鸭β-防御素5基因的分离、鉴定及其生物学作用

为克隆与表达鸭β-防御素5 (AvBD5)基因及测定其生物学特性,采用RT-PCR方法从鸭肺脏组织中扩增到鸭AvBD5,将测得的序列与已发现的禽β-防御素和部分哺乳类动物β-防御素的氨 基酸序列构建进化树进行同源性分析.结果显示鸭AvBD5 cDNA大小为201 bp,编码66个氨基酸残基,内含6个位置保守的半胱...     

Structure and antimicrobial activity of platypus ‘intermediate’ defensin-like peptide

The three-dimensional structure of a chemically synthesized peptide that we have called ‘intermediate’ defensin-like peptide (Int-DLP), from the platypus genome, was determined by nuclear magnetic resonance (NMR) spectroscopy; and its antimicrobial activity was investigated. The overall structural fold of Int-DLP was similar to that of the DLPs and β-defensins, however the presence of a third antiparallel β-strand makes its structure more similar to the β-defensins than the DLPs. Int-DLP displayed potent antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa. The four arginine residues at the N-terminus of Int-DLP did not affect the overall fold, but were important for its antimicrobial potency.     

Three-dimensional NMR Structure of Hen Egg Gallin (Chicken Ovodefensin) Reveals a New Variation of the β-Defensin Fold

Gallin is a 41-residue protein, first identified as a minor component of hen egg white and found to be antimicrobial against Escherichia coli. Gallin may participate in the protection of the embryo during its development in the egg. Its sequence is related to antimicrobial β-defensin peptides.In the present study, gallin was chemically synthesized 1) to further investigate its antimicrobial spectrum and 2) to solve its three-dimensional NMR structure and thus gain insight into structure-function relationships, a prerequisite to understanding its mode(s) of action. Antibacterial assays confirmed that gallin was active against Escherichia coli, but no additional antibacterial activity was observed against the other Gram-positive or Gram-negative bacteria tested. The three-dimensional structure of gallin, which is the first ovodefensin structure to have been solved to date, displays a new five-stranded arrangement. The gallin three-dimensional fold contains the three-stranded antiparallel β-sheet and the disulfide bridge array typical of vertebrate β-defensins. Gallin can therefore be unambiguously classified as a β-defensin. However, an additional short two-stranded β-sheet reveals that gallin and presumably the other ovodefensins form a new structural subfamily of β-defensins. Moreover, gallin and the other ovodefensins calculated by homology modeling exhibit atypical hydrophobic surface properties, compared with the already known vertebrate β-defensins. These specific structural features of gallin might be related to its restricted activity against E. coli and/or to other yet unknown functions. This work provides initial understanding of a critical sequence-structure-function relationship for the ovodefensin family.     

Haplotype structure and copy number polymorphism of the beta‐defensin 7 genes in diverse chicken breeds

Beta‐defensins is a family of avian peptides related to the innate immune system. Copy number variation was recently reported for the avian beta‐defensin 7 gene (AvBD7) between the highly inbred Leghorn and Fayoumi lines. Here, we examined copy number variants in 35 different chicken breeds and found that 31 of them have at least the same representation of the duplicated AvBD7 allele. We also found haplotypes upstream of the AvBD6 regions that are strongly linked to the AvBD7 duplication. We observed a strong linkage disequilibrium spanning of the upstream region of the AvBD6 gene, with two SNPs being flanking markers to detect duplication of the AvBD7.     

鹅β-防御素3基因的分离、鉴定及其表达产物的生物学特性

为了克隆鹅β-防御素(AvBD)3基因,并在原核表达重组鹅AvBD3蛋白,进一步研究鹅AvBD3蛋白的生物学特性,利用RT-PCR方法从鹅脾脏和法氏囊组织中扩增到鹅AvBD3基因片段,其cDNA片段大小为182 bp,编码60个氨基酸残基.经同源性分析发现鹅AvBD3氨基酸序列与鸡AvBD3氨基酸序列同源性最高,为100％.将该基因亚克隆到原核表达载体pGEX-6p-1的BamH Ⅰ和SalⅠ双酶切位点上,构建重组表达质粒pGEX-goose AvBD3.将重组质粒转化大肠杆菌BL21,于37℃用IPTG诱导表达,SDS-PAGE电泳表明,重组鹅AvBD3蛋白在原核高效表达(分子量约31 kDa).该重组蛋白经纯化后测定其体外抗菌活性与理化特性,结果显示,重组鹅AvBD3蛋白具有广谱的抗菌活性,对12种细菌,包括革兰氏阳性菌和革兰氏阴性菌均具有抑菌作用.高盐离子浓度显著降低重组鹅AvBD3蛋白的抗菌活性.此外,该重组蛋白的溶血活性极低,并对酸碱度具有较高的稳定性.     

Antimicrobial activity of human β-defensin 4 analogs: Insights into the role of disulfide linkages in modulating activity

Human β-defensins (HBDs) are cationic antimicrobial peptides that are components of the innate immune system. They are characterized by three disulfide bridges. However, the number of cationic residues as well as the presence of lysine and arginine residues vary. In HBD4, the cationic residues occur predominantly in the N-terminal segment, unlike in HBD1–3. We have examined the antimicrobial activity of peptides spanning the N- and C-terminal segments of HBD4. We have introduced one, two and three disulfide bridges in the peptides corresponding to the N-terminal segments. Peptides corresponding to the N-terminal segment had identical sequences and variation was only in the number and spacing of cysteines and disulfide bridges. Antimicrobial activity to varying extents was observed for all the peptides. When two disulfide bridges were present, decrease in antimicrobial potency as well as sensitivity of activity to salt was observed. Enhanced antimicrobial activity was observed when three disulfide bridges were present. The antimicrobial potency was similar to HBD4 except against Escherichia coli and was attenuated in the presence of salt. While the presence of three disulfide bridges did not constrain the peptide to a rigid β-sheet, the activity was considerably more as compared to the peptides with one or two disulfide bridges. The peptides enter bacterial and fungal cells rapidly without membrane permeabilization and appear to exert their activity inside the cells rather than at the membrane.     

Characterization of β-defensin prepropeptide mRNA from chicken and turkey bone marrow

Four avian β-defensin prepropeptide cDNA sequences [gallinacins: Gal 1 (synonym CHP 1, chicken heterophil peptide 1), and Gal 2; turkey heterophil peptides: THP 1 and THP 2] were amplified from chicken or turkey bone marrow mRNA samples, respectively. Partial chicken β-defensin cDNA sequences were obtained using degenerate primers based on chicken peptide sequences (Gal 1/CHP 1 and Gal 2). The complete cDNA sequences of the chicken β-defensins were then determined by designing specific intrapeptidal primers, from the newly acquired sequence, and pairing one primer with a specific poly A primer tail sequence (3' end) and the other primer with an adapter primer in a 5' rapid amplification of cDNA ends (RACE) reaction. The two, turkey β-defensins were amplified from turkey marrow using primers designed from chicken β-defensin preproregions. The complete amino acid sequences for the prepropeptides were deduced for all four avian β-defensins. Previously, only partial mature peptide sequences for the turkey β-defensins and complete mature peptide sequences for the chicken β-defensins were known. All sequences obtained translated accurately to complete and partial amino acid sequences reported for β-defensins purified from chicken and turkey heterophil granules except for one additional amino acid for Gal 1/CHP 1. The four deduced β-defensin proregions lack the long, negatively charged propiece reported in classical defensin proregions. These regions are thought to stabilize and inactivate the positively charged mature peptide and target the propeptide to the storage granule. Instead, these β-defensin proregions are shorter and similar to storage granule-free β-defensins proregions reported for bovine tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP). These are the first prepropeptide β-defensins from leukocyte granules to be completely characterized.     

Structure-activity relations of parasin I, a histone H2A-derived antimicrobial peptide

The structure-activity relations and mechanism of action of parasin I, a 19-amino acid histone H2A-derived antimicrobial peptide, were investigated. Parasin I formed an amphipathic alpha-helical structure (residues 9-17) flanked by two random coil regions (residues 1-8 and 18-19) in helix-promoting environments. Deletion of the lysine residue at the N-terminal [Pa(2-19)] resulted in loss of antimicrobial activity, but did not affect the alpha-helical content of the peptide. The antimicrobial activity was recovered when the lysine residue was substituted with another basic residue, arginine ([R(1)]Pa), but not with polar, neutral, or acidic residues. Progressive deletions from the C-terminal [Pa(1-17), Pa(1-15)] slightly increased the antimicrobial activity (1-4 microg/ml) without affecting the alpha-helical content of the peptide. However, further deletion [Pa(1-14)] resulted in nearly complete loss of antimicrobial activity and alpha-helical structure. Confocal microscopic analysis and membrane permeabilization assays showed that parasin I and its analogs with comparable antimicrobial activities localized to the cell membrane and subsequently permeabilized the outer and cytoplasmic membranes. Pa(1-14) also localized to the cell membrane, but lost membrane-permeabilizing activity, whereas Pa(2-19) showed poor membrane-binding and -permeabilizing activities. The results indicate that the basic residue at the N-terminal is essential for the membrane-binding activity of parasin I, and among the membrane-binding parasin I analogs, the alpha-helical structure is necessary for the membrane-permeabilizing activity.     

Comparative In-Vitro Functional Analysis of Synthetic Defensins and Their Corresponding Peptide Variants Against HIV-1NL4.3, E. coli, S. aureus and P. aeruginosa

Defensins found in mammals belong to mainly two subfamilies α- and β-defensins. Mammalian defensins are small molecules (18–45 residues) that are cysteine, arginine rich compounds. Antimicrobial activities of these peptides were shown against a wide variety of microbes including bacteria, fungi, viruses and protozoan parasites. To investigate the structure and activity relationship, amino acid substitutions that alter charge were introduced into synthetic defensin peptides by adding 2–2 Arg (RR) and Asp (DD) at both the terminal and tested their effects on HIV-1, E. coli, S. aureus, and P. aeruginosa. In the present study, we have chemically synthesized native defensin peptides and their variants with Arg (RR) and Asp (DD) amino acid residues at N- and C-termini. Later, we assayed their anti-HIV, anti-microbial activities, stability, cytotoxicity and hemolytic properties. We reported that anti-HIV and antimicrobial activities of native defensins is increased significantly by adding Arg (RR) residues at both the termini while the substitution of Arg (RR) with Asp (DD), eliminate anti-HIV and antimicrobial activity against all bacterial species tested. While other physical features i.e. stability, cell toxicity and hemolytic property were not affected by any of the changes in the sequence. The results suggest that the terminal residues in defensins are crucial functional elements that determine their microbicidal potency. The enhanced microbicidal activity observed for defensin peptides with Arg (RR) residues could be due to optimization of amphiphilicity of the structure, which could facilitate specific interactions with the microbial membranes.     

In vitro tests of natural allelic variation of innate immune genes (avian β-defensins) reveal functional differences in microbial inhibition

Allelic variation in immune genes might result from, and contribute to, host-pathogen evolution. Functional allelic variation in the innate immune system has received little attention. Here, we investigate whether naturally occurring allelic variation within the avian innate immune system (β-defensins) is associated with variation in antimicrobial activity. We tested differences in in vitro antimicrobial properties of the synthesized products of two alleles of avian β-defensin 7, both of which occur at high frequency in natural populations of the great tit (Parus major). Only one allele strongly inhibited the growth of the gram-positive bacterium Staphylococcus aureus, but both alleles strongly inhibited growth of the gram-negative bacterium Escherechia coli. Our data demonstrate functional allelic variation in natural defensin genes, and we discuss how differences in efficacy against microbial species might contribute to maintaining this variation.     

鸽β-防御素1基因的克隆及其生物学作用鉴定

【目的】从鸽子组织中克隆鸽子β-防御素1(AvBD1)基因,在大肠杆菌中表达重组鸽子AvBD1蛋白,测定其生物学特性。【方法】应用RT-PCR法从鸽子骨髓组织中扩增鸽子AvBD1基因,采用Real-time PCR法检测该基因在鸽子组织器官中的表达分布。将该基因亚克隆到大肠杆菌原核表达载体pProEX-HTa的EcoR I和Xho I双酶切位点上,构建重组表达质粒pProEX-pigeon AvBD1,将重组质粒进行诱导表达;对该重组蛋白进行纯化,通过菌落计数法测定其体外抗菌活性与理化特性。【结果】从鸽子骨髓组织中克隆到鸽子AvBD1基因,其cDNA大小为198 bp,编码65个氨基酸,经序列相似性分析,鸽子AvBD1与鸭AvBD1氨基酸序列相似性最高(81.5%)。鸽子AvBD1主要分布于免疫系统和消化系统组织中。Tricine-SDS-PAGE电泳结果表明,重组鸽子AvBD1蛋白分子量约8.8 kD,与预期大小一致。该重组蛋白具有广谱抗菌活性,高盐浓度显著降低其抗菌活性。此外,该重组蛋白的溶血活性极低。【结论】从鸽子骨髓组织中克隆到鸽子AvBD1基因,其主要分布在机体的免疫系统和消化系统中。该重组蛋白具有广谱抗菌活性,高盐浓度显著降低其抗菌活性,且该重组蛋白的溶血活性极低。     

Discovery and characterization of Coturnix chinensis avian β‐defensin 10, with broad antibacterial activity

A novel avian β‐defensin (AvBD), AvBD10, was discovered in the liver and bone marrow tissues from Chinese painted quail (Coturnix chinensis) in the present study. The complete nucleotide sequence of quail AvBD10 contains a 207‐bp open reading frame that encodes 68 amino acids. The quail AvBD10 was expressed widely in all the tissues from quails except the tongue, crop, breast muscle, and thymus and was highly expressed in the bone marrow. In contrast to the expression pattern of AvBD10 in tissues from quail, the chicken AvBD10 was expressed in all 21 tissues from the layer hens investigated, with a high level of expression in the kidney, lung, liver, bone marrow, and Harderian glands. Recombinant glutathione S‐transferase (GST)‐tagged AvBD10s of both quail and chicken were produced and purified by expression of the two cDNAs in Escherichia coli, respectively. In addition, peptide according to the respective AvBD10s sequence was synthesized, named synthetic AvBD10s. As expected, both recombinant GST‐tagged AvBD10s and synthetic AvBD10s of quail and chicken exhibited similar bactericidal properties against most bacteria, including Gram‐positive and Gram‐negative forms. However, no significant bactericidal activity was found for quail recombinant GST‐tagged AvBD10 against Salmonella choleraesuis or for chicken recombinant GST‐tagged AvBD10 against Proteus mirabilis. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Evolution of primate α and θ defensins revealed by analysis of genomes

Defensins are endogenous peptides with cysteine-rich antimicrobial ability that contribute to host defence against bacterial, fungal and viral infections. There are three subfamilies of defensins in primates: α, β and θ-defensins. α-defensins are most present in neutrophils and Paneth cells; β-defensins are involved in protecting the skin and the mucous membranes of the respiratory, genitourinary and gastrointestinal tracts; and θ-defensins are physically distinguished as the only known fully-cyclic peptides of animal origin, which are first isolated from rhesus macaques. All three kinds of defensins have six conserved cysteines, three intramolecular disulfide bonds, a net positive charge, and β-sheet regions. α and θ-defensins are closely related, comparative amino acid sequences showed that the difference between them is that θ-defensins have an additional stop codon limits the initial defensin domain peptides to 12 residues. Humans, chimpanzees and gorillas do not produce θ-defensin peptides due to a premature stop codon present in the signal sequence of all θ-defensin pseudogenes. By using comprehensive computational searches, here we report the discovery of complete repertoires of the α and θ-defensin gene family in ten primate species. Consistent with previous studies, our phylogenetic analyses showed all primate θ-defensins evident formed one distinct clusters evolved from α-defensins. β-defensins are ancestors of both α and θ-defensins. Human has two copies of DEFA1 and DEFT1P, and two extra DEFA3 and DEFA10P genes compared with gorilla. As different primates inhabit in quite different ecological niches, the production of species-specific α and θ-defensins and these highly evolved θ-defensins in old world monkeys would presumably allow them to better respond to the specific microbial challenges that they face.     

Defensin γ-thionin from Capsicum chinense has immunomodulatory effects on bovine mammary epithelial cells during Staphylococcus aureus internalization

β-Defensins are members of the antimicrobial peptide superfamily that are produced in various species from different kingdoms, including plants. Plant defensins exhibit primarily antifungal activities, unlike those from animals that exhibit a broad-spectrum antimicrobial action. Recently, immunomodulatory roles of mammal β-defensins have been observed to regulate inflammation and activate the immune system. Similar roles for plant β-defensins remain unknown. In addition, the regulation of the immune system by mammalian β-defensins has been studied in humans and mice models, particularly in immune cells, but few studies have investigated these peptides in epithelial cells, which are in intimate contact with pathogens. The aim of this work was to evaluate the effect of the chemically synthesized β-defensin γ-thionin from Capsicum chinense on the innate immune response of bovine mammary epithelial cells (bMECs) infected with Staphylococcus aureus, the primary pathogen responsible for bovine mastitis, which is capable of living within bMECs. Our results indicate that γ-thionin at 0.1 μg/ml was able to reduce the internalization of S. aureus into bMECs (∼50%), and it also modulates the innate immune response of these cells by inducing the mRNA expression (∼5-fold) and membrane abundance (∼3-fold) of Toll-like receptor 2 (TLR2), as well as by inducing genes coding for the pro-inflammatory cytokines TNF-α and IL-1β (∼14 and 8-fold, respectively) before and after the bacterial infection. γ-Thionin also induces the expression of the mRNA of anti-inflammatory cytokine IL-10 (∼12-fold). Interestingly, the reduction in bacterial internalization coincides with the production of other antimicrobial products by bMECs, such as NO before infection, and the secretion into the medium of the endogenous antimicrobial peptide DEFB1 after infection. The results from this work support the potential use of β-defensins from plants as immunomodulators of the mammalian innate immune response.     

鱼类β-防御素的研究进展

β-防御素是一种具有六个保守的半胱氨酸位点的阳离子两亲性小分子多肽,是抗菌肽的重要成员。随着基因组学研究的不断深入,目前在鱼类中发现了20个β-防御素基因,其基因结构均是由3个外显子和2个内含子组成,并且具有广谱抗菌活性及抗病毒活性。主要从分子结构、组织分布、基因结构、生物学活性以及生物进化等方面对鱼类β-防御素的研究概况进行综述。     

Antimicrobial potentials and structural disorder of human and animal defensins

Defensins are moonlighting peptides which are broadly distributed throughout all the living kingdoms. They play a multitude of important roles in human health and disease, possessing several immunoregulatory functions and manifesting broad antimicrobial activities against viruses, bacteria, and fungi. Based on their patterns of intramolecular disulfide bridges, these small cysteine-rich cationic proteins are divided into three major types, α-, β-, and θ-defensins, with the α- and β-defensins being further subdivided into a number of subtypes. The various roles played by the defensins in the innate (especially mucosal) and adoptive immunities place these polypeptides at the frontiers of the defense against the microbial invasions. Current work analyzes the antimicrobial activities of human and animal defensins in light of their intrinsic disorder propensities.     

Invariant gly residue is important for α-defensin folding, dimerization, and function: a case study of the human neutrophil α-defensin HNP1

The human α-defensins (HNP) are synthesized in vivo as inactive prodefensins, and contain a conserved glycine, Gly(17), which is part of a β-bulge structure. It had previously been shown that the glycine main chain torsion angles are in a D-configuration, and that d-amino acids but not L-alanine could be substituted at that position to yield correctly folded peptides without the help of a prodomain. In this study, the glycine to L-alanine mutant defensin was synthesized in the form of a prodefensin using native chemical ligation. The ligation product folded correctly and yielded an active peptide upon CNBr cleavage. The L-Ala(17)-HNP1 crystal structure depicted a β-bulge identical to wild-type HNP1. However, dimerization was perturbed, causing one monomer to tilt with respect to the other in a dimerization model. Inhibitory activity against the anthrax lethal factor showed a 2-fold reduction relative to wild-type HNP1 as measured by the inhibitory concentration IC(50). Self-association was slightly reduced, as detected by surface plasmon resonance measurements. According to the results of the virtual colony count assay, the antibacterial activity against Escherichia coli, Staphylococcus aureus, and Bacillus cereus exhibited a less than 2-fold reduction in virtual lethal dose values. Prodefensins with two other L-amino acid substitutions, Arg and Phe, at the same position did not fold, indicating that only small side chains are tolerable. These results further elucidate the factors governing the region of the β-bulge structure that includes Gly(17), illuminating why glycine is conserved in all mammalian α-defensins.     

Comparative <Emphasis Type="Italic">in vivo</Emphasis> infection models yield insights on early host immune response to Campylobacter in chickens

Salmonella typhimurium and Campylobacter jejuni pose significant risks to human health and poultry are a major vector for infection. Comparative in vivo infection models were performed to compare the avian host immune response to both bacterial species. Forty-five commercial broiler chickens were orally challenged with either C. jejuni or S. typhimurium whilst 60 similar control birds were mock challenged in parallel. Birds were sacrificed at 0, 6, 20 and 48 h post-infection and cloacal swabs, blood and tissue samples taken. Peripheral blood leukocytes were isolated for flow cytometric analyses and RNA was extracted for gene expression profiling. Colonisation patterns were markedly different between the two bacterial species, with systemic colonisation of Campylobacter outside the gastrointestinal tract. Salmonella infection induced significant changes in circulating heterophil and monocyte/macrophage populations, whilst Campylobacter infection had no effect on the heterophil numbers but caused a significant early increase in circulating monocytes/macrophages. Toll-like receptor 1 (TLR1) gene expression was decreased, and avian β-defensin (AvBD) gene expression (AvBD3, AvBD10 and AvBD12) was significantly increased in response to Salmonella infection (P < 0.05). In contrast, Campylobacter infection induced increased TLR21 gene expression but significantly reduced expression of seven antimicrobial peptide (AMP) genes (AvBD3, AvBD4, AvBD8, AvBD13, AvBD14, CTHL2 and CTHL3; P < 0.05). Considered together, microbiological, cellular and gene expression profiles indicate that the innate immune system responds differently to Salmonella and to Campylobacter infection. Furthermore, reduction in the expression of AMPs may play a role in the persistence of high level colonisation of the host by Campylobacter. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.