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PubMed2003

Alpha-defensins in the gastrointestinal tract.

The human intestinal tract is constantly exposed to an enormous indigenous bacterial flora. It has recently been recognised that antimicrobial peptides of the defensin family likely play a role in protection against microbial invasion at a variety of mucosal epithelial surfaces, including that of the intestinal tract. To date, six alpha-defensins have been identified in humans. Four of these, designated Human Neutrophil Peptides (HNP) 1,2,3 and 4, form part of the armoury of neutrophils, where they participate in systemic innate immunity. The remaining two, Human Defensin (HD) 5 and 6, are expressed in intestinal Paneth cells, and probably contribute to innate defense of the GI mucosal surface. Murine intestinal alpha-defensins (the 'cryptdins') have been extensively studied, but less is known about their human counterparts. The putative mature HD-5 was chemically synthesised and used to raise polyclonal antiserum. Using this anti-HD-5 antiserum, the expression of HD-5 in normal and inflamed intestinal mucosal samples was studied using immunohistochemistry. HD-5 is expressed in Paneth cells and also in some villous epithelial cells in normal duodenum, jejunum and ileum. HD-5 is not expressed in the normal stomach or colon. In cases of gastritis, colonic Crohn's disease and ulcerative colitis, HD-5 is expressed in metaplastic Paneth cells. Utilizing the anti-HD-5 antiserum, native HD-5 was isolated and purified from acid extracts of normal terminal ileal mucosal epithelial cells using cation exchange and reverse phase high pressure liquid chromatography. The purified peptide was characterised using N-terminal amino acid sequence and mass spectral analysis. Antimicrobial activity of the peptide was assessed using a sensitive colony forming unit antimicrobial assay. HD-5 is stored in the predicted precursor form in Paneth cells, and this form does not have antimicrobial activity against a defensin sensitive Salmonella. Potential processing of the precursor form of the HD-5 peptide into a mature active form, was studied by stimulating Paneth cell granule secretion in freshly isolated, cultured ileal crypts. A truncated form of the precursor form of HD-5, but not the predicted mature form, was present in the culture supernatant. Recently published studies suggest that further processing of the molecule occurs in vivo. The expression of HNP 1-3 in the normal intestinal mucosa and in cases of inflammatory bowel disease was studied. In the normal intestinal mucosa, HNP are expressed only in sparse lamina propria neutrophils, and not in Paneth cells. In cases of active ulcerative colitis and Crohn's disease, scattered surface epithelial cells, as well as numerous lamina propria neutrophils, were seen to express HNP. In conclusion, HD-5 is stored only in its precursor form in normal ileal Paneth cells, and partial processing of the peptide to a mature form occurs during and/or after secretion. In inflammatory bowel disease, HD-5 is expressed in metaplastic Paneth cells in the colon, and HNP is expressed by some surface epithelial cells. These studies suggest that antimicrobial defensin peptides may be important in protection of the host against microbial invasion in states of intestinal inflammation.

Amino Acid Sequence↗
PubMed2003

Rapid sequence divergence in mammalian beta-defensins by adaptive evolution.

beta-Defensin genes encode broad spectrum antimicrobial cationic peptides. We have analysed the largest murine and human clusters of these genes, which localise to mouse and human chromosome 8. Using hidden Markov models, we identified novel mouse and human beta-defensin genes. We subsequently found full-length expressed transcripts for these novel genes. Expression in the mouse was high in brain and reproductive tissues. Fourteen murine beta-defensins could be grouped into two clear sub-groups by virtue of their position and high signal sequence (exon 1 encoded) identity. In contrast, there was a very low level of sequence conservation in the exon 2 region encoding the mature antimicrobial peptide. Evolutionary analysis revealed strong evidence that following gene duplication, exon 1 and surrounding non-coding DNA show little divergence within subfamilies. The focus for rapid sequence divergence is localised in the DNA encoding the mature peptide and this is driven by accelerated positive selection. In the human we also conclude that the locus has evolved by successive rounds of duplication followed by substantial divergence involving positive selection, to produce a diverse cluster of paralogous genes prior to human-baboon divergence. This mechanism of adaptive evolution is consistent with the role of this gene family as defence against bacterial pathogens. In order to look at function of these rapidly evolving genes, we characterised one of the novel mouse beta-defensin genes. This gene deviates from the canonical six cysteine motif present in the mature functional peptide of all other beta defensins. This defensin related gene (Defr1) is most highly expressed in testis and heart and the genomic organisation is highly similar to Defb3-6. A synthetic Defr1 peptide was shown to exist as a dimer and yet displayed both antimicrobial and chemotactic activity. The antimicrobial activity of Defr1 against S. aureus, E. coli and B. cepacia was found to be reduced in raised concentration of NaCl, but its action against P. aeruginosa was independent of NaCl concentration. These data have major implications on the structure and functions of these important host defence molecules.

Adaptation, Physiological↗
PubMed2003

The time-dependent expression of fibronectin, MRP8, MRP14 and defensin in surgically treated human skin wounds.

Fibronectin, MRP8, MRP14 and defensin were detected immunohistochemically in 46 surgically treated, fresh (hours old) human wounds and in wounds of 13 individuals who died immediately from fatal trauma (airplane crashes or train rollovers). In immediate fatal trauma, it was not possible to detect fibronectin outside of bleeding areas nor could inflammatory cells be visualized in the interstitium using MRP8, MRP14, or defensin antibodies. Fibronectin staining could be regularly demonstrated in wounds at least 20 min-old. Granulocytic infiltrates limited to the perivasal space could be detected 20-30 min after infliction of the wound expressing MRP8, MRP14 and defensin. It was also possible to detect fibronectin networks and MRP8-, MRP14-, and defensin-positive granulocytes and macrophages in particular wounds up to 30 days-old. No differences between the expression of MRP8 and MRP14 could be demonstrated in the wounds, the majority of which were only several hours old. As wound age increased, the number of defensin-positive granulocytes detected decreased. The immunohistochemical detection of fibronectin is a useful way to demonstrate vitality in fresh wounds, beginning about 20-30 min post-trauma. However, detection of MRP8, MRP14 and defensin provides no advantage over the routine histological detection of granulocytes and macrophages in wounds under 1-2 days old.

Adolescent↗
PubMed1995

Refined three-dimensional solution structure of insect defensin A.

BACKGROUND: Insect defensin A is a basic 4 kDa protein secreted by Phormia terranovae larvae in response to bacterial challenges or injuries. Previous biological tests suggest that the bacterial cytoplasmic membrane is the target of defensin A. The structural study of this protein is the first step towards establishing a structure-activity relationship and forms the basis for understanding its antibiotic activity at the molecular level. RESULTS: We describe a refined model of the three-dimensional structure of defensin A derived from an extensive analysis of 786 inter-proton nuclear Overhauser effects. The backbone fold involves an N-terminal loop and an alpha-helical fragment followed by an antiparallel beta-structure. The helix and the beta-structure are connected by two of the three disulphide bridges present in defensin A, forming a so-called 'cysteine-stabilized alpha beta' (CS alpha beta) motif. The N-terminal loop, which is locally well defined, can occupy different positions with respect to the other moieties of the molecule. CONCLUSIONS: The CS alpha beta motif, which forms the core of the defensin A structure, appears to be a common organization for several families of small proteins with toxic properties. The distribution of amino acid side chains in the protein structure creates several hydrophobic or hydrophilic patches. This leads us to propose that the initial step in the action of positively charged defensin A molecules with cytoplasmic membranes may involve interactions with acidic phospholipids.

Amino Acid Sequence↗
PubMed2003

Isolation and characterization of Neurospora crassa mutants resistant to antifungal plant defensins.

Twenty-five Neurospora crassa mutants obtained by chemical mutagenesis were screened for increased resistance to various antifungal plant defensins. Plant defensin-resistant N. crassa mutants were further tested for their cross-resistance towards other families of structurally different antimicrobial peptides. Two N. crassa mutants, termed MUT16 and MUT24, displaying resistance towards all plant defensins tested but not to structurally different antimicrobial peptides were selected for further characterization. MUT16 and MUT24 were more resistant towards plant defensin-induced membrane permeabilization as compared to the N. crassa wild-type. Based on the previously demonstrated key role of fungal sphingolipids in the mechanism of growth inhibition by plant defensins, membrane sphingolipids of MUT16 and MUT24 were analysed. Membranes of these mutants contained structurally different glucosylceramides, novel glycosylinositolphosphorylceramides, and an altered level of steryl glucosides. Evidence is provided to link these clear differences in sphingolipid profiles of N. crassa mutants with their resistance towards different plant defensins.

Anti-Infective Agents↗
PubMed2002

Mammalian defensins in immunity: more than just microbicidal.

Mammalian defensins are small, cationic, antimicrobial peptides encoded by the host that are considered to be important antibiotic-like effectors of innate immunity. By using chemokine receptors on dendritic cells and T cells, defensins might also contribute to the regulation of host adaptive immunity against microbial invasion. Defensins have considerable immunological adjuvant activity and linkage of beta-defensins or selected chemokines to an idiotypic lymphoma antigen has yielded potent antitumor vaccines. The functional overlap between defensins and chemokines is reinforced by reports that some chemokines have antimicrobial activities. Although showing similarity in activity and overall tertiary structure, the evolutionary relationship between defensins and chemokines remains to be determined.

Animals↗
PubMed1999

Immunomagnetic recovery of human neutrophil defensins from the human gingival crevice.

The human neutrophilic protein defensins are cationic, antimicrobial, cytotoxic, corticostatic chemotactic, opsonic peptides found in azurophilic granules and constitute about 5% of the total protein in human neutrophils. In the present study, defensins were recovered from the human gingival crevice using paramagnetic microspheres (beads), coated with anti-defensin antibodies. The bead-bound defensins were assayed using an enzyme-linked immunosorbent assay developed in this laboratory. Twenty sites were sampled; defensin was found in 100% of the sites and ranged in amount from 270-2000 ng/site. The large local concentrations of defensins, estimated in the mg/ml range, may have major effects on the microbiology of the gingival crevice.

Adult↗
PubMed1999

Insect immunity: molecular cloning, expression, and characterization of cDNAs and genomic DNA encoding three isoforms of insect defensin in Aedes aegypti.

Aedes aegypti were immune activated by injection with bacteria, and the expression of insect defensins was measured over time. Northern analyses indicated that defensin transcriptional activity continued for at least 21 days after bacterial injection, and up to 10 days after saline inoculation. Mature defensin levels in the haemolymph reached approximately 45 microM at 24 h post inoculation. cDNAs encoding the preprodefensins of three previously described mature Ae. aegypti defensins were amplified by PCR, cloned and sequenced. Genomic clones were amplified using primers designed against the cDNA sequence. Sequence comparison indicates that there is significant inter- and intra-isoform variability in the signal peptide and prodefensin sequences of defensin genes. Preprodefensin sequences of isoforms A and B are very similar, consisting of a signal peptide region of twenty amino acids, a prodefensin region of thirty-eight amino acids and a forty amino acid mature peptide domain. The sequence encoding isoform C is significantly different, comprising a signal peptide region of twenty-three amino acids, a prodefensin region of thirty-six amino acids, and the mature protein domain of forty amino acids. Analysis of the genomic clones of each isoform revealed one intron spatially conserved in the prodefensin region of all sequences. The intron in isoforms A and B is 64 nt long, and except for a 4 nt substitution in one clone, these intron sequences are identical. The intron in isoform C is 76 nt long and does not share significant identity with the intron sequences of isoforms A or B. The defensin gene mapped to chromosome 3, between two known loci, blt and LF168.

Aedes↗
PubMed2005

Reduced Paneth cell alpha-defensins in ileal Crohn's disease.

The pathogenesis of Crohn's disease (CD), an idiopathic inflammatory bowel disease, is attributed, in part, to intestinal bacteria that may initiate and perpetuate mucosal inflammation in genetically susceptible individuals. Paneth cells (PC) are the major source of antimicrobial peptides in the small intestine, including human alpha-defensins HD5 and HD6. We tested the hypothesis that reduced expression of PC alpha-defensins compromises mucosal host defenses and predisposes patients to CD of the ileum. We report that patients with CD of the ileum have reduced antibacterial activity in their intestinal mucosal extracts. These specimens also showed decreased expression of PC alpha-defensins, whereas the expression of eight other PC products either remained unchanged or increased when compared with controls. The specific decrease of alpha-defensins was independent of the degree of inflammation in the specimens and was not observed in either CD of the colon, ulcerative colitis, or pouchitis. The functional consequence of alpha-defensin expression levels was examined by using a transgenic mouse model, where we found changes in HD5 expression levels, comparable to those observed in CD, had a pronounced impact on the luminal microbiota. Thus, the specific deficiency of PC defensins that characterizes ileal CD may compromise innate immune defenses of the ileal mucosa and initiate and/or perpetuate this disease.

Analysis of Variance↗
PubMed2004

Solution structure of spheniscin, a beta-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.

Amino Acid Motifs↗
PubMed2000

Evidence for the existence of insect defensin-like peptide in scorpion venom.

Insect defensin refers to a group of antibacterial peptides derived from a variety of insect species as well as from scorpion and possessing a three-dimensional structure highly similar to that of scorpion toxins. A full-length cDNA encoding an insect defensin-like peptide was isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch. The precursor, the overall organization of which is similar to that of insect defensins, consists of 61 amino acid residues with a putative signal peptide of 15 residues, a propeptide of 7 residues, and a mature peptide of 39 residues (named BmTXKS2). The positions of six cysteines and a conserved glycine in mature BmTXKS2 are the same as those in LqDef, the first defensin found in scorpions, which suggests these peptides should present a similar cysteine-stabilized alphabetamotif. Phylogenetic analysis further shows that the structure of BmTXKS2 is closer to that of ancient defensins (e.g., LqDef and AaDef, two insect defensins present in the scorpion hemolymph) than to scorpion toxins.

Amino Acid Sequence↗
PubMed2002

Association between elevated neutrophil defensin levels and endometritis.

The role of host defenses in the pathogenesis of pelvic inflammatory disease (PID) remains largely uncharacterized. The antimicrobial peptides defensins are important components of innate host defense. To explore the relationship between neutrophil defensins and upper genital tract infection, 377 women who were at risk for PID were enrolled in a study. Women infected with Neisseria gonorrhoeae, Trichomonas vaginalis, or Chlamydia trachomatis had higher median levels of neutrophil defensins (human neutrophil peptides 1-3) in the vagina than did uninfected women. Neutrophil defensins were strongly associated with the presence of endometritis after the analysis was controlled for the presence of sexually transmitted diseases. Vaginal neutrophils were associated with endometritis only in the presence of elevated defensin levels, which highlights the importance of neutrophil activation, rather than the presence of neutrophils alone, in this inflammatory process. Neutrophil defensins appear to participate in the host defense in ascending pelvic infection and the pathogenesis of PID.

Adolescent↗
PubMed2006

The novel beta-defensin DEFB123 prevents lipopolysaccharide-mediated effects in vitro and in vivo.

Defensins are a family of secreted antimicrobial peptides proposed to directly interfere with bacterial membranes. Here we show a functional analysis of the novel beta-defensin DEFB123. A peptide comprising the beta-defensin core region was synthesized and used for our analysis. Like other beta-defensins, DEFB123 exerted antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative bacteria, which was assessed by microbroth dilution assay and radial diffusion zone assay. In addition, the peptide showed lipopolysaccharide (LPS)-binding activity in a Limulus amoebocyte lysate (LAL) assay. Moreover, DEFB123 prevented LPS-induced tumor necrosis factor (TNF)-alpha secretion in a murine monocyte cell line (RAW264.7). Accordingly, DEFB123 abolished LPS-mediated MAPK induction in these cells. Protection against LPS-mediated effects was then investigated in a murine model of acute sepsis. Our experiments show that synthetic beta-defensin DEFB123 prevents LPS-induced mortality in C57BL/6 mice in a therapeutic approach. We propose that the physiological role of beta-defensins may include interference with LPS-action on macrophages, a function formerly thought to be restricted to the family of cathelicidins, a structurally unrelated group of antimicrobial peptides.

Animals↗
PubMedDate not supplied

Expression of human beta defensin 4 in genetically modified keratinocytes enhances antimicrobial activity.

Defensins are cationic peptides of the innate host defense system with antimicrobial activity against many of the microorganisms commonly found in burn units. Beta defensins are variably expressed in the epithelia of skin and other organs. Human beta defensin 4 reportedly has antimicrobial activity against Pseudomonas aeruginosa and is not normally expressed in intact skin. Genetic modification was used to ectopically express human beta defensin 4 in cultured primary epidermal keratinocytes. Keratinocytes expressing human beta defensin 4 showed significantly elevated antimicrobial activity against clinically-isolated P. aeruginosa compared with controls. These results suggest that genetic modification of keratinocytes can increase their resistance to microbial contamination. Bioengineered skin replacements containing human beta defensin 4-modified keratinocytes may be useful for transplantation to contaminated burn wounds.

Adult↗
PubMed2004

Differential antifungal and calcium channel-blocking activity among structurally related plant defensins.

Plant defensins are a family of small Cys-rich antifungal proteins that play important roles in plant defense against invading fungi. Structures of several plant defensins share a Cys-stabilized alpha/beta-motif. Structural determinants in plant defensins that govern their antifungal activity and the mechanisms by which they inhibit fungal growth remain unclear. Alfalfa (Medicago sativa) seed defensin, MsDef1, strongly inhibits the growth of Fusarium graminearum in vitro, and its antifungal activity is markedly reduced in the presence of Ca(2+). By contrast, MtDef2 from Medicago truncatula, which shares 65% amino acid sequence identity with MsDef1, lacks antifungal activity against F. graminearum. Characterization of the in vitro antifungal activity of the chimeras containing portions of the MsDef1 and MtDef2 proteins shows that the major determinants of antifungal activity reside in the carboxy-terminal region (amino acids 31-45) of MsDef1. We further define the active site by demonstrating that the Arg at position 38 of MsDef1 is critical for its antifungal activity. Furthermore, we have found for the first time, to our knowledge, that MsDef1 blocks the mammalian L-type Ca(2+) channel in a manner akin to a virally encoded and structurally unrelated antifungal toxin KP4 from Ustilago maydis, whereas structurally similar MtDef2 and the radish (Raphanus sativus) seed defensin Rs-AFP2 fail to block the L-type Ca(2+) channel. From these results, we speculate that the two unrelated antifungal proteins, KP4 and MsDef1, have evolutionarily converged upon the same molecular target, whereas the two structurally related antifungal plant defensins, MtDef2 and Rs-AFP2, have diverged to attack different targets in fungi.

Amino Acid Sequence↗
PubMed1994

Characterization and transcriptional profiles of a Drosophila gene encoding an insect defensin. A study in insect immunity.

Insect defensins are a family of 4-kDa, cationic, inducible antibacterial peptides which bear six cysteine residues engaged in three intramolecular disulfide bridges. They owe their name to certain sequence similarities with defensins from mammalian neutrophiles and macrophages. We report the characterization of a novel defensin isoform from Drosophila and the cloning of the gene encoding a preprodefensin. The gene, which is intronless and present in a single copy/haploid genome, maps at position 46CD on the right arm of the second chromosome. The analysis of the upstream region of the gene reveals the presence of multiple putative cis-regulatory sequences similar to mammalian regulatory motifs of acute-phase-response genes. Transcriptional profiles indicate that the Drosophila defensin gene is induced by bacterial challenge with acute-phase kinetics. It is also expressed in the absence of immune challenge during metamorphosis. These and other data on the Drosophila defensin gene lead us to suggest that insect and mammalian defensins have evolved independently.

Amino Acid Sequence↗
PubMed1995

[A study of defensins in bronchoalveolar lavage fluid in patients with diffuse panbronchiolitis].

We estimated defensins, antimicrobial and cytotoxic peptides localized in azurophil granules of neutrophils, in bronchoalveolar lavage fluid (BALF) in patients with diffuse panbronchiolitis (DPB). BALF from DPB patients contained a higher concentration of defensins than those from patients with idiopathic pulmonary fibrosis and healthy volunteers. A significant correlation was observed between the concentration of defensins and the number of neutrophils, the concentration of interleukin-8 or neutrophil elastase in BALF of DPB patients. An immunohistochemical defensins in neutrophils and mucinous exudates in the airways and in the surface of bronchiolar epithelial cells. After treatment with macrolide antibiotics, significant reductions in the concentrations of defensins, IL-8 and neutrophil numbers in BALF of DPB patients were observed. These findings suggest that the lung injury in DPB could be caused by defensins released by neutrophils accumulated in the airways.

Adult↗
PubMed1990

Polymorphic expression of defensins in neutrophils from outbred rats.

We isolated and characterized a rat neutrophil defensin, RatNP-2, that differs from the previously described defensin RatNP-1 by containing Ser-7 in place of Arg-7. Although the resulting charge difference rendered RatNP-2 easily distinguishable from RatNP-1 on polyacrylamide gel electrophoresis gels, the two defensins exhibited very similar antimicrobial efficacies against Salmonella typhimurium, Staphylococcus aureus, and Candida albicans. The polymorphonuclear leukocytes of Sprague-Dawley rats obtained from one of two breeders also showed a marked polymorphism for defensin RatNP-4. This defensin was absent in two of seven animals and present in 1x or 2x relative amounts in the others. These observations indicate that a striking degree of defensin polymorphism exists in the polymorphonuclear leukocytes of outbred rodents.

Amino Acids↗