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PubMed1999

Permeabilization of fungal membranes by plant defensins inhibits fungal growth.

We used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in fungi. Membrane permeabilization induced by plant defensins in Neurospora crassa was biphasic, depending on the plant defensin dose. At high defensin levels (10 to 40 microM), strong permeabilization was detected that could be strongly suppressed by cations in the medium. This permeabilization appears to rely on direct peptide-phospholipid interactions. At lower defensin levels (0.1 to 1 microM), a weaker, but more cation-resistant, permeabilization occurred at concentrations that correlated with the inhibition of fungal growth. Rs-AFP2(Y38G), an inactive variant of the plant defensin Rs-AFP2 from Raphanus sativus, failed to induce cation-resistant permeabilization in N. crassa. Dm-AMP1, a plant defensin from Dahlia merckii, induced cation-resistant membrane permeabilization in yeast (Saccharomyces cerevisiae) which correlated with its antifungal activity. However, Dm-AMP1 could not induce cation-resistant permeabilization in the Dm-AMP1-resistant S. cerevisiae mutant DM1, which has a drastically reduced capacity for binding Dm-AMP1. We think that cation-resistant permeabilization is binding site mediated and linked to the primary cause of fungal growth inhibition induced by plant defensins.

Antifungal Agents↗
PubMed1999

Induction of a rat enteric defensin gene by hemorrhagic shock.

Multicellular organisms utilize a battery of extracellular and cellular mechanisms to defend against microbial infiltration. Among the armamentarium used by the small intestine to defend against microbial invasion are antimicrobial peptides called defensins. We previously have shown that gut barrier function is impaired following hemorrhagic shock, resulting in translocation of bacteria or endotoxin. Using a rat model, we examined the effect of hemorrhagic shock on alpha-defensin expression. We utilized the anchored reverse transcriptase PCR strategy to isolate a rat enteric defensin cDNA. The cDNA is 406 bases in length and encodes a putative prepro-enteric defensin that we have named rat defensin 5 (RD-5). RD-5 expression is restricted to the small intestine and is specifically localized by in situ hybridization to the Paneth cells. A 10-fold increase in its steady state levels was observed in the distal intestine immediately after the termination of shock. This is the first study to show that enteric defensins are inducible following injury. We suggest that enteric defensins may contribute to the complex and integrated barrier function of the intestinal mucosal surface.

Amino Acid Sequence↗
PubMed2004

Differential regulation of beta-defensin gene expression during Cryptosporidium parvum infection.

Invasion of enterocytes by pathogenic microbes evokes both innate and adaptive immune responses, and microbial pathogens have developed strategies to overcome the initial host immune defense. beta-Defensins are potentially important endogenous antibiotic-like effectors of innate immunity expressed by intestinal epithelia. In this study, the interplay between the enteric protozoan parasite Cryptosporidium parvum and host epithelial beta-defensin expression was investigated. Using human and murine models of infection, we demonstrated that C. parvum infection differentially regulates beta-defensin gene expression. Downregulation of murine beta-defensin-1 mRNA and protein was observed in both in vitro and in vivo models of infection. Infection of the human colonic HT29 cell line with the parasite resulted in differential effects on various members of the defensin gene family. Partial reduction in human beta-defensin-1 (hBD-1), induction of hBD-2, and no effect on hBD-3 gene expression was observed. Recombinant hBD-1 and hBD-2 peptides exhibited significant antimicrobial activity against C. parvum sporozoites in vitro. These findings demonstrate that C. parvum infection of enterocytes may affect the expression of various defensins in different ways and suggest that the overall outcome of the effect of antimicrobial peptides on early survival of the parasite may be complex.

Animals↗
PubMed1991

Mechanism of mammalian cell lysis mediated by peptide defensins. Evidence for an initial alteration of the plasma membrane.

Defensins induce ion channels in model lipid bilayers and permeabilize the membranes of Escherichia coli. We investigated whether similar membrane-active events occur during defensin-mediated cytolysis of tumor cells. Although defensin-treated K562 targets did not release chromium-labeled cytoplasmic components for 5-6 h, they experienced a rapid collapse (within minutes) of the membrane potential, efflux of rubidium, and influx of trypan blue. Defensin treatment also blunted the subsequent acidification response induced by nigericin, thereby further supporting the notion of enhanced transmembrane ion flow during exposure. These initial effects on the plasma membrane were not sufficient for subsequent lysis; a second phase of injury was required which involved the continued presence of defensin. The rapid membrane permeabilization phase was inhibited by azide/2-deoxyglucose, cytochalasin B, and increased concentrations of extracellular potassium and was unaffected by actinomycin-D, cycloheximide, and varying the calcium concentration. In contrast, the second phase was unaffected by cytochalasin B, inhibited by azide/2-deoxyglucose, enhanced by actinomycin D and cycloheximide, and varied with calcium concentration. These results indicate the initial adverse effect of defensins on mammalian cells occurs at the cell membrane. It is possible that the second phase of injury is mediated intracellularly by defensin that has been internalized through this leaky membrane.

Blood Proteins↗
PubMed2007

Alpha-defensins block the early steps of HIV-1 infection: interference with the binding of gp120 to CD4.

Alpha-defensins are antibiotic peptides that act as natural inhibitors of HIV-1 infection. However, the mechanisms of such inhibition are still unclear. Here we demonstrate that alpha-defensins block the earliest steps in the viral infectious cycle, as documented using an HIV-1 envelope-mediated cell-fusion assay. A broad-spectrum inhibitory activity was observed on primary and laboratory-adapted HIV-1 isolates irrespective of their coreceptor specificity and genetic subtype. A primary mechanism of such inhibition was identified as the ability of alpha-defensins to bind specifically both to the primary HIV-1 cellular receptor, CD4, and to the viral envelope glycoprotein, gp120. Moreover, treatment of CD4+ T cells with alpha-defensins caused a dramatic downmodulation of CD4 expression. By monoclonal antibody competition, the regions of interaction with alpha-defensins were mapped to the D1 domain of CD4 and to a surface contiguous to the CD4- and coreceptor-binding sites of gp120. Consistent with these findings, alpha-defensins inhibited the binding of gp120 to CD4. These data demonstrate that alpha-defensins specifically block the initial phase of the HIV infectious cycle and modulate the expression of CD4, a critical receptor in the physiology of T-cell activation.

Binding Sites↗
PubMed2003

Duplication and selection in the evolution of primate beta-defensin genes.

BACKGROUND: Innate immunity is the first line of defense against microorganisms in vertebrates and acts by providing an initial barrier to microorganisms and triggering adaptive immune responses. Peptides such as beta-defensins are an important component of this defense, providing a broad spectrum of antimicrobial activity against bacteria, fungi, mycobacteria and several enveloped viruses. Beta-defensins are small cationic peptides that vary in their expression patterns and spectrum of pathogen specificity. Disruptions in beta-defensin function have been implicated in human diseases, including cystic fibrosis, and a fuller understanding of the variety, function and evolution of human beta-defensins might form the basis for novel therapies. Here we use a combination of laboratory and computational techniques to characterize the main human beta-defensin locus on chromosome 8p22-p23. RESULTS: In addition to known genes in the region we report the genomic structures and expression patterns of four novel human beta-defensin genes and a related pseudogene. These genes show an unusual pattern of evolution, with rapid divergence between second exon sequences that encode the mature beta-defensin peptides matched by relative stasis in first exons that encode signal peptides. CONCLUSIONS: We conclude that the 8p22-p23 locus has evolved by successive rounds of duplication followed by substantial divergence involving positive selection, to produce a diverse cluster of paralogous genes established before the human-baboon divergence more than 23 million years ago. Positive selection, disproportionately favoring alterations in the charge of amino-acid residues, is implicated as driving second exon divergence in these genes.

Amino Acid Sequence↗
PubMed1999

Defensin promotes the binding of lipoprotein(a) to vascular matrix.

Retention of lipoproteins within the vasculature is a central event in the pathogenesis of atherosclerosis. However, the signals that mediate this process are only partially understood. Prompted by putative links between inflammation and atherosclerosis, we previously reported that alpha-defensins released by neutrophils are present in human atherosclerotic lesions and promote the binding of lipoprotein(a) [Lp(a)] to vascular cells without a concomitant increase in degradation. We have now tested the hypothesis that this accumulation results from the propensity of defensin to form stable complexes with Lp(a) that divert the lipoprotein from its normal cellular degradative pathways to the extracellular matrix (ECM). In accord with this hypothesis, defensin stimulated the binding of Lp(a) to vascular matrices approximately 40-fold and binding of the reactants to the matrix was essentially irreversible. Defensin formed stable, multivalent complexes with Lp(a) and with its components, apoprotein (a) and low-density lipoprotein (LDL), as assessed by optical biosensor analysis, gel filtration, and immunoelectron microscopy. Binding of defensin/Lp(a) complexes to matrix was inhibited (>90%) by heparin and by antibodies to fibronectin (>70%), but not by antibodies to vitronectin or thrombospondin. Defensin increased the binding of Lp(a) (10 nmol/L) to purified fibronectin more than 30-fold. Whereas defensin and Lp(a) readily traversed the endothelial cell membranes individually, defensin/Lp(a) complexes lodged on the cell surface. These studies demonstrate that alpha-defensins released from activated or senescent neutrophils stimulate the binding of an atherogenic lipoprotein to the ECM of endothelial cells, a process that may contribute to lipoprotein accumulation in atherosclerotic lesions.

Apolipoproteins↗
PubMed2000

Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells.

Defensins, a family of cationic, structurally related, antimicrobial peptides, contribute to host defense by disrupting the cytoplasmic membrane of microbes. Here we show that human neutrophil defensins selectively induce the migration of human CD4+/CD45RA+ naive and CD8+, but not CD4+/CD45RO+ memory, T cells. Moreover, human neutrophil defensins are chemotactic for immature human dendritic cells derived from either CD34+ progenitors or peripheral blood monocytes. Upon maturation induced by treatment with tumor necrosis factor alpha (TNF-alpha), dendritic cells lose their responsiveness to human neutrophil defensins. The chemotactic effect of human neutrophil defensins on both T and dendritic cells is pertussis toxin-sensitive, suggesting that a G(ialpha) protein-coupled receptor is responsible. Human neutrophil defensins are also chemotactic for immature murine dendritic cells. These data suggest that, in addition to their antimicrobial role, human neutrophil defensins also contribute to adaptive immunity by mobilizing T cells and dendritic cells.

Adult↗
PubMedDate not supplied

Defensins: natural anti-HIV peptides.

Mammalian defensins are small cationic antimicrobial peptides predominantly found in leukocytes and epithelial cells engaged in host defense. These peptides act as effector molecules in innate immunity as well as regulators in adaptive immunity. Increasing evidence indicates that defensins are effective inhibitors of HIV-1. While the level of defensins in HIV-1 infected individuals has not been determined, neutropenia and neutrophil dysfunction associated with HIV disease progression may result in altered alpha-defensin production. This review provides an overview of the structure and function of defensins, and focuses on the anti-HIV-1 activity of defensins and the mechanism of this activity. Although many questions remain, studying the complex function of defensins in innate immunity against HIV has implications for our further understanding of disease progression and for the development of novel approaches to prevention and therapy.

Amino Acid Sequence↗
PubMed2005

[Cloning and prokaryotic expression of Anopheles sinensis defensin gene and preliminary bioactivity evaluation of the recombinant product].

OBJECTIVE: To clone the defensin gene of Anopheles sinensis for its prokaryotic expression and preliminary bioactivity evaluation of the recombinant protein. METHODS: Two pairs of primer were designed and synthesized based on the published defensin sequences, and PCR amplification was performed using the cDNA of An.sinensis as the template. The PCR products were cloned and sequenced. A truncated defensin fragment was cloned into pET-32(a) plasmid and expressed with the induction by IPTG. The expressed product was purified and its bioactivity evaluated with agarose diffusion assay. RESULTS: The PCR product 270 bp in size was identified as the coding sequence for defensin after sequence analysis, which had 85%; homology with the defensin sequence from An.gambiae. The truncated defensin (162 bp) cloned was subcloned into the expression plasmid pET-32(a) and the molecular weight of the target fusion protein was 26 000 after IPTG induction. Preliminary experiment, however, failed to demonstrate bacteriostatic effect of the purified recombinant protein. CONCLUSION: The coding sequence for defensin gene of An.sinensis has been successfully cloned and the truncated fraction can be highly expressed as a soluble fusion protein, which, however, does not possess bacteriostatic activity.

Amino Acid Sequence↗
PubMed1989

Purification and characterization of human neutrophil peptide 4, a novel member of the defensin family.

Primary (azurophil) granules of neutrophils contain proteins which play a major role in the killing and digestion of bacteria in the phagolysosome. We have isolated and characterized a novel antimicrobial peptide from the azurophil granule fraction of discontinuous Percoll gradients. We have named this peptide human neutrophil peptide 4 (HNP-4) based on its structural similarity to a group of antimicrobial polypeptides known as defensins (HNP 1-3). Using size exclusion and reverse-phase high performance liquid chromatography, HNP-4 was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequence analysis. The amino acid sequence determined from isolated HNP-4 and from tryptic fragments of reduced and alkylated peptide is: NH2-Val-Cys-Ser-Cys-Arg-Leu-Val-Phe-Cys-Arg-Arg-Thr-Glu- Leu-Arg-Val-Gly-Asn-Cys-Leu-Ile-Gly-Gly-Val-Ser-Phe-Thr-Tyr-Cys-Cys-Thr- Arg-Val - COOH. Based on this sequence, HNP-4 has a calculated molecular weight of 3715 and a theoretical pI of 8.61. HNP-4 shows structural similarity to the family of three human defensins. HNP-4 and the defensins have identical cysteine backbones and, like the defensins, HNP-4 is rich in arginine (15.2 mol %). However, the amino acids at 22 of the 33 positions differ between HNP-4 and human defensins. Further, HNP-4 is significantly more hydrophobic than the defensins, as determined by its retention time on reverse-phase high performance liquid chromatography. In vitro, purified HNP-4 was shown to kill Escherichia coli, Streptococcus faecalis, and Candida albicans. Compared to a mixture of the other human defensins, HNP-4 was found to be approximately 100 times more potent against E. coli and four times more potent against both S. faecalis and C. albicans.

Amino Acid Sequence↗
PubMed1996

Widespread expression of beta-defensin hBD-1 in human secretory glands and epithelial cells.

We compared the expression of human alpha- and beta-defensins by various human tissues. mRNA for alpha-defensins HNP1-3, abundant in bone marrow, was detected in peripheral blood leukocytes, spleen and thymus by RT-PCR, which revealed alpha-defensins HD5 and HD6 only in the small intestine. In contrast, the pancreas and kidney expressed high levels of hBD-1 and lower levels of this beta-defensin were found in many organs by RT-PCR (salivary gland > trachea > prostate and placenta > thymus, testis, small intestine). hBD-1 mRNA was produced constitutively by cultured normal human epithelial cells derived from the trachea, bronchi, small airways and the mammary gland. These largely non-overlapping tissue distributions of human alpha- and beta-defensins suggest that hBD-1 may be positioned to defend epithelial cells and mucosae from infection, whereas expression of HNP1-3 in neutrophils and HD5 and HD6 in Paneth cells allows these alpha-defensins to participate in systemic and small intestinal host defenses, respectively.

Amino Acid Sequence↗
PubMed1992

Enteric defensins: antibiotic peptide components of intestinal host defense.

Five intestinal defensins, termed cryptdins 1-5, have been purified from mouse small bowel, sequenced, and localized to the epithelium by immunohistochemistry. Although identified as members of the defensin peptide family by peptide sequencing, enteric defensins are novel in that four cryptdins have amino termini which are three to six residues longer than those of leukocyte-derived defensins. A fifth cryptdin is the first defensin to diverge from the previously invariant spacing of cysteines in the peptide structure. The most abundant enteric defensin, cryptdin-1, had antimicrobial activity against an attenuated phoP mutant of Salmonella typhimurium but was not active against the virulent wild-type parent. Immunohistochemical localization demonstrated that cryptdin-1, and probably cryptdins 2 and 3, occur exclusively in Paneth cells, where the peptides appear to be associated with cytoplasmic granules. Biochemical and immunologic analysis of the luminal contents of the small intestine suggest that cryptdin peptides are secreted into the lumen, similar to Paneth cell secretion of lysozyme. The presence of several enteric defensins in the intestinal epithelium, evidence of their presence in the lumen, and the antibacterial activity of cryptdin-1 suggest that these peptides contribute to the antimicrobial barrier function of the small bowel mucosa.

Amino Acid Sequence↗
PubMed1992

Cryptdins: antimicrobial defensins of the murine small intestine.

Paneth cells are specialized small intestine epithelial cells that contain lysozyme, possess phagocytic properties, and secrete cytoplasmic granules into the intestinal crypt lumen after the entry of bacteria. Recent studies by Ouellette and associates (A. J. Ouellette, R. M. Greco, M. James, D. Frederick, J. Naftilan, and J. T. Fallon, J. Cell Biol. 108:1687-1695, 1989) indicated that murine Paneth cells produce prodefensin mRNA, but the properties of its peptide product were not reported. We purified two closely related defensins, cryptdin 1 and cryptdin 2, from a subcellular fraction of murine small intestine cells that was enriched in Paneth cells. Both peptides contained 35 amino acid residues, including the characteristic defensin "signature" of six invariantly conserved cysteines. Cryptdins 1 and 2 were approximately 90 to 95% homologous to each other and to the carboxy-terminal domain of the 93-amino-acid defensin precursor, cryptdin A, described by Ouellette and associates (Ouellette et al., J. Cell Biol. 108:1687-1695, 1989). Both cryptdins exerted bactericidal activity against Listeria monocytogenes EGD and Escherichia coli ML-35p in vitro. Their potency exceeded that of human neutrophil defensin HNP-1 but was considerably lower than that of NP-1, a defensin produced by rabbit neutrophils and alveolar macrophages. Both cryptdins killed mouse-avirulent Salmonella typhimurium 7953S (phoP) much more effectively than its phoP+, mouse-virulent, isogenic counterpart, S. typhimurium 14028S. Our data indicate that mouse intestinal prodefensins are processed into 35-amino-acid mature defensins (cryptdins) with broad-spectrum antimicrobial properties. The production of defensins and lysozyme by Paneth cells may enable them to protect the small intestine from bacterial overgrowth by autochthonous flora and from invasion by potential pathogens that cause infection via the peroral route, such as L. monocytogenes and Salmonella species.

Amino Acid Sequence↗
PubMed2006

Lipopolysaccharide induced-in vivo increases in beta-defensins of the rat parotid gland.

Antimicrobial beta-defensins are thought to protect epithelial surfaces. Their mobilization in response to inflammation was studied in the rat parotid gland using an ELISA assay. Bacterial lipopolysaccharide (LPS), injected into the parotid duct on one side, induced a marked local inflammatory response in the parotid gland as judged by several fold increases in myeloperoxidase activity and, in histological sections, infiltration of neutrophils. Three hours after the injection, beta-defensin 1 and 3 were increased (by 41% and 15%, respectively, P<0.01) as compared to the contralateral gland. Though still elevated 6h after the injection, the percentage figures for beta-defensin 1 were, at this time, somewhat lower (30%) compared to the situation at 3h, while those for defensin 3 were significantly higher 65% (P<0.01); neither at the early nor at the late time of observation were any changes in the level of beta-defensin 2 observed. The beta-defensins under study were not detected in submandibular and sublingual glands, neither were they detected in the inflamed submandibular gland, showing also here several fold increases in myeloperoxidase activity and, in addition, the presence of inflammatory cells, following ductal injection of LPS towards the gland.

Animals↗
PubMed2001

Expression profile of human defensins and antimicrobial proteins in oral tissues.

Antimicrobial peptides and proteins are an important part of the innate host defense. In the present study, the expression profile of three human alpha-defensins, of two human beta-defensins (hBD) and of phospholipase A-2 (PLA-2) and lysozyme was determined by reverse transcription-polymerase chain reaction (RT-PCR) in 56 non-inflamed and 18 inflamed oral tissue samples and primary oral keratinocytes and fibroblasts. The transcripts for hBD-1 and -2 as well as for PLA-2 and lysozyme were found to be widely expressed. In the group of the alpha-defensins, the message for the human neutrophil peptide-1 (HNP-1) was frequently detected, whereas an expression of human Paneth's cell defensin-5 (HD-5) was identified in only a minority of samples. Transcripts for HD-6 were not detectable in any sample. Oral keratinocytes but not fibroblasts contained transcripts for the beta-defensins, suggesting that these defensins are produced in the epithelial compartment. In contrast, mRNA expression of neutrophil-derived HNP-1 and PLA-2 was not observed in any of these cells. These results suggest an important role for hBD-1 and hBD-2 in the innate oral epithelial host defense.

DNA, Complementary↗
PubMed2000

Engineered salt-insensitive alpha-defensins with end-to-end circularized structures.

We designed a retro-isomer and seven circularized "beta-tile" peptide analogs of a typical rabbit alpha-defensin, NP-1. The analogs retained defensin-like architecture after the characteristic end-to-end, Cys(3,31) (C I:C VI), alpha-defensin disulfide bond was replaced by a backbone peptide bond. The retro-isomer of NP-1 was as active as the parent compound, suggesting that overall topology and amphipathicity governed its antimicrobial activity. A beta-tile design with or without a single cross-bracing disulfide bond sufficed for antimicrobial activity, and some of the analogs retained activity against Escherichia coli and Salmonella typhimurium in NaCl concentrations that rendered NP-1 inactive. The new molecules had clustered positive charges resembling those in protegrins and tachyplesins, but were less cytotoxic. Such simplified alpha-defensin analogs minimize problems encountered during the oxidative folding of three-disulfide defensins. In addition, they are readily accessible to a novel thia zip cyclization procedure applicable to large unprotected peptide precursors of 31 amino acids in aqueous solutions. Collectively, these findings provide new and improved methodology to create salt-insensitive defensin-like peptides for application against bacterial diseases.

Amino Acid Sequence↗
PubMed2005

Oesophageal defensin expression during Candida infection and reflux disease.

OBJECTIVE: Alpha (alpha-) and beta (beta-) defensins are major constituents of the innate defence system, providing rapid antimicrobial action. The expression of alpha- and beta-defensins in the oesophagus and stomach by quantitative, real-time, polymerase chain reaction (PCR) in healthy individuals was studied to define the influence of oesophageal Candida infection on their expression in comparison to oesophageal reflux disease. MATERIAL AND METHODS: Biopsy samples were taken from the upper gastrointestinal tract, mRNA was extracted, reverse transcribed into cDNA and real-time reverse transcription PCR (RT-PCR) analysis measuring transcript number of a-defensins and ss-defensins performed. Standard curves allowed quantification of gene copies per weight of mRNA. RESULTS: hBD-1, hBD-2 and hBD-3 had their highest expression levels in the oesophagus with factor 3 to 5 lower in the stomach. Candida oesophagitis resulted in massive up-regulation of hBD-2 (800-fold), while hBD-1 and hBD-3 expression were slightly increased. In addition, expression of HNP 1-3 was detected, indicating infiltration of neutrophil granulocytes. In reflux disease, an up-regulation of hBD-2 (20-fold) and hBD-3 (50-fold) could also be observed, while hBD-1, hBD-4 and HD5 remained unaffected. Cytokine expression of interleukin-1beta, interleukin-6 and interleukin-8 were increased in both groups, while interleukin-10 expression was elevated only in reflux lesions. CONCLUSIONS: Candida colonization induced a high expression of antimicrobial peptides. In oesophageal reflux disease, induction of defensin expression could also be observed but to a lower degree. Therefore, up-regulation of defensins might protect against invasive candidiasis and keep the Candida infection limited to the mucosal surface.

Adult↗