against the Gram-negative bacteria Escherichia coli and Pseudomonas entomophila or against the yeast Saccharomyces cerevisiae. All three defensins were transcriptionally upregulated following a bacterial challenge, suggesting a key role in the immunity of T. castaneum against bacterial pathogens. Phylogenetic analysis showed that defensins from T. castaneum, mealworms, Udo longhorn beetle and houseflies cluster within a well-defined clade of insect defensins. We conclude that T. castaneum defensins are primarily active against Gram-positive bacteria and that other AMPs may play a more prominent role against Gram-negative species.

Uropathogenic Escherichia coli (UPEC) induced antimicrobial gene expression in the male reproductive tract of rat: evaluation of the potential of Defensin 21 to limit infection.

Escherichia coli (E. coli) is a common pathogen in epididymitis, which represents a prevalent entity in male reproductive tract infections (RTI). Although current treatment regimens using antibiotics are satisfactory, development of antimicrobial resistance by the pathogen represents a challenge in the management of RTI. Hence, identification of antimicrobial peptides as alternatives to antibiotics has gained importance. We demonstrate that in a rat epididymo-orchitis model induced with uropathogenic E. coli (UPEC) strain MTCC 729, the expression of defensins and defensin-like Spag11 genes are induced in the epididymis and testes. The induction of antimicrobial gene expression is paralleled by phosphorylation of the NF-kB subunit p65 and the inhibitor of NFkB (IkB-alpha), decreased levels of histone deacetylase 1 and increased methylation of Histone 3, indicating the role of classical Toll-like receptor mediated signaling and epigenetic regulation. Recombinant Defensin 21, when administered to UPEC-infected rats, substantially reduced the bacterial load in the epididymis and testis and proved to be more effective than gentamycin. The ability of Defensin 21 to limit RTI provides support that antibacterial proteins of the male reproductive tract may be used as potential alternatives to antibiotics in treatment of this disease.