Coexpression of a defensin gene and a thionin-like gene via different signal transduction pathways in pepper and Colletotrichum gloeosporioides interactions

The anthracnose fungus, Colletotrichum gloeosporioides, interacts incompatibly with the ripe fruit of pepper (Capsicum annuum). It interacts compatibly with the unripe-mature fruit. We isolated a defensin gene, j1-1, and a thionin-like gene, PepThi, expressed in the incompatible interaction by using an mRNA differential display method. Both genes were developmentally regulated during fruit ripening, organ-specifically regulated, and differentially induced during the compatible and incompatible interactions. Expression of the PepThi gene was rapidly induced in the incompatible-ripe fruit upon fungal infection. The fungus-inducible PepThi gene is highly inducible only in the unripe fruit by salicylic acid. In both ripe and unripe fruit, it was induced by wounding, but not by jasmonic acid. Expression of the j1-1 gene is enhanced by jasmonic acid in the unripe fruit but suppressed in the ripe fruit. These results suggest that both small and cysteine-rich protein genes are induced via different signal transduction pathways during fruit ripening to protect the reproductive organs against biotic and abiotic stresses.     

Synthesis and structure-activity relationship of beta-defensins, multi-functional peptides of the immune system.

beta-defensins are a large family of multiple disulfide-bonded peptides occurring in mammals and birds. They play an important role in the innate immune system, directly killing microbial organisms. Recent research has demonstrated that beta-defensins are important for other biological functions beyond antimicrobial effects, including inhibition of viral infection, interaction with Toll-like receptors, chemotactic effects, and sperm function. The corresponding broad spectrum of activities makes this peptide class an important subject and tool in immunologic research. In this review, we summarize the current status of the routes to obtain synthetic beta-defensins, their major structural properties and structure-activity relationship.     

Structure and dynamics of the neutrophil defensins NP-2, NP-5, and HNP-1: NMR studies of amide hydrogen exchange kinetics

The exchange kinetics for the slowly exchanging amide hydrogens in three defensins, rabbit NP-2, rabbit NP-5, and human HNP-1, have been measured over a range of pH at 25°C using 1D and 2D NMR methods. These NHs have exchange rates 10² to 10⁵ times slower than rates from unstructured model peptides. The observed distribution of exchange rates under these conditions can be rationalized by intramolecular hydrogen bonding of the individual NHs, solvent accessibility of the NHs, and local fluctuations in structure. The temperature dependencies of NH chemical shifts (NH temperature coefficients) were measured for the defensins and these values are consistent with the defensin structure. A comparison is made between NH exchange kinetics, NH solvent accessibility, and NH temperature coefficients of the defensins and other globular proteins. Titration of the histidine side chain in NP-2 was examined and the results are mapped to the three-dimensional structure. © 1994 Wiley-Liss, Inc.     

Interactions between human defensins and lipid bilayers: evidence for formation of multimeric pores.

Defensins comprise a family of broad-spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP-2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP-2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP-2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions. Because vesicle aggregation and fusion can confound studies of the interaction of HNP-2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP-2, and the POPG:POPC ratio. Vesicles (300 microM POPG) readily aggregated at HNP-2 concentrations above 1 microM, but no mixing of vesicle contents could be detected for concentrations as high as 2 microM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi-fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 microM POPG vesicles, 50% of entrapped solute is released by 0.7 microM HNP-2. We introduce a simple method for determining whether leakage from vesicles is graded or all-or-none. We show by means of this fluorescence "requenching" method that native HNP-2 induces vesicle leakage in an all-or-none manner, whereas reduced HNP-2 induces partial, or graded, leakage of vesicle contents. At HNP-2 concentrations that release 100% of small (approximately 400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900-Da dextran is mostly retained. These results suggest that HNP-2 can form pores that have a maximum diameter of approximately 25 A. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.     

Effect of the rearing diet on gene expression of antimicrobial peptides in Hermetia illucens (Diptera: Stratiomyidae)

Insect proteins have been proposed for human and animal food production. Safeguarding the health status of insects in mass rearing allows to obtain high-quality products and to avoid severe economic losses due to entomopathogens. Therefore, new strategies for preserving insect health must be implemented. Modulation of the insect immune system through the diet is one such strategy. We evaluated gene expression of two antimicrobial peptides (one defensin and one cecropin) in Hermetia illucens (L.) (Diptera: Stratiomyidae) reared on different diets. Analyses were performed on prepupae and 10-day-old larvae reared on cereal- and municipal organic waste-based diets and on only prepupae reared on a cereal-based diet supplemented with sunflower, corn, or soybean oil. The inclusion of sunflower oil at different points in the cereal-based diet was also evaluated. Moreover, diet-driven differences in the inhibitory activity of the hemolymph were tested against Escherichia coli DH5α and Micrococcus yunnanensis HI55 using diffusion assays in solid media. Results showed that a municipal organic waste-based diet produced a significant overexpression of antimicrobial peptides only in prepupae. Inclusion of vegetable oils caused an upregulation of at least one peptide, except for the corn oil. Higher expression of both genes was observed when sunflower oil was added 5 days before pupation. All hemolymph samples showed an inhibitory activity against bacteria colonies. Our results suggest that municipal organic waste-based diet and vegetable oil-added diet may successfully impact the immune system of H. illucens. Such alternatives may also exist for other species of economic interest.     

猪防御素基因PBD-I在大肠杆菌中的重组和融合表达

用RT-PCR方法获得PBD-I基因,将该基因插入原核表达载体PinPoint^TMXa-3中,重组质粒转化大肠杆菌JM109,经IPTG诱导后以融合蛋白形式表达。SDS-PAGE电泳显示PBD-I基因在目标位置17kDa处获得了表达。PBD-I基因的成功表达为进一步研究其抗菌活性、抗菌机理及应用研究形成基础。     

Unique Properties of Human β-Defensin 6 (hBD6) and Glycosaminoglycan Complex: SANDWICH-LIKE DIMERIZATION AND COMPETITION WITH THE CHEMOKINE RECEPTOR 2 (CCR2) BINDING SITE*

Defensins are components of the innate immune system that promote the directional migration and activation of dendritic cells, thereby modulating the adaptive immune response. Because matrix glycosaminoglycan (GAG) is known to be important for these functions, we characterized the structural features of human β-defensin 6 (hBD6) and GAG interaction using a combination of structural and in silico analyses. Our results showed that GAG model compounds, a pentasaccharide (fondaparinux, FX) and an octasaccharide heparin derivative (dp8) bind to the α-helix and in the loops between the β2 and β3 strands, inducing the formation of a ternary complex with a 2:1 hBD6:FX stoichiometry. Competition experiments indicated an overlap of GAG and chemokine receptor CCR2 binding sites. An NMR-derived model of the ternary complex revealed that FX interacts with hBD6 along the dimerization interface, primarily contacting the α-helices and β2-β3 loops from each monomer. We further demonstrated that high-pressure NMR spectroscopy could capture an intermediate stage of hBD6-FX interaction, exhibiting features of a cooperative binding mechanism. Collectively, these data suggest a “sandwich-like” model in which two hBD6 molecules bind a single FX chain and provide novel structural insights into how defensin orchestrates leukocyte recruitment through GAG binding and G protein-coupled receptor activation. Despite the similarity to chemokines and hBD2, our data indicate different properties for the hBD6-GAG complex. This work adds significant information to the currently limited data available for the molecular structures and dynamics of defensin carbohydrate binding.     

Locus-specific protocol for nine different innate immune genes (antimicrobial peptides: β-defensins) across passerine bird species reveals within-species coding variation and a case of trans-species polymorphisms

We present a locus-specific protocol suitable for the investigation, from extracted DNA, into natural inter- and intra-specific genetic variation in a group of nine innate immune genes, all belonging to the β-defensin gene family. The products of these genes encode peptides with antimicrobial properties and form part of the innate immune system. The protocol amplifies the exon coding for the peptide that interacts with invading pathogens and is applicable across a wide range of passerine bird species, although with varying success depending on species. In several species tested, we found individuals to be heterozygous at several of the genes, highlighting the existence of coding genetic variation in this part of the immune system. Furthermore, for several of the genes, alleles have been conserved at the amino acid level across taxonomically distant bird species. In one case, we observed the existence of trans-species polymorphisms, often taken as evidence of balancing selection. The method will make it possible to investigate a part of the immune system for which there exists very little information of the genetic variation in wild vertebrate populations, thus making it possible to start investigating the selective forces under which the genes are evolving and the extent to which the found genetic variation is associated with pathogen susceptibility in wild populations.     

Effects of intravesical liposome-mediated human beta-defensin-2 gene transfection in a mouse urinary tract infection model

The purpose of this study was to assess human β-defensin-2 (hBD-2) gene transfection in human bladder epithelial cells and its therapeutic efficacy in a rat urinary tract infection (UTI) model via liposome mediated gene transfer. A large amount of hBD2 production (36.5 ± 3.2 ng/10(6) cells) was demonstrated in transfected cells' supernatants. In addition, a detectable amount of hBD-2 was identified in rats' urine (4.77 ± 1.4 ng/mL) by ELISA. Expression of the transgene hBD-2 in transfected cells and rats' bladders was also confirmed by RT-PCR and Western blotting. Immunohistochemistry revealed that the transgene hBD-2 expressed in the entire epithelial layer of the transduced bladders. Numbers of bacterial colony-forming units in urine and bladders from hBD2 gene treated UTI rats were significantly lower than those from the UTI rats administered PBS at 24, 36, and 72 hr after infection (P < 0.05). In addition, in vivo expression of hBD-2 reduced mucosal damage, interstitial edema and inflammatory cell infiltration in UTI animals. The results indicate that successful inhibition of UTI progression can be produced by hBD2 gene therapy. The liposome-mediated hBD2 plasmid DNA transfection system appears to be a promising method for antimicrobial gene therapy of UTI.     

Structure-dependent functional properties of human defensin 5

The mucosal epithelium secretes a variety of antimicrobial peptides that act as part of the innate immune system to protect against invading microbes. Here, we describe the functional properties of human defensin (HD) 5, the major antimicrobial peptide produced by Paneth cells in the ileum, in relation to its structure. The antimicrobial activity of HD-5 against Escherichia coli proved to be independent of its structure, whereas the unstructured peptide showed greatly reduced antimicrobial activity against Staphylococcus aureus. We find that HD-5 binds to the cell membrane of intestinal epithelial cells and induced secretion of the chemokine interleukin (IL)-8 in a concentration- and structure-dependent fashion. Incubation of HD-5 in the presence of tumor necrosis factor alpha further increased IL-8 secretion synergistically, suggesting that HD-5 may act as a regulator of the intestinal inflammatory response.