A genome-wide screen identifies a single β-defensin gene cluster in the chicken: implications for the origin and evolution of mammalian defensins

Background  

Defensins comprise a large family of cationic antimicrobial peptides that are characterized by the presence of a conserved cysteine-rich defensin motif. Based on the spacing pattern of cysteines, these defensins are broadly divided into five groups, namely plant, invertebrate, α-, β-, and θ-defensins, with the last three groups being mostly found in mammalian species. However, the evolutionary relationships among these five groups of defensins remain controversial.     

Bioinformatic and expression analysis of novel porcine β-defensins

β-Defensins are a major group of mammalian antimicrobial peptides. Although more than 30 β-defensins have been identified in humans, only one porcine β-defensin has been reported. In this article we report the identification and initial characterization of 11 novel porcine β-defensins (pBD). Using bioinformatic approaches, we screened 287,821 porcine expressed sequence tags for similarity of their predicted peptides to known human β-defensins and identified full-length or partial sequences for the 11 novel pBDs. Similar to the previously identified pBD1, all of these peptides have a consensus β-defensin motif. A differential expression pattern for these newly identified genes was found. For example, unlike most β-defensins, pBD2 and pBD3 were expressed in bone marrow and in other lymphoid tissues including thymus, spleen, lymph nodes, duodenum, and liver. Including pBD2 and pBD3, six porcine β-defensins were expressed in lung and skin. Several newly identified porcine β-defensins, including pBD123, pBD125, and pBD129, were expressed in male reproductive tissues, including lobuli testis and some segments of the epididymis. Phylogenetic analysis indicates that in most cases the evolutionary relationship between individual porcine β-defensins and their human orthologs is closer than the relationship among β-defensins in the same species. These findings establish the existence of multiple porcine β-defensins and suggest that the pig may be an ideal model for the characterization of β-defensin diversity and function. The nucleotide sequence data reported in this article have been submitted to GenBank.     

Mouse beta defensin-1 is a functional homolog of human beta defensin-1

Defensin are 3–4 kDa antimicrobial peptides of which three distinct families have been identified; α-defensin, β-defensins, and insect defensins. Recent investigations have shown that β-defensins are present in the human airways and may be relevant to the pathogenesis of cystic fibrosis (CF) lung disease. We report here the further characterization of a recently identified mouse β-defensin gene, Defb1, sometimes referred to as mBD-1, which is homologous to the human airway beta defensin hBD-1. We report that Defb1 is expressed in a variety of tissues including the airways and, similar to hBD-1, is not upregulated by lipopolysaccharide (LPS). Defb1 was found to consist of two small exons separated by a 16-kb intron and cytogenetic, and physical mapping linked it to the alpha defensin gene cluster on mouse Chromosome (Chr) 8. Functional studies demonstrate that, like hBD-1, Defb1 demonstrates a salt-sensitive antimicrobial activity against Pseudomonas aeruginosa. Of relevance to CF lung disease is the fact that neither the hBD-1 nor the mBD-1 peptides are active against Burkholderia cepacia. Received: 3 December 1997 / Accepted: 17 February 1998     

Roles of human β-defensins in innate immune defense at the ocular surface: arming and alarming corneal and conjunctival epithelial cells

Human β-defensins are cationic peptides produced by epithelial cells that have been proposed to be an important component of immune function at mucosal surfaces. In this study, the expression and inducibility of β-defensins at the ocular surface were investigated in vitro and in vivo. Expression of human β-defensins (hBD) was determined by RT-PCR and immunohistochemistry in tissues of the ocular surface and lacrimal apparatus. Cultured corneal and conjunctival epithelial cells were stimulated with proinflammatory cytokines and supernatants of different ocular pathogens. Real-time PCR and ELISA experiments were performed to study the effect on the inducibility of hBD2 and 3. Expression and inducibility of mouse β-defensins-2, -3 and -4 (mBD2–4) were tested in a mouse ocular surface scratch model with and without treatment of supernatants of a clinical Staphylococcus aureus (SA) isolate by means of immunohistochemistry. Here we show that hBD1, -2, -3 and -4 are constitutively expressed in conjunctival epithelial cells and also partly in cornea. Healthy tissues of the ocular surface, lacrimal apparatus and human tears contain measurable amounts of hBD2 and -3, with highest concentrations in cornea and much lower concentrations in all other tissues, especially tears, suggesting intraepithelial storage of β-defensins. Exposure of cultured human corneal and conjunctival epithelial cells to proinflammatory cytokines and supernatants of various bacteria revealed that IL-1β is a very strong inductor of hBD2 and Staphylococcus aureus increases both hBD2 and hBD3 production in corneal and conjunctival epithelial cells. A murine corneal scratch model demonstrated that β-defensins are only induced if microbial products within the tear film come into contact with a defective epithelium. Our finding suggests that the tear film per se contains so much antimicrobial substances that epithelial induction of β-defensins occurs only as a result of ocular surface damage. These findings widen our knowledge of the distribution, amount and inducibility of β-defensins at the ocular surface and lacrimal apparatus and show how β-defensins are regulated specifically.     

Human Antimicrobial Peptides: Defensins, Cathelicidins and Histatins

Antimicrobial peptides, which have been isolated from many bacteria, fungi, plants, invertebrates and vertebrates, are an important component of the natural defenses of most living organisms. The isolated peptides are very heterogeneous in length, sequence and structure, but most of them are small, cationic and amphipathic. These peptides exhibit broad-spectrum activity against Gram-positive and Gram-negative bacteria, yeasts, fungi and enveloped viruses. A wide variety of human proteins and peptides also have antimicrobial activity and play important roles in innate immunity. In this review we discuss three important groups of human antimicrobial peptides. The defensins are cationic non-glycosylated peptides containing six cysteine residues that form three intramolecular disulfide bridges, resulting in a triple-stranded β-sheet structure. In humans, two classes of defensins can be found: α-defensins and β-defensins. The defensin-related HE2 isoforms will also be discussed. The second group is the family of histatins, which are small, cationic, histidine-rich peptides present in human saliva. Histatins adopt a random coil conformation in aqueous solvents and form α-helices in non-aqueous solvents. The third group comprises only one antimicrobial peptide, the cathelicidin LL−37. This peptide is derived proteolytically from the C-terminal end of the human CAP18 protein. Just like the histatins, it adopts a largely random coil conformation in a hydrophilic environment, and forms an α-helical structure in a hydrophobic environment.     

Evolution, expression and effectiveness in a cluster of novel bovine β-defensins

The anti-microbial peptides β-defensins constitute a large family of innate immune effector molecules, conserved across a wide species range. In this paper, we describe a systematic search of the sequenced bovine genome to characterise this extensive gene family in Bos taurus, providing an insight into the pattern of conservation of β-defensin genes between species. We have sequenced a sub-set of these newly discovered bovine β-defensin genes and also report expression data for these genes across a range of tissues. We have synthesised the peptide product of one of these genes, bovine β-defensin 123, and found it to be a potent inhibitor of several pathogenic microbes, particularly Escherichia coli and Listeria monocytogenes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Background  

Pre-elafin/trappin-2 is a human innate defense molecule initially described as a potent inhibitor of neutrophil elastase. The full-length protein as well as the N-terminal "cementoin" and C-terminal "elafin" domains were also shown to possess broad antimicrobial activity, namely against the opportunistic pathogen P. aeruginosa. The mode of action of these peptides has, however, yet to be fully elucidated. Both domains of pre-elafin/trappin-2 are polycationic, but only the structure of the elafin domain is currently known. The aim of the present study was to determine the secondary structures of the cementoin domain and to characterize the antibacterial properties of these peptides against P. aeruginosa.     

Tumor necrosis factor and norepinephrine lower the levels of human neutrophil peptides 1-3 secretion by mixed synovial tissue cultures in osteoarthritis and rheumatoid arthritis

Introduction  

Neutrophils and monocytes play an important role in overt inflammation in chronic inflammatory joint diseases such as rheumatoid arthritis (RA). The sympathetic nervous system (SNS) inhibits many neutrophil/monocyte functions and macrophage tumor necrosis factor (TNF), but because of the loss of sympathetic nerve fibers in inflamed tissue, sympathetic control is attenuated. In this study, we focused on noradrenergic and TNF regulation of human neutrophil peptides 1-3 (HNP1-3), which are proinflammatory bactericidal α-defensins.     

Biotransformation using <Emphasis Type="Italic">Mucor rouxii</Emphasis> for the production of oleanolic acid derivatives and their antimicrobial activity against oral pathogens

The goal of this study is to produce oleanolic acid derivatives by biotransformation process using Mucor rouxii and evaluate their antimicrobial activity against oral pathogens. The microbial transformation was carried out in shake flasks at 30°C for 216 h with shaking at 120 rpm. Three new derivatives, 7β-hydroxy-3-oxo-olean-12-en-28-oic acid, 7β,21β-dihydroxy-3-oxo-olean-12-en-28-oic acid, and 3β,7β,21β-trihydroxyolean-12-en-28-oic acid, and one know compound, 21β-hydroxy-3-oxo-olean-12-en-28-oic acid, were isolated, and the structures were elucidated on the basis of spectroscopic analyses. The antimicrobial activity of the substrate and its transformed products was evaluated against five oral pathogens. Among these compounds, the derivative 21β-hydroxy-3-oxo-olean-12-en-28-oic acid displayed the strongest activity against Porphyromonas gingivalis, which is a primary etiological agent of periodontal disease. In an attempt to improve the antimicrobial activity of the derivative 21β-hydroxy-3-oxo-olean-12-en-28-oic acid, its sodium salt was prepared, and the minimum inhibitory concentration against P. gingivalis was reduced by one-half. The biotransformation process using M. rouxii has potential to be applied to the production of oleanolic acid derivatives. Research and antimicrobial activity evaluation of new oleanolic acid derivatives may provide an important contribution to the discovery of new adjunct agents for treatment of dental diseases such as dental caries, gingivitis, and periodontitis.     

Antimicrobial peptides effectively kill a broad spectrum of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> strains independently of origin,sub-type,or virulence factor expression

Background  

Host defense peptides (HDPs), or antimicrobial peptides (AMPs), are important components of the innate immune system that bacterial pathogens must overcome to establish an infection and HDPs have been suggested as novel antimicrobial therapeutics in treatment of infectious diseases. Hence it is important to determine the natural variation in susceptibility to HDPs to ensure a successful use in clinical treatment regimes.     

Expression and Identification of Porcine β-Defensin 1 in Escherichia coli and Up-Regulation by Streptococcus Infection in Porcine Tongue In Vivo

Defensins are members of a major family of antimicrobial peptides that play an important role in the innate immune response, which are of interest as potential novel pharmaceutical agents. We successfully constructed expression system of porcine β-defensin 1 (pBD-1) with his-tag in Escherichia coli Rossetta (DE3) cells and investigated the effect of Streptococcus ATCC 19714 infection on the mRNA expression of pBD-1 in porcine tongue in vivo. The results showed that active pBD-1 not to be affected by the presence of his-tag was obtained and displayed a high antimicrobial activity against Streptococcus ATCC 19714 at a concentration of 42 ± 5.2 μg/ml. The mRNA level of pBD-1 after infection in the tongue mucosa was initially changed with significant up-regulation at 3 h and reached the highest level at 6 h (about tenfold higher than 0 h), thereafter reduced to normal level at 12 h. The results indicated that pBD-1 is shown as a potent antimicrobial activity, and the expression level of pBD-1 against Streptococcus ATCC 19714 is up-regulation in the porcine tongue.     

Potential therapeutic efficacy of a bactericidal–immunomodulatory fusion peptide against methicillin-resistant Staphylococcus aureus skin infection

To enhance the potential therapeutic efficacy of an antimicrobial peptide human β-defensin 3, two fusion peptides, a bactericidal–immunomodulatory fusion peptide human β-defensin 3-mannose-binding lectin and a bactericidal–bactericidal fusion peptide human β-defensin 3-lysozyme were synthesized and the bactericidal activities in vitro and in vivo against methicillin-resistant Staphylococcus aureus N315 were demonstrated in this study. Peptide human β-defensin 3-lysozyme showed the best bactericidal activity in vitro, but human β-defensin 3-mannose-binding lectin showed a significant improvement in angiogenesis and tissue reconstruction. Our results illustrated that outstanding bactericidal activity in vitro is not essential in the development of antimicrobial peptides. Fusion strategy and immunomodulatory factors should be utilized in novel antimicrobial peptide development.     

Vv-AMP1, a ripening induced peptide from <Emphasis Type="Italic">Vitis vinifera</Emphasis> shows strong antifungal activity

Background  

Latest research shows that small antimicrobial peptides play a role in the innate defense system of plants. These peptides typically contribute to preformed defense by developing protective barriers around germinating seeds or between different tissue layers within plant organs. The encoding genes could also be upregulated by abiotic and biotic stimuli during active defense processes. The peptides display a broad spectrum of antimicrobial activities. Their potent anti-pathogenic characteristics have ensured that they are promising targets in the medical and agricultural biotechnology sectors.     

Antistaphylococcal and biofilm inhibitory activities of acetyl-11-keto-β-boswellic acid from <Emphasis Type="Italic">Boswellia serrata</Emphasis>

Background  

Boswellic acids are pentacyclic triterpenes, which are produced in plants belonging to the genus Boswellia. Boswellic acids appear in the resin exudates of the plant and it makes up 25-35% of the resin. β-boswellic acid, 11-keto-β-boswellic acid and acetyl-11-keto-β-boswellic acid have been implicated in apoptosis of cancer cells, particularly that of brain tumors and cells affected by leukemia or colon cancer. These molecules are also associated with potent antimicrobial activities. The present study describes the antimicrobial activities of boswellic acid molecules against 112 pathogenic bacterial isolates including ATCC strains. Acetyl-11-keto-β-boswellic acid (AKBA), which exhibited the most potent antibacterial activity, was further evaluated in time kill studies, postantibiotic effect (PAE) and biofilm susceptibility assay. The mechanism of action of AKBA was investigated by propidium iodide uptake, leakage of 260 and 280 nm absorbing material assays.     

The heme sensing response regulator HssR in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> but not the homologous RR23 in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> modulates susceptibility to the antimicrobial peptide plectasin

Background  

Host defence peptides (HDPs), also known as antimicrobial peptides (AMPs), have emerged as potential new therapeutics and their antimicrobial spectrum covers a wide range of target organisms. However, the mode of action and the genetics behind the bacterial response to HDPs is incompletely understood and such knowledge is required to evaluate their potential as antimicrobial therapeutics. Plectasin is a recently discovered HDP active against Gram-positive bacteria with the human pathogen, Staphylococcus aureus (S. aureus) being highly susceptible and the food borne pathogen, Listeria monocytogenes (L. monocytogenes) being less sensitive. In the present study we aimed to use transposon mutagenesis to determine the genetic basis for S. aureus and L. monocytogenes susceptibility to plectasin.     

Bovine β-defensins: Identification and characterization of novel bovine β-defensin genes and their expression in mammarygland tissue

-Defensin genes code for multifunctional peptides with a broad-range antimicrobial activity. In this project we hypothesized that -defensin genes may be candidate genes for resistance to mastitis. In this article we describe the identification and genomic characterization of eight bovine -defensin genes, including six novel defensin genes and two pseudogenes. Expression in the bovine mammary gland of one of the novel genes, DEFB401, has been demonstrated, as well as the expression of LAP, TAP, DEFB1, BNBD3, BNBD9, and BNBD12. For genomic characterization, 20 BACs from two different bovine BAC libraries (RZPD numbers 750 and 754) were isolated by PCR screening with -defensin consensus primers derived from published sequences. PCR products from BACs generated with consensus primers have been subcloned and sequenced, revealing a total of 16 genes and two pseudogenes. Six novel -defensin genes share the typical exon–intron structure and are highly homologous to published bovine -defensin genes. They are named DEFB401–DEFB405 and LAP-like, and two novel pseudogenes are named EBD-P and EBD-P2. Analysis of mammary gland tissue-derived cDNA from nine cows with different clinical findings demonstrated the expression of several -defensin genes mentioned above. First results indicate that the lactational status of the cow presumably has no influence on gene expression. Competent knowledge of antimicrobial activity of -defensins from literature, the abundance of -defensin mRNA in the bovine mammary gland, and the inducibility of some genes give first evidence that -defensins may play a role in local host defense during udder infections.The nucleotide sequence data reported in this article have been submitted to EMBL and have been assigned the accession numbers AJ563279–AJ563283, AJ567353–AJ567365, AJ567990–AJ567993, and AJ620296.     

Reproductive tissue-specific expression profiling and genetic variation across a 19 gene bovine β-defensin cluster

β-defensins are small cationic peptides, with potent immunoregulatory and antimicrobial activity which are produced constitutively and inducibly by eukaryotic cells. This study profiles the expression of a cluster of 19 novel defensin genes which spans 320 kb on chromosome 13 in Bos taurus. It also assesses the genetic variation in these genes between two divergently selected cattle breeds. Using quantitative real-time PCR (qRT-PCR), all 19 genes in this cluster were shown to be expressed in the male genital tract and 9 in the female genital tract, in a region-specific manner. These genes were sequenced in Norwegian Red (NR) and Holstein-Friesian (HF) cattle for population genetic analysis. Of the 17 novel single nucleotide polymorphisms (SNPs) identified, 7 were non-synonymous, 6 synonymous and 4 outside the protein coding region. Significant frequency differences in SNPs in bovine β-defensins (BBD) 115, 117, 121, and 122 were detected between the two breeds, which was also reflected at the haplotype level (P < 0.05). There was clear segregation of the haplotypes into two blocks on chromosome 13 in both breeds, presumably due to historical recombination. This study documents genetic variation in this β-defensin gene cluster between Norwegian Red and Holstein-Friesian cattle which may result from divergent selection for production and fertility traits in these two breeds. Regional expression in the epididymis and fallopian tube suggests a potential reproductive-immunobiology role for these genes in cattle.     

Expression of the antimicrobial peptide cecropin fused with human lysozyme in Escherichia coli

Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in host defense. It targets the β (1–4) glycosidic bond between N-acetylglucosamine and N-acetylmuramic residues that make up peptidoglycan, making lysozyme highly active against Gram-positive bacteria. However, lysozyme alone is inactive against Gram-negative bacteria because it cannot reach the peptidoglycan layer. Cecropins are cationic molecules with a wide range of antimicrobial activities. The main target for these peptides is the cytoplasmic membrane. We resume that cecopin may disrupt the outer membrane, giving the enzyme access to the peptidoglycan in cell wall. So in the present study, novel hybrid protein combining Musca domestica cecropin (Mdc) with human lysozyme (Hly) was designed. The DNA sequence encoding recombination fusion protein Mdc–hly was cloned into the pET-32a vector for protein expression in Escherichia coli strain BL21 (DE3). The protein was expressed as a His-tagged fusion protein, and the Mdc–hly was released from the fusion by enterokinase cleavage and separated from the carrier thioredoxin. Antimicrobial activity assays showed that the recombinant fusion protein Mdc–hly has improved in vitro antimicrobial activity and action spectrum compared to Mdc and hly. Mdc–hly may have important potential application as a future safely administered human drug and food additive.     

The GraRS regulatory system controls <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> susceptibility to antimicrobial host defenses

Background  

Modification of teichoic acids with D-alanine by the products of the dlt operon protects Gram-positive bacteria against major antimicrobial host defense molecules such as defensins, cathelicidins, myeloperoxidase or phospholipase. The graRS regulatory genes have recently been implicated in the control of D-alanylation in Staphylococcus aureus.     

Tertiary structure-related activity of tick defensin (persulcatusin) in the taiga tick, <Emphasis Type="Italic">Ixodes persulcatus</Emphasis>

Defensins are small cysteine-rich cationic proteins found in both vertebrates and invertebrates constituting the front line of host innate immunity. To examine the importance of the tertiary structure of tick defensin in its antimicrobial activity, we synthesized two types of the peptides with tertiary structure or primary one on basis of the information of the sequence in the defensin originated from the taiga tick, Ixodes persulcatus. Chemically synthesized peptides were used to investigate the activity spectrum against Staphylococcus aureus, Borrelia garinii and flora-associated bacteria. Both synthetic peptides showed antimicrobial activity against S. aureus in short-time killing within 1 h, but they do not show the activity against B. garinii, Stenotrophomonas maltophila and Bacillus spp., which were frequently isolated from the midgut of I. persulcatus. The teriary structure brought more potent activity to S. aureus than primary one in short-time killing. We also examined its antimicrobial activity by evaluation of growth inhibition in the presence of the synthetic peptides. Minimum inhibitory concentration (MIC) was ranged from 1.2 to 5.0 μg/ml in tertiary peptide and from 10 to 40 μg/ml in primary peptide, when 10 strains of S. aureus were used. From the curve of cumulative inhibition rates, MIC50 (MIC which half of the strains showed) to S. aureus is about 1.2 μg/ml in the peptide with tertiary structure and about 10 μg/ml in the linear one. Corynebacterium renale is 10 times or more sensitive to tertiary peptide than primary one. In conclusion, the presence of 3 disulfide bridges, which stabilize the molecule and maintain the tertiary structure, is considered to have an effect on their antimicrobial activities against Gram-positive bacteria such as S. aureus.