Mammalian Beta-defensins Research Articles

Molecular analysis of crossbreeding experiment to improve immune genetic response against salmonella in poultry (Abstract)

Gallinacinsare functional analogues of the mammalian beta-defensins and play an important role in the innate immunity against microbial infections in chickens. Salmonella Typhimurium is a gram-negative bacterium that adversely affects both human and animal health. A crossbreeding experiment in chickens was conducted between an Egyptian breed (Fayoumi; F) and an exotic breed (Rhode Island Red; R) to improve the immune genetic response against salmonella in F1 chicks of ½F½R and their reciprocal cross of ½R½F. This study aimed to assess the association between four Gallinacins polymorphic genes (Gal2, Gal3, Gal4 and Gal5) and cecal salmonella bacterial count or antibodies titer (IgA, IgG and IgM). The cecal bacterial count and the antibodies titer were determined in both F1 chicks after pathogenic with S. Typhimurium. The polymorphic genes of Gal2, Gal3, Gal4 and Gal5 were assessed by the restriction fragment length polymorphism technique (PCR-RFLP) using HpyCH4IV, AvaI, AluI and HinfI restriction enzymes, respectively. The Gal2 gene was homozygous for the four genetic groups (FCF, RCR, FCR and RCF), so it was excluded from the association study. Among the four genes studied, the GAL3 gene was found to be significantly (p<0.05) associated with the salmonella cecal bacterial count and the antibodies produced. This gene encourages us to use it in any marker-assisted selection in order to improve and enhance the genetic immune response against salmonella infection in chickens.

Expression of mouse beta-defensin-3 in MDCK cells and its anti-influenza-virus activity

Influenza (flu) pandemics have presented a threat to human health in the past century. Because of outbreaks of avian flu in humans in some developing countries in recent years, humans are more eager to find a way to control flu. Mammalian beta-defensins (beta-defensins) are associated primarily with mucosal and skin innate immunity. Previous studies have demonstrated antimicrobial properties of a variety of defensin peptides. We have identified the presence of mouse beta-defensin 1, 2, and 3 genes (Mbd-1, 2, and 3) in trachea and lung tissues by RT-PCR before and after infection with influenza virus. We constructed a eukaryotic expression plasmid containing Mbd-3, pcDNA 3.1(+)/MBD-3, and the plasmid was introduced into Madin-Darby canine kidney (MDCK) cells by transfection. The expression of Mbd-3 in MDCK cells was verified by immunofluorescence test, RT-PCR, and Western blot. The pcDNA 3.1(+)/MBD-3 plasmid was injected into mice to observe its effect against influenza A virus (IAV) in vivo. Mouse beta-defensin genes could be expressed in trachea and lung tissues before IAV infection, but expression of Mbd-2 and Mbd-3 was increased significantly after IAV infection. The survival rate of mice with MBD-3 against IAV challenge was 71.43%, and MDCK cells with MBD-3 could clearly inhibit IAV replication. The results demonstrated that mouse beta-defensins possess anti-influenza virus activity, suggesting that mouse beta-defensins might be used as agents to prevent and treat influenza.

Analyses of Five gallinacin genes and the Salmonella enterica serovar Enteritidis response in poultry.

Gallinacins in poultry are functional equivalents of mammalian beta-defensins, which constitute an integral component of the innate immune system. Salmonella enterica serovar Enteritidis is a gram-negative bacterium that negatively affects both human and animal health. To analyze the association of genetic variations of the gallinacin genes with the phenotypic response to S. enterica serovar Enteritidis, an F1 population of chickens was created by crossing four outbred broiler sires to dams of two highly inbred lines. The F1 chicks were evaluated for bacterial colonization after pathogenic S. enterica serovar Enteritidis inoculation and for circulating antibody levels after inoculation with S. enterica serovar Enteritidis bacterin vaccine. Five candidate genes were studied, including gallinacins 2, 3, 4, 5, and 7. Gene fragments were sequenced from the founder individuals of the resource population, and a mean of 13.2 single-nucleotide polymorphisms (SNP) per kilobase was identified. One allele-defining SNP per gene was utilized to test for statistical associations of sire alleles with progeny response to S. enterica serovar Enteritidis. Among the five gallinacin genes evaluated, the Gal3 and Gal7 SNPs in broiler sires were found to be associated with antibody production after S. enterica serovar Enteritidis vaccination. Utilization of these SNPs as molecular markers for the response to S. enterica serovar Enteritidis may result in the enhancement of the immune response in poultry.

Human defensins

Antimicrobial peptides are small, cationic, amphiphilic peptides of 12-50 amino acids with microbicidal activity against both bacteria and fungi. The eukaryotic antimicrobial peptides may be divided into four distinct groups according to their structural features: cysteine-free alpha-helices, extended cysteine-free alpha-helices with a predominance of one or two amino acids, loop structures with one intramolecular disulfide bond, and beta-sheet structures which are stabilised by two or three intramolecular disulfide bonds. Mammalian defensins are part of the last-mentioned group. The mammalian defensins can be subdivided into three main classes according to their structural differences: the alpha-defensins, beta-defensins and the recently described theta-defensins. Mammalian alpha-defensins are predominantly found in neutrophils and in small intestinal Paneth cells, whereas mammalian beta-defensins have been isolated from both leukocytes and epithelial cells. Recently, two novel human beta-defensins, human beta-defensin-3 (HBD-3), and human beta-defensin-4 (HBD-4) have been discovered. Similar to HBD-1 and HBD-2, HBD-3 has microbicidal activity towards the Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli) and the yeasts Candida albicans and Malassezia furfur. In addition, HBD-3 kills Gram-positive bacteria such as Streptococcus pyogenes or Staphylococcus aureus, including multi-resistant S. aureus strains, and even vancomycin-resistant Enterococcus faecium. In contrast to HBD-1 and HBD-2, significant expression of HBD-3 has been demonstrated in non-epithelial tissues, such as leukocytes, heart and skeletal muscle. HBD-4 is expressed in certain epithelia and in neutrophils. Its bactericidal activity against P. aeruginosa is stronger than that of the other known beta-defensins. Here we present an overview of human antimicrobial peptides with some emphasis on their antifungal properties.

Solution Structure of Spheniscin, a β-Defensin from the Penguin Stomach

Recently two beta-defensins, named spheniscins, have been isolated from the stomach content of the king penguin (Aptenodytes patagonicus), which is capable of preserving food for several weeks during egg incubation (Thouzeau, C., Le Maho, Y., Froget, G., Sabatier, L., Le Bohec, C., Hoffmann, J. A., and Bulet, P. (2003) J. Biol. Chem. 278, 51053-51058). It has been proposed that, in combination with other antimicrobial peptides, spheniscins may be involved in this long term preservation of food in the bird's stomach. To draw some structure/function features, the three-dimensional structure in aqueous solution of the most abundant spheniscin (Sphe-2) was determined by two-dimensional NMR and molecular modeling techniques. The overall fold of Sphe-2 includes a three-stranded antiparallel beta-sheet stabilized by three disulfide bridges with a pairing typical of beta-defensins. In addition, the N-terminal segment shows helical features on most structures. Sphe-2 is highly cationic, and its surface displays a hydrophobic patch. Comparative modeling revealed that this patch is preserved in avian defensins. The activity of Sphe-2 against a pathogenic Gram-positive strain was retained in vitro in the conditions of osmolarity found in penguin stomach content and also in different salt concentrations and compositions up to those reported for seawater. Comparison with structurally related mammalian beta-defensins showed that the hydrophobic patch is not preserved in mammalian beta-defensins and that the high cationicity of Sphe-2 is presumably the critical factor for its retained activity in high salt concentrations. Such peculiarities, in addition to a broad activity spectrum, suggest that penguin defensins may represent interesting probes for the design of highly efficient antibiotics to fight off pathogens that develop in relatively salt-rich body fluids.

Murine beta-defensin-3 is an inducible peptide with limited tissue expression and broad-spectrum antimicrobial activity.

Beta-defensins are cationic peptides produced by epithelial cells that have been proposed to be an important component of immune function at mucosal surfaces. Similarities between mammalian beta-defensins may permit the use of murine models to further define the role of these peptides in innate host defense. Murine beta-defensin-3 (mBD-3) is a peptide that exhibits homology at the gene level to human beta-defensin-2 (hBD-2), one of four beta-defensins identified in man. The purpose of this study was to determine the antimicrobial activity of mBD-3, the tissue distribution of mBD-3 expression, and the effect of gram-negative bacterial infection on mBD-3 expression. Based on the sequence deduced from mBD-3 cDNA, a 40-amino acid peptide was assembled using automated [n-(9-fluorenyl)methoxycarbonyl] solid-phase synthesis. The antimicrobial activity of synthetic mBD-3 was evaluated in microdilution broth assays using bacterial and fungal organisms. mBD-3 mRNA expression was assayed by polymerase chain reaction (PCR) using cDNA derived from a panel of tissues. Expression of mBD-3 was also evaluated in tissues obtained from mice 24 h after intraperitoneal infection with Escherichia coli using reverse transcriptase (RT)-PCR. Synthetic mBD-3 inhibited the growth of E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans at concentrations from 25 to 50 microg/mL. Constitutive expression of mBD-3 mRNA was not consistently found in any organ using RT-PCR. In an E. coli peritonitis model, expression of mBD-3 mRNA was upregulated only in the esophagus and tongue. We conclude that mBD-3 is an inducible peptide with limited tissue expression during E. coli peritonitis. Because it exhibits broad-spectrum antimicrobial activity, this peptide may serve as an innate defense against microbial invasion at specific mucosal surfaces in the mouse.

Properties of the prophenoloxidase activating enzyme of the freshwater crayfish, Pacifastacus leniusculus.

The prophenoloxidase activating enzyme (ppA), a serine proteinase catalyzing the conversion of prophenoloxidase to an active phenoloxidase, has a molecular mass of about 36 kDa in its active form. This protein was cloned from a blood cell cDNA library and its corresponding cDNA of 1736 base pairs encodes a zymogenic protein (proppA) of 468 amino acids. An antibody raised against a synthetic peptide derived from a region of the cDNA sequence could efficiently inhibit the beta-1,3-glucan triggered activation of prophenoloxidase in vitro. The C-terminal half of the proppA is composed of a typical serine proteinase domain, with a sequence similar to other invertebrate and vertebrate serine proteinases. The N-terminal half contains a cationic glycine-rich domain, a cationic proline-rich domain and a clip-domain, in which the disulfide-bonding pattern is likely to be identical to those of the horseshoe crab big defensin and mammalian beta-defensins. Antibodies made against both the C- and the N-terminal halves recognize two proppAs under reducing conditions. However, under nonreducing conditions only the anti-C antibody recognized the two proppAs, which suggests that a conformational change takes place upon reduction that allows the anti-N to react with the N-terminal half of proppA. The recombinant clip-domain in crayfish proppA was overexpressed in Escherichia coli and the resulting peptide exhibited antibacterial activity against Gram-positive bacterial strains such as Micrococcus luteus Ml11 and Bacillus megaterium Bm11 with 50% growth inhibitory concentrations of 1.43 microM and 17.9 microM, respectively. These results suggest that the clip-domains in proppAs may function as antibacterial peptides.

Cloning and Characterization of the Gene for a New Epithelial β-Defensin: GENOMIC STRUCTURE, CHROMOSOMAL LOCALIZATION, AND EVIDENCE FOR ITS CONSTITUTIVE EXPRESSION

Mammalian beta-defensins are endogenous cysteine-rich peptide antibiotics that are produced either by epithelial cells lining the respiratory, digestive, and urogenital tracts or by granulocytes and macrophages. A growing body of evidence has implicated these peptides in host defense, particularly mucosal innate immunity. We previously reported the cloning of the full-length cDNA for a porcine beta-defensin (pBD-1), which was found to be expressed throughout the airway and oral mucosa. Here, we provide the structural organization of the pBD-1 gene, showing that the entire gene spans approximately 1.9 kilobases with two short exons separated by a 1.5-kilobase intron. Fluorescence in situ hybridization mapped the pBD-1 gene to porcine chromosome 15q14-q15. 1 within a region of conserved synteny to the chromosomal locations of human and mouse alpha- and beta-defensins. We also provide several independent lines of evidence showing that the pBD-1 gene is expressed constitutively during inflammation and infection, despite its resemblance to many inducible epithelial beta-defensins in amino acid sequence, genomic structure, and sites of expression. First, stimulation of primary porcine tongue epithelial cells with lipopolysaccharide, tumor necrosis factor-alpha, and interleukin (IL)-1beta failed to up-regulate the expression of pBD-1 mRNA. Second, pBD-1 gene expression was not enhanced in either digestive or respiratory mucosa of pigs following a 2-day infection with Salmonella typhimurium or Actinobacillus pleuropneumoniae. Last, direct transfection of the pBD-1 gene promoter into NIH/3T3 cells showed no difference in reporter gene activity in response to stimulation by lipopolysaccharide and IL-1beta. The constitutive expression of pBD-1 in airway and oral mucosa, which is consistent with a lack of consensus binding sites for nuclear factor-kappaB or NF-IL-6 in its promoter region, suggests that it may play a surveillance role in maintaining the steady state of microflora on mucosal surfaces.