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Last updated: 13th August 2010

Resonance Assignment and Three-Dimensional Structure Determination of a Human Alpha-Defensin, HNP-1, by Solid-State NMR.
Zhang Y, Doherty T, Li J, Lu W, Barinka C, Lubkowski J, Hong M
Department of Chemistry, Iowa State University, Ames, IA, 50011, USA.
J Mol Biol. 2010 Mar 26;397(2):408-22.
Human alpha-defensins (HNPs) are immune defense mini-proteins that act by disrupting microbial cell membranes. Elucidating the three-dimensional structures of HNPs in lipid membranes is important for understanding their mechanisms of action. Using solid-state NMR, we have determined the three-dimensional structure of HNP-1 in a microcrystalline state outside the lipid membrane, which provides benchmarks for structure determination and comparison with the membrane-bound state. From a suite of 2D and 3D magic-angle spinning experiments, (13)C and (15)N chemical shifts were obtained that yielded torsion angle constraints while inter-residue distances were obtained to restrain the three-dimensional fold. Together, these constraints led to the first high-resolution SSNMR structure of a human defensin. The SSNMR structure has close similarity to the crystal structures of the HNP family, with the exception of the loop region between the first and second beta-strands. The difference, which is partially validated by direct torsion angle measurements of selected loop residues, suggests possible conformational variation and flexibility of this segment of the protein, which may regulate HNP interaction with the phospholipid membrane of microbial cells.
Expression and Purification of Recombinant alpha-Defensins and alpha-Defensin Precursors in Escherichia coli.
Figueredo S, Mastroianni JR, Tai KP, Ouellette AJ
Department of Pathology and Laboratory Medicine, School of Medicine, College of Health Sciences, University of California, Irvine, CA, USA.
Methods Mol. Biol. 2010 ;618 47-60
Recombinant expression of alpha-defensins can be obtained at efficient levels in Escherichia coli. Amplified alpha-defensin or pro-alpha-defensin coding cDNA sequences are cloned directionally between EcoRI and SalI sites of the pET-28a expression vector and expressed in E. coli BL21 RIS cells. Cells growing exponentially in nutrient-rich liquid medium are induced to express the recombinant protein by addition of 50 mM isopropyl beta-D: -1-thiogalactopyranoside for 3-6 h. After bacterial cells collected by centrifugation are lysed in 6 M guanidine-HCl under non-reducing conditions, the expressed defensin fused to its 6xHis-34 amino acid N-terminal fusion partner is purified by affinity chromatography on nickel-NTA columns. A Met codon introduced at the N terminus of expressed Met-free peptides provides a unique CNBr cleavage site, enabling release of the alpha-defensin free of ancillary residues by sequential C18 RP-HPLC. Molecular masses of C18 RP-HPLC purified peptides are confirmed by MALDI-TOF mass spectrometry, and peptide homogeneity is assessed using analytical RP-HPLC and acid-urea polyacrylamide gel electrophoresis. alpha-Defensins prepared in this manner are biochemically equivalent to the natural molecules.
Purification of antimicrobial peptides from human skin.
Schröder JM
Clinical Research Unit "Cutaneous Inflammation", Department of Dermatology, University Hospital Schleswig-Holstein, Kiel, Germany,
Methods Mol. Biol. 2010 ;618 15-30
Human skin is a rich source of human antimicrobial peptides. Its cellular source is the keratinocyte, which terminally differentiates in the uppermost parts of the skin, eventually forming the stratum corneum, the horny layer. The easy availability of human stratum corneum makes it possible to identify and characterize human antimicrobial peptides with a biochemical approach. Moreover, the availability of lesional scales of patients with psoriasis, an inflammatory skin disease, allows the identification of human-inducible peptide antibiotics, which are absent in healthy skin. With this strategy, the beta-defensins hBD-2 and hBD-3, RNase-7 as well as psoriasin/S100A7 have been discovered as human antimicrobial peptides and proteins.A detailed description of the strategies and methods is presented, which allowed a successful identification and characterization of human antimicrobial peptides. We used various HPLC techniques, combined with antimicrobial testing as read-out system. In parallel, SDS-PAGE analyses as well as electrospray ionization mass spectrometry were used for further biochemical characterization as well as purity assessment.
Neutrophil {alpha}-Defensins Cause Lung Injury by Disrupting the Capillary-Epithelial Barrier.
Bdeir K, Higazi AA, Kulikovkaya I, Christofidou-Solomidou M, Vinogradov SA, Allen TC, Idell S, Linzmeier R, Ganz T, Cines DB
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA l9l04, USA.
Am J Respir Crit Care Med. 2010 May 1;181(9):935-46
RATIONALE: The involvement of neutrophil activation in the sentinel, potentially reversible, events in the pathogenesis of acute lung injury is only partially understood. alpha-defensins are the most abundant proteins secreted by activated human neutrophils, but their contribution to acute lung injury in mouse models is hindered by their absence from murine neutrophils and the inability to study their effects in isolation in other species. OBJECTIVES: To study the role of alpha-defensins in the pathogenesis of acute lung injury in a clinically relevant setting using mice transgenic for PMN expression of alpha-defensins. METHODS: Transgenic mice expressing PMN alpha-defensins were generated. ALI was induced by acid aspiration. Pulmonary vascular permeability was studied in vivo using labeled dextran and fibrin deposition. The role of the low-density lipoprotein-related receptor in permeability was examined. MEASUREMENTS AND MAIN RESULTS: Acid aspiration-induced neutrophil migration and release of alpha-defensins into lung parenchyma and airways. Acute lung injury was more severe in alpha-defensin-expressing mice than wild-type mice by inspection, influx of neutrophils into the interstitial space and airways, histological evidence of epithelial injury, interstitial edema, extravascular fibrin deposition, impaired oxygenation and reduced survival. Within four hours of insult, alpha-defensin-expressing mice showed greater disruption of capillary-epithelial barrier function and acute lung injury that was attenuated by systemic or intratracheal administration of specific inhibitors of the low-density lipoprotein-related receptor. CONCLUSIONS: alpha-defensins mediate acute lung injury through low-density lipoprotein-related receptor-mediated loss of capillary-epithelial barrier function, suggesting a potential new approach to intervention.
Expression and purification of moricin CM4 and human beta-defensins 4 in Escherichia coli using a new technology.
Shen Y, Ai HX, Song R, Liang ZN, Li JF, Zhang SQ
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
Microbiological research 2010 Jan
Different strategies have been developed to produce small antimicrobial peptides using recombinant techniques. Here we report a new technology of biosynthesis of moricin CM4 and human beta-defensins 4 (HbetaD4) in the Escherichia coli. The CM4 and HbetaD4 gene were cloned into a vector containing the tags elastin-like peptide (ELP) and intein to construct the expression vector pET-EI-CM4 and pET-EI-HbetaD4. All the peptides, expressed as soluble fusions, were isolated from the protein debris by the method called inverse transition cycling (ITC) rather than traditional immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by self-cleavage. Fully reduced peptides that were purified exhibited expected antimicrobial activity. The approach described here is a low-cost, convenient and potential way for generating small antimicrobial peptide.
Lactobacillus delbrueckii subsp lactis (strain CIDCA 133) resists the antimicrobial activity triggered by molecules derived from enterocyte-like Caco-2 cells.
Hugo AA, De Antoni GL, Pérez PF
Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA-CCT CONICET), UNLP, La Plata, Argentina.
Lett Appl Microbiol. 2010 Apr;50(4):335-40
AIMS: The aim of the present study was to assess the ability of a potentially probiotic strain to resist, in vitro, the effect of intestinal antimicrobial molecules. METHODS AND RESULTS: Strain CIDCA 133 of Lactobacillus delbrueckii subsp lactis was studied. Lactobacillus delbrueckii subsp bulgaricus as well as other gram-positive and gram-negative bacteria were used for comparison purposes. The effect of different antimicrobial extracts was determined by diffusion assays, viable counts and growth kinetics. Human-defensins (h beta D1 and h beta D2) were also included in the study. Two types of cellular fractions from Caco-2 cells were tested: (i) cytosolic fractions, obtained by sonication of cultured human enterocytes and (ii) cationic fraction, obtained by batch extraction of the cytosolic fraction with a weak cation exchange resin. In addition, the effect of Caco-2-secreted factors was studied. Strain CIDCA 133 was neither inhibited by Caco-2 secreted, cytosolic nor cationic fractions. Of note, human-defensins were inactive against strain CIDCA 133. In contrast, a related lactobacilli: Lactobacilli delbrueckii subsp bulgaricus (strain CIDCA 331) and other species of gram-positive or gram-negative bacteria were strongly inhibited. CONCLUSIONS: Strain CIDCA 133 is able to survive and grow in the presence of enterocyte-derived antimicrobial molecules. This ability is not a general property of lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: Results could provide a new insight into the mechanisms of the probiotic effect and encourage further studies on this field. Resistance to antimicrobial peptides can be relevant to understand the interaction of potentially probiotic strains with the host's immune system. This ability can be also relevant as a selection criterion for new probiotic strains.
EST-Database Search of Plant Defensins - An Example using Sugarcane, a Large and Complex Genome.
Belarmino C, Capriles PV, Crovella S, Dardene LE, Benko-Iseppon AM
Federal University of Pernambuco, Genetics Dept., Recife, Brazil.
Curr Protein Pept Sci. 2010 May 1;11(3):248-54.
EST (Expressed Sequence Tags) databases are increasing in number and size, especially regarding cultivated plants. Sugarcane is one of the most important tropical and subtropical crops, presenting a complex polyploid genome of hybrid origin, bearing a challenge for the understanding of genetic processes in higher plants. In the present work a general search was carried out on the largest Sugarcane EST Database (SUCEST) that includes 237,954 ESTs aiming to identify defensin antimicrobial peptides - a class of small, basic, cysteine-rich peptides distributed throughout the kingdoms. Using a computational approach 17 new defensin isoforms could be identified. Main steps for the search, characterization and evaluation of the defensin expression profile are presented. Prevalent expression tissues were leaf roll, lateral bark, root apex, base of inflorescence, developing seed, and calli. Bioinformatics and phylogenetic analysis of the primary structure of the sugarcane defensin candidates as well as the 3D structures obtained by comparative modeling support their role as antimicrobials.
Techniques for Plant Defensin Production.
Padovan L, Crovella S, Tossi A, Segat L
Department of Life Sciences, University of Trieste - Via Giorgieri, 1 - 34127 Trieste, Italy.
Curr Protein Pept Sci. 2010 May 1;11(3):231-5.
To defend themselves from attack by pathogens, plants can rely only on their innate defense systems. Defensins are antimicrobial peptides that contribute to plant immunity by displaying a direct cidal activity against various pathogens, some of which are responsible for plant diseases. These determine a significant decrease in the quality and safety of agricultural products, especially among food crops, and cause significant economic losses. There is consequently an increasing interest for antimicrobial compounds such as the defensins, which might be applied in different ways to protect important food or bio-fuel crops. In this review we analyse the techniques that have been reported in the literature for the production of isolated plant defensins of adequate quality and sufficient quantity for potential use in plant protection. For research purposes, defensins have been heterogously expressed in diverse hosts, such as bacteria, yeasts, fungi and plants. Chemical synthesis is instead not commonly used for their production, due to structural characteristics that makes it difficult to obtain the correct protein folding. To consider the possibility of producing plant defensins in a large scale, cost-effective methods guaranteeing high quality product are required. Future studies may thus focus on the development of more stable compounds, as well as decreasing production costs by improving preparative syntheses or biotechnological procedures such as using transgenic crops as plant factories.
Bioinformatics-Coupled Molecular Approaches for Unravelling Potential Antimicrobial Peptides Coding Genes in Brazilian Native and Crop Plant Species.
Pestana-Calsa MC, Ribeiro IL, Calsa T
Laboratory of Molecular Genetics, Department of Genetics, Center of Biological Sciences, Federal University of Pernambuco - Av. Prof. Moraes Rego 1235, Cidade Universitária, 50670-901, Recife, PE, Brazil.
Curr Protein Pept Sci. 2010 May 1;11(3):199-209.
As eukaryotes, plants include in innate defense antimicrobial peptides (AMP), usually small cysteine or glycine-rich peptides effective against a wide range of pathogens. The main classes of AMPs are represented by alpha/beta-defensins, lipid-transfer proteins, thionins, cyclotides, snakins and hevein-like, according to amino acid sequence homology. In spite of increasing number of described AMPs from plants, last decade advances in methodologies for gene expression and the huge amounts of genomic, proteomic and other "-omics" data lead to new prospection strategies of novel potential candidates. Organised user-friendly databases are available to be searched and enlarged with newly discovered plant-derived AMPs. Bioinformatics has allowed the application of in silico-associated molecular tools aiming to screen and identify genes coding for these peptides, starting from genome, transcriptomes, proteome or metabolome from various cultivated or wild plants. As expected, crop plants have been the main target for AMP research and application, also because the higher availability of molecular data. However, wild plant species biodiversity and results for AMP search have increased the importance of characterization in native plants. Enormous plant diversity in Brazilian ecosystems summed to croplands provides potential targets to identify novel candidates for plant AMP. Despite these opportunities, bioinformatics tools are restricted to species whose "-omics" are available, otherwise only heterology-based analyses are feasible, as it has been the case of most Brazilian plant AMP prospection research groups. Still rare, but promising results indicate that this research field on Brazilian crop/native species presents a growing trend of application in agriculture, medicine and industry.
Haemophilus ducreyi SapA Contributes to Cathelicidin Resistance and Virulence in Humans.
Mount KL, Townsend CA, Rinker SD, Gu X, Fortney KR, Zwickl BW, Janowicz DM, Spinola SM, Katz BP, Bauer ME
Departments of Microbiology and Immunology, Medicine, and Pathology and Laboratory Medicine, and Center for Immunobiology, Indiana University School of Medicine, 635 Barnhill Drive, Room MS 420, Indianapolis, IN, 46202-5124.
Infect Immun. 2010 Mar;78(3):1176-84
Haemophilus ducreyi is an extracellular pathogen of human epithelial surfaces that resists human antimicrobial peptides. The organism´s genome contains homologs of the sensitive to antimicrobial peptides (sap) operon of nontypeable H. influenzae. In this study, we characterized the sap-containing loci of H. ducreyi 35000HP and demonstrated that sapA is expressed in broth cultures and H. ducreyi-infected tissue; sapA is also conserved among both class I and class II H. ducreyi strains. We constructed a nonpolar sapA mutant of H. ducreyi 35000HP, designated 35000HPsapA, and compared the percent survival of wild type 35000HP and 35000HPsapA exposed to several human APs, including alpha-defensins, beta-defensins, and the cathelicidin LL-37. Unlike an H. influenzae sapA mutant, 35000HPsapA was not more susceptible than 35000HP to defensins. However, we observed a significant decrease in survival of 35000HPsapA after exposure to LL-37, which was complemented by introducing sapA in trans. Thus, the Sap transporter plays a role in resistance of H. ducreyi to LL-37. We next compared 35000HPsapA with 35000HP for their ability to cause disease in human volunteers. Although both strains caused papules to form at similar rates, the pustule formation rate at sites inoculated with 35000HPsapA was significantly lower than that of sites inoculated with 35000HP (33.3% vs. 66.7%; P = 0.007). Together, these data establish SapA both a virulence factor and one mechanism for H. ducreyi to resist killing by antimicrobial peptides. To our knowledge, this is the first demonstration that an antimicrobial peptide resistance mechanism contributes to bacterial virulence in humans.