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The structural similarity between defensins and scorpion neurotoxins suggests that they might have evolved from a common ancestor. However, there is no direct experimental evidence demonstrating a functional link between scorpion neurotoxins and defensins. The scorpion defensin BmKDfsin4 from Mesobuthus martensii Karsch contains 37 amino acid residues and a conserved cystine-stabilized α/β structural fold. The recombinant BmKDfsin4, a classical defensin, has been found to have inhibitory activity against Gram-positive bacteria such as Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteus as well as methicillin-resistant Staphylococcus aureus. Interestingly, electrophysiological experiments showed that BmKDfsin4,like scorpion potassium channel neurotoxins, could effectively inhibit Kv1.1, Kv1.2, and Kv1.3 channel currents, and its IC50 value for the Kv1.3 channel was 510.2 nm. Similar to the structure-function relationships of classical scorpion potassium channel-blocking toxins, basic residues (Lys-13 and Arg-19) of BmKDfsin4 play critical roles in peptide-Kv1.3 channel interactions. Furthermore, mutagenesis and electrophysiological experiments demonstrated that the channel extracellular pore region is the binding site of BmKDfsin4, indicating that BmKDfsin4adopts the same mechanism for blocking potassium channel currents as classical scorpion toxins. Taken together, our work identifies scorpion BmKDfsin4 as the first invertebrate defensin to block potassium channels. These findings not only demonstrate that defensins from invertebrate animals are a novel type of potassium channel blockers but also provide evidence of a functional link between defensins and neurotoxins.

Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche. This repertoire represents a rich and inexhaustible source for antibiotics and fungicides. Antimicrobial peptides are an emerging class of fungal defense molecules that are promising candidates for pharmaceutical applications. Based on a co-cultivation system, we studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly produced in Pichia pastoris, and the three-dimensional structure was solved by NMR. The cysteine stabilized α/β-fold with a unique disulfide connectivity, and an N-terminal pyroglutamate rendered copsin extremely stable against high temperatures and protease digestion. Copsin was bactericidal against a diversity of Gram-positive bacteria, including human pathogens such as Enterococcus faecium and Listeria monocytogenes. Characterization of the antibacterial activity revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore interfered with the cell wall biosynthesis. In particular, and unlike lantibiotics and other defensins, the third position of the lipid II pentapeptide is essential for effective copsin binding. The unique structural properties of copsin make it a possible scaffold for new antibiotics.

Defensins are components of the innate immune system that promote the directional migration and activation of dendritic cells, thereby modulating the adaptive immune response. Because matrix glycosaminoglycan (GAG) is known to be important for these functions, we characterized the structural features of human β-defensin 6 (hBD6) and GAG interaction using a combination of structural and in silico analyses. Our results showed that GAG model compounds, a pentasaccharide (fondaparinux, FX) and an octasaccharide heparin derivative (dp8) bind to the α-helix and in the loops between the β2 and β3 strands, inducing the formation of a ternary complex with a 2:1 hBD6:FX stoichiometry. Competition experiments indicated an overlap of GAG and chemokine receptor CCR2 binding sites. An NMR-derived model of the ternary complex revealed that FX interacts with hBD6 along the dimerization interface, primarily contacting the α-helices and β2-β3 loops from each monomer. We further demonstrated that high-pressure NMR spectroscopy could capture an intermediate stage of hBD6-FX interaction, exhibiting features of a cooperative binding mechanism. Collectively, these data suggest a “sandwich-like” model in which two hBD6 molecules bind a single FX chain and provide novel structural insights into how defensin orchestrates leukocyte recruitment through GAG binding and G protein-coupled receptor activation. Despite the similarity to chemokines and hBD2, our data indicate different properties for the hBD6-GAG complex. This work adds significant information to the currently limited data available for the molecular structures and dynamics of defensin carbohydrate binding.

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

Antimicrobial peptides are a new class of antibiotics that are promising for pharmaceutical applications because they have retained efficacy throughout evolution. One class of antimicrobial peptides are the defensins, which have been found in different species. Here we describe a new fungal defensin, eurocin. Eurocin acts against a range of Gram-positive human pathogens but not against Gram-negative bacteria. Eurocin consists of 42 amino acids, forming a cysteine-stabilized α/β-fold. The thermal denaturation data point shows the disulfide bridges being responsible for the stability of the fold. Eurocin does not form pores in cell membranes at physiologically relevant concentrations; it does, however, lead to limited leakage of a fluorophore from small unilamellar vesicles. Eurocin interacts with detergent micelles, and it inhibits the synthesis of cell walls by binding equimolarly to the cell wall precursor lipid II.

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

Human β-defensins (hBDs) are small, cationic antimicrobial peptides, secreted by mucosal epithelial cells that regulate adaptive immune functions. We previously reported that Fusobacterium nucleatum, a ubiquitous Gram-negative bacterium of the human oral cavity, induces human β-defensin 2 (hBD2) upon contact with primary oral epithelial cells. We now report the isolation and characterization of an F. nucleatum (ATCC 25586)-associated defensin inducer (FAD-I). Biochemical approaches revealed a cell wall fraction containing four proteins that stimulated the production of hBD2 in human oral epithelial cells (HOECs). Cross-referencing of the N-terminal sequences of these proteins with the F. nucleatum genome revealed that the genes encoding the proteins were FadA, FN1527, FN1529, and FN1792. Quantitative PCR of HOEC monolayers challenged with Escherichia coli clones expressing the respective cell wall proteins revealed that FN1527 was most active in the induction of hBD2 and hence was termed FAD-I. We tagged FN1527 with a c-myc epitope on the C-terminal end to identify and purify it from the E. coli clone. Purified FN1527 (FAD-I) induced hBD2 mRNA and protein expression in HOEC monolayers. F. nucleatum cell wall and FAD-I induced hBD2 via TLR2. Porphorymonas gingivalis, an oral pathogen that does not induce hBD2 in HOECs, was able to significantly induce expression of hBD2 in HOECs only when transformed to express FAD-I. FAD-I or its derivates offer a potentially new paradigm in immunoregulatory therapeutics because they may one day be used to bolster the innate defenses of vulnerable mucosae.

Three oyster defensin variants (Cg-Defh1, Cg-Defh2, and Cg-Defm) were produced as recombinant peptides and characterized in terms of activities and mechanism of action. In agreement with their spectrum of activity almost specifically directed against Gram-positive bacteria, oyster defensins were shown here to be specific inhibitors of a bacterial biosynthesis pathway rather than mere membrane-active agents. Indeed, at lethal concentrations, the three defensins did not compromise Staphylococcus aureus membrane integrity but inhibited the cell wall biosynthesis as indicated by the accumulation of the UDP-N-acetylmuramyl-pentapeptide cell wall precursor. In addition, a combination of antagonization assays, thin layer chromatography, and surface plasmon resonance measurements showed that oyster defensins bind almost irreversibly to the lipid II peptidoglycan precursor, thereby inhibiting the cell wall biosynthesis. To our knowledge, this is the first detailed analysis of the mechanism of action of antibacterial defensins produced by invertebrates. Interestingly, the three defensins, which were chosen as representative of the oyster defensin molecular diversity, bound differentially to lipid II. This correlated with their differential antibacterial activities. From our experimental data and the analysis of oyster defensin sequence diversity, we propose that oyster defensin activity results from selective forces that have conserved residues involved in lipid II binding and diversified residues at the surface of oyster defensins that could improve electrostatic interactions with the bacterial membranes.

We performed a comprehensive alanine scan of human alpha-defensin HNP1 and tested the ability of the resulting analogs to kill Staphylococcus aureus, inhibit anthrax lethal factor, and bind human immunodeficiency virus-1 gp120. By far, the most deleterious mutation for all of these functions was W26A. The activities lost by W26A-HNP1 were restored progressively by replacing W26 with non-coded, straight-chain aliphatic amino acids of increasing chain length. The hydrophobicity of residue 26 also correlated with the ability of the analogs to bind immobilized wild type HNP1 and to undergo further self-association. Thus, the hydrophobicity of residue 26 is not only a key determinant of the direct interactions of HNP1 with target molecules, but it also governs the ability of this peptide to form dimers and more complex quaternary structures at micromolar concentrations. Although all defensin peptides are cationic, their amphipathicity is at least as important as their positive charge in enabling them to participate in innate host defense.

Proteolytic processing of defensins is a critical mode of posttranslational regulation of peptide activity. Because mouse alpha-defensin precursors are cleaved and activated by matrix metalloproteinase-7 (MMP-7), we determined if additional defensin molecules, namely human neutrophil defensin pro-HNP-1 and beta-defensins, are targets for MMP-7. We found that MMP-7 cleaves within the pro-domain of the HNP-1 precursor, a reaction that does not generate the mature peptide but produces a 59-amino acid intermediate. This intermediate, which retains the carboxyl-terminal end of the pro-domain, had antimicrobial activity, indicating that the residues important for masking defensin activity reside in the amino terminus of this domain. Mature HNP-1 was resistant to processing by MMP-7 unless the peptide was reduced and alkylated, demonstrating that only the pro-domain of alpha-defensins is normally accessible for cleavage by this enzyme. From the 47-residue HBD-1 precursor, MMP-7 catalyzed removal of 6 amino acids from the amino terminus. Neither a 39-residue intermediate form of HBD-1 nor the mature 36-residue form of HBD-1 was cleaved by MMP-7. In addition, both pro-HBD-2, with its shorter amino-terminal extension, and pro-HBD-3 were resistant to MMP-7. However, human and mouse beta-defensin precursors that lack disulfide bonding contain a cryptic MMP-7-sensitive site within the mature peptide moiety. These findings support and extend accumulating evidence that the native three-dimensional structure of both alpha- and beta-defensins protects the mature peptides against proteolytic processing by MMP-7. We also conclude that sites for MMP-7 cleavage are more common at the amino termini of alpha-defensin rather than beta-defensin precursors, and that catalysis at these sites in alpha-defensin pro-domains results in acquisition of defensin activity.

Mammalian defensins are cationic antimicrobial peptides that play a central role in host innate immunity and as regulators of acquired immunity. In animals, three structural defensin subfamilies, designated as alpha, beta, and , have been characterized, each possessing a distinctive tridisulfide motif. Mature alpha- and beta-defensins are produced by simple proteolytic processing of their prepropeptide precursors. In contrast, the macrocyclic -defensins are formed by the head-to-tail splicing of nonapeptides excised from a pair of prepropeptide precursors. Thus, elucidation of the -defensin biosynthetic pathway provides an opportunity to identify novel factors involved in this unique process. We incorporated the -defensin precursor, proRTD1a, into a bait construct for a yeast two-hybrid screen that identified rhesus macaque stromal cell-derived factor 2-like protein 1 (SDF2L1), as an interactor. SDF2L1 is a component of the endoplasmic reticulum (ER) chaperone complex, which we found to also interact with alpha- and beta-defensins. However, analysis of the SDF2L1 domain requirements for binding of representative alpha-, beta-, and -defensins revealed that alpha- and beta-defensins bind SDF2L1 similarly, but differently from the interactions that mediate binding of SDF2L1 to pro--defensins. Thus, SDF2L1 is a factor involved in processing and/or sorting of all three defensin subfamilies.

Hydroxyproline-rich glycopeptides (HypSys peptides) are recently discovered 16-20-amino acid defense signals in tobacco and tomato leaves that are derived from cell wall-associated precursors. The peptides are powerful wound signals that activate the expression of defensive genes in tobacco and tomato leaves in response to herbivore attacks. We have isolated a cDNA from petunia (Petunia hybrida) leaves encoding a putative protein of 214 amino acids that is a homolog of tobacco and tomato HypSys peptide precursors and is inducible by wounding and MeJA. The deduced protein contains a leader sequence and four predicted proline-rich peptides of 18-21 amino acids. Three of the four peptides were isolated from leaves, and each peptide contained hydroxylated prolines and glycosyl residues. Each of the peptides has a -GR- motif at its N terminus, indicating that it may be the substrate site for a processing enzyme. The peptides were active in a petunia suspension culture bioassay at nanomolar concentrations, but they did not induce the expression of defense genes that are directed against herbivores, as found in tobacco and tomato leaves. They did, however, activate expression of defensin 1, a gene associated with inducible defense responses against pathogens.

Defensins are small (30-45 amino acid residues) cationic proteins with broad antimicrobial activity against many bacteria and fungi, some enveloped viruses, and other activities such as chemoattraction of a range of different cell types to the sites of inflammation. These proteins represent attractive targets for developing novel antimicrobial agents and modulators of immune responses with therapeutic applicability. In this report, we present the results of functional and structural studies of 26 single-site mutants of human beta-defensin 1 (hBD1). All mutants were assayed for antimicrobial activity against Escherichia coli (ATCC strain 25922) and for chemotactic activity with CCR6-transfected HEK293 cells. To analyze the structural implications of mutagenesis and to verify the correctness of the disulfide connectivity, we used x-ray crystallography to conduct complete structural studies for 10 mutants in which the topology of disulfides was the same as in the native hBD1. Mutations did not induce significant changes of the tertiary structure, suggesting that the observed alterations of biological properties of the mutants were solely associated with changes in the respective side chains. We found that cationic residues located near the C terminus (Arg(29), Lys(31), Lys(33), and Lys(36)) of hBD1 define most of the anti-E. coli in vitro activity of this protein. In turn, nearly all mutations altering the CCR6-mediated chemotaxis are located at one area of the protein, defined by the N-terminal alpha-helical region (Asp(1)... Ser(8)) and a few topologically adjacent residues (Lys(22), Arg(29), and Lys(33)). These experimental results allow for the first time drafting of the CCR6-epitope for a defensin molecule.

Defensins are cationic antimicrobial mini-proteins that play important roles in the innate immune defense against microbial infection. Six invariant Cys residues in each defensin form three structurally indispensable intramolecular disulfide bridges. The only other residue invariant in all known mammalian defensins is a Gly. Structural studies indicate that the invariant Gly residue is located in an atypical, classic-type beta-bulge with the backbone torsion angles (Phi, Psi) disallowed for L-amino acids but permissible for D-enantiomers. We replaced the invariant Gly17 residue in human neutrophil alpha-defensin 2 (HNP2) by L-Ala or one of the D-amino acids Ala, Glu, Phe, Arg, Thr, Val, or Tyr. Although L-Ala17-HNP2 could not be folded, resulting in massive aggregation, all of the D-amino acid-substituted analogs folded with high efficiency. The high resolution x-ray crystal structures of dimeric D-Ala17-HNP2 were determined in three different crystal forms, showing a well preserved beta-bulge identical to those found in other defensins. The seven D-analogs of HNP2 exhibited highly variable bactericidal activity against Gram-positive and Gram-negative test strains, consistent with the premise that interplay between charge and hydrophobicity dictates how amphiphilic defensins kill. Further, the bactericidal activity of these d-amino acid analogs of HNP2 correlated well with their ability to induce leakage from large unilamellar vesicles, supporting membrane permeabilization as the lethal event in microbial killing by HNP2. Our findings identify a conformational prerequisite in the beta-bulge of defensins essential for correct folding and native structure, thereby explaining the molecular basis of the Gly-Xaa-Cys motif conserved in all mammalian defensins.

Defensins are cationic antimicrobial peptides that have a characteristic six-cysteine motif and are important components of the innate immune system. We recently described a beta-defensin-related peptide (Defr1) that had potent antimicrobial activity despite having only five cysteines. Here we report a relationship between the structure and activity of Defr1 through a comparative study with its six cysteine-containing analogue (Defr1 Y5C). Against a panel of pathogens, we found that oxidized Defr1 had significantly higher activity than its reduced form and the oxidized and reduced forms of Defr1 Y5C. Furthermore, Defr1 displayed activity against Pseudomonas aeruginosa in the presence of 150 mm NaCl, whereas Defr1 Y5C was inactive. By using nondenaturing gel electrophoresis and Fourier transform ion cyclotron resonance mass spectrometry, we observed Defr1 and Defr1 Y5C dimers. Two complementary fragmentation techniques (collision-induced dissociation and electron capture dissociation) revealed that Defr1 Y5C dimers form by noncovalent, weak association of monomers that contain three intramolecular disulfide bonds. In contrast, Defr1 dimers are resistant to collision-induced dissociation and are only dissociated into monomers by reduction using electron capture. This is indicative of Defr1 dimerization being mediated by an intermolecular disulfide bond. Proteolysis and peptide mass mapping revealed that Defr1 Y5C monomers have beta-defensin disulfide bond connectivity, whereas oxidized Defr1 is a complex mixture of dimeric isoforms with as yet unknown inter- and intramolecular connectivities. Each isoform contains one intermolecular and four intramolecular disulfide bonds, but because we were unable to resolve the isoforms by reverse phase chromatography, we could not assign each isoform with a specific antimicrobial activity. We conclude that the enhanced activity and stability of this mixture of Defr1 dimeric isoforms are due to the presence of an intermolecular disulfide bond. This first description of a covalently cross-linked member of the defensin family provides further evidence that the antimicrobial activity of a defensin is linked to its ability to form stable higher order structures.

Defensins are important elements in innate immunity that can also trigger adaptive immune responses. The defensins form a family of small cationic antimicrobial peptides with six characteristic cysteine residues, whose pairing pattern in forming three intramolecular disulfide bonds defines the alpha- and beta-defensin subfamilies. In a search for new beta-defensin genes, we performed computational analysis using the Celera mouse genome data base and found exons encoding 23 different beta-defensins, including the eight previously characterized members of this family. Among the new beta-defensins, nine of them form two groups of phylogenetically related sequences that were characterized in greater detail. Northern blot, reverse transcription PCR, and in situ hybridization analysis showed that expression of these genes is restricted to the epididymis, with a specific regional expression pattern. One of the new beta-defensins (Defb38) was chemically synthesized; in in vitro assays on Gram-positive and -negative bacterial strains, Defb38 showed the characteristic salt-dependent antimicrobial activity of beta-defensins. The results demonstrate the existence of a relatively large number of beta-defensins with specific expression in distinct regions of the murine epididymis and suggest complex roles for these proteins in host defense and other physiological processes of the male reproductive tract.

Defensins are a class of small cationic peptides found in higher organisms that serve as both antimicrobial and cell signaling molecules. The exact mechanism of the antimicrobial activity of defensins is not known, but two models have been postulated, one involving pore formation and the other involving nonspecific electrostatic interaction with the bacterial membrane. Here we report the high resolution structures of human β-defensin-1 (hBD1) in two crystallographic space groups. The structure of a single molecule is very similar to that of human β-defensin-2 (hBD2), confirming the presence of an N-terminal α-helix. However, while the packing of hBD1 is conserved across both space groups, there is no evidence for any larger quaternary structure similar to octameric hBD2. Furthermore, the topology of hBD1 dimers that are formed between monomers in the asymmetric unit is distinct from both hBD2 and other mammalian α-defensins. The structures of hBD1 and hBD2 provide a first step toward understanding the structural basis of antimicrobial and chemotactic properties of human β-defensins.

The growing public health problem of infections caused by multiresistant Gram-positive bacteria, in particular Staphylococcus aureus, prompted us to screen human epithelia for endogenous S. aureus-killing factors. A novel 5-kDa, nonhemolytic antimicrobial peptide (human beta-defensin-3, hBD-3) was isolated from human lesional psoriatic scales and cloned from keratinocytes. hBD-3 demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes including multiresistant S. aureus and vancomycin-resistant Enterococcus faecium. Ultrastructural analyses of hBD-3-treated S. aureus revealed signs of cell wall perforation. Recombinant hBD-3 (expressed as a His-Tag-fusion protein in Escherichia coli) and chemically synthesized hBD-3 were indistinguishable from naturally occurring peptide with respect to their antimicrobial activity and biochemical properties. Investigation of different tissues revealed skin and tonsils to be major hBD-3 mRNA-expressing tissues. Molecular cloning and biochemical analyses of antimicrobial peptides in cell culture supernatants revealed keratinocytes and airway epithelial cells as cellular sources of hBD-3. Tumor necrosis factor alpha and contact with bacteria were found to induce hBD-3 mRNA expression. hBD-3 therefore might be important in the innate epithelial defense of infections by various microorganisms seen in skin and lung, such as cystic fibrosis.

The use of germ-free mice offers the possibility to study antibacterial components in a gut uncolonized by bacteria. We have developed a method to extract and high pressure liquid chromatography-fractionate the antibacterial factors present in the small intestine of a single mouse. By mass spectrometry and sequence analyses of fractions exhibiting antimicrobial activity, we identified and characterized the defensin region in germ-free mice as well as in colonized mice. Defensins made up around 15% of the total antibacterial activity both in germ-free and colonized mice. The intestine of germ-free mice exhibited the same set of mature enteric defensins (defensins 1, 2, 3, 4, and 6) as mice colonized by a normal microflora. Mature defensins are generated through processing of larger precursors by enzymatic removal of a signal peptide and a propiece. We found that all prodefensins were cleaved at a Ser/Ala-Leu bond, giving 34-residue propiece peptides and only trace amounts of the predicted 39-residue peptide. This first step must be followed by the removal of a residual peptide to render the mature defensins, indicating that the processing is more complex than previously anticipated. The same propieces were found in both germ-free and colonized mice, suggesting that the same processing operates independent of bacterial presence in the intestine.

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.

NF-kappaB/Rel family proteins regulate genes that are critical for many cellular processes including apoptosis, inflammation, immune response, and development. NF-kappaB/Rel proteins function as homodimers or heterodimers, which recognize specific DNA sequences within target promoters. We examined the activity of different Drosophila Rel-related proteins in modulating Drosophila immunity genes by expressing the Rel proteins in stably transfected cell lines. We also compared how different combinations of these transcriptional regulators control the activity of various immunity genes. The results show that Rel proteins are directly involved in regulating the Drosophila antimicrobial response. Furthermore, the drosomycin and defensin expression is best induced by the Relish/Dif and the Relish/Dorsal heterodimers, respectively, whereas the attacin activity can be efficiently up-regulated by the Relish homodimer and heterodimers. These results illustrate how the formation of Rel protein dimers differentially regulate target gene expression.

Cell lines from the malaria vector Anopheles gambiae have been established as a tool for the study of the mosquito innate immune system in vitro. Here, we describe the first continuous insect cell line that produces prophenoloxidase (PPO). This cell line (4a-3B) expresses constitutively six PPO genes, three of which are novel (PPO4, PPO5, and PPO6). The PPO genes show distinct temporal expression profiles in the intact mosquito, spanning stages from the embryo to the adult in an overlapping manner. Transient induction of larva-specific PPO genes in blood-fed adult females suggests that the developmental hormone 20-hydroxyecdysone may be involved in PPO gene regulation. Indeed, exposure of 4a-3B cells to 20-hydroxyecdysone in culture results in induction of those PPO genes that are mainly expressed in early developmental stages, and repression of PPO5, which is preferentially expressed at the adult stage. The cell line shows bacteria-induced immune transcripts that encode defensin and Gram-negative bacteria-binding protein, but no induction of PPO transcripts. This cell line most likely derives from a hemocyte lineage, and represents an appropriate in vitro model for the study of the humoral and cellular immune defenses of A. gambiae.

Lepidoptera have been reported to produce several antibacterial peptides in response to septic injury. However, in marked contrast to other insect groups, no inducible antifungal molecules had been described so far in this insect order. Surprisingly, also cysteine-rich antimicrobial peptides, which predominate in the antimicrobial defense of other insects, had not been discovered in Lepidoptera. Here we report the isolation from the hemolymph of immune induced larvae of the lepidopteran Heliothis virescensof a cysteine-rich molecule with exclusive antifungal activity. We have fully characterized this antifungal molecule, which has significant homology with the insect defensins, a large family of antibacterial peptides directed against Gram-positive strains. Interestingly, the novel peptide shows also similarities with the antifungal peptide drosomycin from Drosophila. Thus, Lepidoptera appear to have built their humoral immune response against bacteria on cecropins and attacins. In addition, we report that Lepidoptera have conferred antifungal properties to the well conserved structure of antibacterial insect defensins through amino acid replacements.

Positively charged antimicrobial peptides with membrane-damaging activity are produced by animals and humans as components of their innate immunity against bacterial infections and also by many bacteria to inhibit competing microorganisms. Staphylococcus aureus and Staphylococcus xylosus, which tolerate high concentrations of several antimicrobial peptides, were mutagenized to identify genes responsible for this insensitivity. Several mutants with increased sensitivity were obtained, which exhibited an altered structure of teichoic acids, major components of the Gram-positive cell wall. The mutant teichoic acids lacked D-alanine, as a result of which the cells carried an increased negative surface charge. The mutant cells bound fewer anionic, but more positively charged proteins. They were sensitive to human defensin HNP1-3, animal-derived protegrins, tachyplesins, and magainin II, and to the bacteria-derived peptides gallidermin and nisin. The mutated genes shared sequence similarity with the dlt genes involved in the transfer of D-alanine into teichoic acids from other Gram-positive bacteria. Wild-type strains bearing additional copies of the dlt operon produced teichoic acids with higher amounts of D-alanine esters, bound cationic proteins less effectively and were less sensitive to antimicrobial peptides. We propose a role of the D-alanine-esterified teichoic acids which occur in many pathogenic bacteria in the protection against human and animal defense systems.

The human HNP-defensin-1 gene encodes a peptide antibiotic found exclusively in neutrophils and is key to elimination of microbes. Expression is a marker for the granulocytic lineage and for certain stages of differentiation and is not known to be inducible in mature cells under physiological conditions. Low level of transcription also occurs in HL-60 promyelocytic leukemia cells and is greatly activated upon drug-induced granulocytic maturation and by low doses of retinoic acid, in a strictly cell-specific manner (Herwig, S., Su, Q., Ma, Y., and Tempst, P. (1996) Blood 87, 350-364). We have analyzed a 10-kilobase pair region, upstream of the defensin-1 cap site, for the presence of control elements, and we describe a minimal promoter (position -83 to +82) required to drive transcription in HL-60 cells in a quasi cell-specific manner. Our data also suggest the presence of negative regulatory elements in the -416/-191 region that may further contribute to cell specificity in a chromosomal context. The basal promoter contains two functionally essential, ETS-like (GGAA core sequence) elements. The proximal site (-22/-19) constitutively binds the PU.1 transcription factor in vitro and could function, together perhaps with an adjacent TA-rich sequence (-32/-25), in assembly of a myeloid-restricted, basal transcription factor complex. The distal site (-62/-59) interacts in vitro with an unidentified activity, distinct from PU.1, ETS-1, PEA3, and ELK-1 (factors with definite binding site similarities), and is greatly stimulated by phosphorylation during granulocytic differentiation of HL-60 cells. Identification of this protein will be important to resolve the molecular mechanisms controlling temporal, granulocytic restricted gene expression.

We have examined the interactions of the six known rabbit neutrophil defensin antimicrobial peptides with large unilamellar vesicles (LUV) made from various lipid mixtures based on the lipid composition of Escherichia coli membranes. We find that the permeabilization of LUV made from E. coli whole lipid extracts differs dramatically from that of single-component LUV made from palmitoyl-oleoyl-phosphatidylglycerol (POPG). Specifically, defensins NP-1, NP-2, NP-3A, NP-3B, and a natural mixture of the six defensins cause fast nonpreferential leakage of high molecular weight dextrans as well as the low molecular weight fluorophore/quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX) from E. coli whole lipid LUV through large, transient membrane lesions. In contrast, release of ANTS/DPX from POPG LUV induced by the defensins is slow and graded with preference for DPX (Hristova, K., Selsted, M. E., and White, S. H. (1996) Biochemistry 35, 11888-11894). Interestingly, defensins NP-4 and NP-5 alone do not induce leakage from E. coli whole lipid LUV, whereas only NP-4 is ineffective with POPG LUV. Examination of the sequences of the six defensins suggests that the inactivity of NP-4 and NP-5 may be due to their lower net positive charge and/or the substitution of a Thr for the Arg or Lys that follows the fourth Cys residue. We found the presence of three major lipid components of E. coli whole lipid to be essential for creation of the large lesions observed in LUV: phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Cardiolipin appears to play a key role because no leakage can be induced when only phosphatidylglycerol and phosphatidylethanolamine are present. These results indicate the importance of membrane lipid composition in the permeabilization of cell membranes by rabbit defensins.

We have isolated from the blood of immune-challenged and untreated mussels (Mytilus edulis) antibacterial and antifungal peptides. We have characterized two isoforms of a novel 34-residue, cysteine-rich, peptide with potent bactericidal activity and partially characterized a novel 6.2-kDa antifungal peptide containing 12 cysteines. We report the presence of two members of the insect defensin family of antibacterial peptides and provide a phylogenetic analysis that indicates that mollusc and arthropod defensins have a common ancestry. Our data argue that circulating antimicrobial peptides represent an ancient host defense mechanism that predated the separation between molluscs and arthropods at the root of the Cambrian, about 545 million years ago.

Paneth cells, secretory epithelial cells of the small intestinal crypts, are proposed to contribute to local host defense. Both mouse and human Paneth cells express a collection of antimicrobial proteins, including members of a family of antimicrobial peptides named defensins. In this study, data from an anchored polymerase chain reaction (PCR) strategy suggest that only two defensin mRNA isoforms are expressed in the human small intestine, far fewer than the number expressed in the mouse. The two isoforms detected by this PCR approach were human defensin family members, HD-5 and HD-6. The gene encoding HD-6 was cloned and characterized. HD-6 has a genomic organization similar to HD-5, and the two genes have a striking pattern of sequence similarity localized chiefly in their proximal 5′-flanking regions. Analysis of human fetal RNA by reverse transcriptase-PCR detected enteric defensin HD-5 mRNA at 13.5 weeks of gestation in the small intestine and the colon, but by 17 weeks HD-5 was restricted to the small intestine. HD-6 mRNA was detectable at 13.5-17 weeks of gestation in the small intestine but not in the colon. This pattern of expression coincides with the previously described appearance of Paneth cells as determined by ultrastructural approaches. Northern analysis of total RNA from small intestine revealed quantifiable enteric defensin mRNA in five samples from 19-24 weeks of gestation at levels approximately 40-250-fold less than those observed in the adult, with HD-5 mRNA levels greater than those of HD-6 in all samples. In situ hybridization analysis localized expression of enteric defensin mRNA to Paneth cells at 24 weeks of gestation, as is seen in the newborn term infant and the adult. Consistent with earlier morphological studies, the ratio of Paneth cell number per crypt was reduced in samples at 24 weeks of gestation compared with the adult, and this lower cell number partially accounts for the lower defensin mRNA levels as determined by Northern analysis. Low levels of enteric defensin expression in the fetus may be characteristic of an immaturity of local defense, which is thought to predispose infants born prematurely to infection from intestinal microorganisms.

It is unclear whether the six known human defensin peptides are all encoded by separate genes or whether some of them are allelic. Three of the peptides, HP-1, HP-2, and HP-3, differ by only one amino acid, and it is thought that HP-2 may represent a proteolytic product of HP-1 and/or HP-3. To help determine the relationship of these three proteins, we isolated a nearly full-length cDNA encoding HP-1 with a sequence very similar to, but different from, the previously isolated HP-1 and -3 cDNAs. Gene copy number experiments established that there were at least two but fewer than five defensin genes with a high level of similarity to the HP-1 cDNA (HP-1/3-like). Three genomic clones were isolated that contained two different configurations of the HP-1/3-like sequences. Sequencing established that one encoded the HP-1 peptide, whereas the other encoded HP-3. Analysis of DNAs obtained from 18 unrelated individuals by Southern blot analysis revealed the expected fragments as well as additional fragments that were not present in the genomic clones. This suggested the possibility of alleles; however, when DNAs from families were examined, these fragments did not segregate in an obvious Mendelian fashion. The HP-1/3-like defensin genes are on human chromosome 8. Surprisingly, somatic cell hybrid mapping showed that the number of HP-1/3-like genes on isolated copies of chromosome 8 was variable. We conclude that individuals can inherit versions of chromosome 8 harboring either two or three copies of the genes that encode the HP-1, HP-2, and/or HP-3 peptides.

An inhibitor of tissue-type plasminogen activator (tPA)-mediated and plasminogen-dependent fibrinolysis was isolated from human neutrophils. On a G-50 gel filtration column, the antifibrinolytic activity present in neutrophil homogenates comigrated with proteins of < 13 kDa. The inhibitory fraction had only a slight effect on urokinase with plasminogen- or plasmin-mediated fibrinolysis and no effect on urokinase- or plasmin-mediated cleavage of H-D-valyl-L-leucyl-L-lysine-p-nitroanilide (S-2251). The neutrophil-derived fraction inhibited tPA with plasminogen activity on S-2251 but not on H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288). The inhibition of tPA-mediated and plasminogen-dependent fibrinolysis or S-2251 cleavage showed a competitive pattern and could be relieved by increasing the concentration of plasminogen. The same fraction also inhibited binding of plasminogen to fibrin. Consecutive purification steps revealed that the molecular mass of the inhibitor was 1-5-kDa. Polylysine-Sepharose affinity chromatography indicated that the inhibitor is a protein of 4 kDa, migrating as one band on SDS-polyacrylamide gel electrophoresis. Amino acid sequence analysis of this band showed the presence of two sequences, differing by one amino acid, which are identical to defensin I and II. Comparison of the sequences of plasminogen and defensin showed homology of defensin to the plasminogen kringles known to contain the lysine binding sites. The close structural similarity between defensin and plasminogen kringles and the ability of defensin to compete with plasminogen on binding to fibrin explain the ability of defensin to inhibit tPA-mediated, plasminogen-dependent fibrinolysis. These results suggest that the antifibrinolytic activity of defensin may have a biological function in preventing the spread of infection.

The cell-free immune repertoire of honeybees (Apis mellifera) consists of four polypeptides that are induced by bacterial infection and, through complementarity, provide broad-spectrum antibacterial defense. apidaecin is overproduced by a combination of low threshold transcriptional activation and a unique, genetically encoded amplification mechanism. In contrast, sizable experimental infections are required for induction of the normally silent hymenoptaecin, abaecin, and bee defensin genes; even so, bee defensin transcription is minimal and delayed, and only minute quantities of corresponding peptide are produced. The specific, temporal organization of the multi-component immune response in bees has therefore likely been selected to cope with infection of prevalent, plant-associated Gram-negative bacteria. Post-translational processing and modifications are substantially different for each of the four antibacterial peptides. While no similarities were observed among precursor structures of the various bee peptides, surprisingly, the signal sequences of abaecin (bee) and drosocin (Drosophila) shared unmistakable homology, possibly indicating common ancestral secretion/processing mechanisms. Finally, we report that bee defensin contains a typical disulfide-rich structure (40 amino acids) but also a unique, amphipathic, putatively amidated carboxyl-terminal tail (10 amino acids). We speculate that this structure is a "co-drug," assembled by fusing "disulfide-rich" and "alpha-helical" class peptide antibiotics, a novel concept in naturally occurring antibacterials.

As part of our ongoing search for novel antimicrobial agents and their use in singular or combined drug therapy, we have isolated a series of polypeptides from the lymph fluid of honeybees. These polypeptides are synthesized de novo, following experimental infection of the insect with live Escherichia coli cells, and confer a broad-spectrum antibacterial defense to the host. We have dissected this humoral "immune" system into its constituent components. In addition to the previously characterized apidaecins and abaecin, we also isolated a member of the defensin family of peptide antibiotics and, now, a novel 93-amino acid long, cationic polypeptide, termed hymenoptaecin. Detailed analysis established the complete chemical structure, including a 2-pyrrolidone-5-carboxylic acid at the N terminus, and indicated major differences with all known antibacterial polypeptides. Under physiological conditions, it inhibits viability of Gram-negative and Gram-positive bacteria, including several human pathogens. Lethal effects against E. coli are secondary to sequential permeabilization of outer and inner membrane. In combination, the six-constituent "peptide antibiotics" of bee lymph provide wide-spectrum antibacterial protection in vitro by virtue of complementarity rather than synergism.

A new family of cysteine-rich antimicrobial peptides from bovine neutrophils was isolated and characterized. Thirteen structurally homologous peptides were purified to homogeneity from a granule-rich cytoplasmic fraction of purified blood neutrophils. The complete sequences of the peptides were determined by a combination of enzymatic digestion, Edman degradation, and additional biochemical characterization of the carboxyl termini. The peptides are characterized by a highly cationic 38-42-residue chain which includes 6 invariantly spaced cysteines which form three disulfides. They share a highly conserved consensus sequence which is also found in a recently described epithelial antimicrobial peptide from bovine trachea. The in vitro antibacterial activities of the 13 neutrophil peptides, determined in assays using Staphylococcus aureus and Escherichia coli as test organisms, demonstrated that each peptide possessed antimicrobial activity, and that several were as active as the most potent neutrophil defensin, rabbit NP-1. Though the structural and functional attributes of the bovine neutrophil peptides are similar to those of defensins, the two peptide families are distinguished by their unique consensus sequences and additionally by differing tridisulfide motifs. We therefore propose that this new defensin-like antimicrobial peptide family be named beta-defensins.

Mucosal surfaces of several organ systems are important interfaces for host defense against microbes. Recent evidence suggests that antimicrobial peptides contribute to the defense of these surfaces. Defensins are one family of antimicrobial peptide, but their known distribution in humans has been limited to four members found in cells of myeloid origin. We sought to determine if the human defensin family was more complex. We found that the family of human defensins is diverse and is not restricted to expression in leukocytes. Southern blot and genomic clone analyses reveal that numerous defensin-related sequences are present in the human genome. A gene for a new human defensin family member was characterized. This gene, designated human defensin-5, is highly expressed in Paneth cells of the small intestine. This is the first example of an antimicrobial peptide gene expressed in an epithelial cell in humans. The data support the hypotheses that epithelial defensins equip the human small bowel with a previously unrecognized defensive capability which would augment other antimicrobial defenses.

Cryptdin is a Paneth cell corticostatin/defensin in the mouse small bowel. To help define the intestinal role of cryptdin, cryptdin-related sequence (CRS) mRNAs have been characterized with respect to developmental regulation, sequence homology, putative coding function, and occurrence in myeloid cells. Cryptdin, CRS1C, and CRS4C mRNAs are transcribed from separate genes, occur at equivalent abundance in small intestine, and appear in the small bowel in concert during the 2nd and 3rd weeks postpartum. Cryptdin and CRS1C mRNAs are not detectable in adult mouse bone marrow, but probes specific for the 5'- or the 3'-untranslated regions of CRS4C mRNA hybridize to a moderately abundant 1.05-kilobase bone marrow mRNA in contrast to a highly abundant 0.75-kilobase mRNA in small intestine. Nucleotide sequences corresponding to the deduced prepro-coding regions of cryptdin, CRS1C, and CRS4C mRNAs contain a highly conserved 200-base pair region of 92% sequence similarity (CSE.2), but the mRNAs are not homologous otherwise. The deduced CRS1C and CRS4C polypeptides are apparent precursors of secreted, cationic, proline- and cysteine-rich peptides that contain Cys-Pro-X repeats. Unlike cryptdin, however, the proposed CRS1C and CRS4C mature peptide regions lack the structural motif characteristic of defensins. Attempts to find homologies between the putative CRS peptides and existing protein sequences have been unsuccessful, leading us to speculate that CRS1C and CRS4C represent a new family of nondefensin antimicrobial peptides in the mouse small bowel.

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.

HNP-2 is a 29-residue peptide present in human neutrophils and is a member of the defensin family of antimicrobial peptides. All defensins contain an invariant disulfide infrastructure comprised of 6 half-cystine residues. The disulfide structure of HNP-2 was determined using a novel method to identify the cross-links involving the amino- and carboxyl-terminal cysteine residues. A derivative of HNP-2 was synthesized by covalent modification of the terminal cysteine residues. This derivative was purified, characterized, and subjected to exhaustive proteolytic digestion. Characterization of purified proteolytic fragments by amino acid analysis and/or sequence analysis identified an oligopeptide containing all 6 cystine residues. This oligopeptide was subjected to a single cycle of Edman degradation to cleave the peptide bond linking 2 adjacent cysteines. Purification and characterization of the Edman reaction products allowed for assignment of the disulfide array in HNP-2, revealing a cystine motif unique to the defensin peptide family. Further, the covalent structure of HNP-2 was found to be cyclic as one disulfide links the amino- and carboxyl-terminal cysteine residues. HNP-2 is the only polypeptide known to possess such a configuration.

Two orthorhombic crystal forms (P2(1)2(1)2, a = 30.5 A, b = 44.5 A, c = 40.5 A; I2(1)2(1)2(1) (or I222), a = 30.1 A, b = 66.5 A, c = 35.5 A) of human neutrophil cationic peptide 1 have been characterized. The P2(1)2(1)2 form contains two peptides (Mr = 3425) per asymmetric unit; the I2(1)2(1)2(1) form contains one peptide per asymmetric unit. Both crystal forms diffract to beyond 1.8 A resolution.