ABSTRACT
Antimicrobial proteins stored in lysosome-like granules of neutrophils and macrophages probably play an important role in killing phagocytosed microbes after delivery to the phagolysosome. Among the granules' antimicrobial armamentarium are defensins, peptides that kill a broad spectrum of microorganisms in vitro. Antimicrobial defensins were recently also isolated from non-phagocytic granulocytes of the mouse small intestinal epithelium, from where they are secreted into the lumen to function extracellularly. Clarification of the antimicrobial mechanisms of defensins in intracellular and extracellular environments will provide a key to understanding peptide-mediated host defence.
ABSTRACT
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.
Subject(s)
Intestinal Mucosa/chemistry , Intestine, Small/chemistry , Protein Precursors/isolation & purification , Proteins/isolation & purification , Amino Acid Sequence , Amino Acids/analysis , Animals , Cytoplasmic Granules/chemistry , Epithelium/chemistry , Immunoenzyme Techniques , Intestinal Mucosa/cytology , Intestine, Small/cytology , Male , Mice , Molecular Sequence Data , Protein Precursors/analysis , Protein Precursors/chemistry , Protein Precursors/pharmacology , Proteins/analysis , Proteins/chemistry , Proteins/pharmacology , Salmonella typhimurium/drug effectsABSTRACT
Defensins (molecular weight 3500 to 4000) act in the mammalian immune response by permeabilizing the plasma membranes of a broad spectrum of target organisms, including bacteria, fungi, and enveloped viruses. The high-resolution crystal structure of defensin HNP-3 (1.9 angstrom resolution, R factor 0.19) reveals a dimeric beta sheet that has an architecture very different from other lytic peptides. The dimeric assembly suggests mechanisms by which defensins might bind to and permeabilize the lipid bilayer.
Subject(s)
Blood Proteins/ultrastructure , alpha-Defensins , Amino Acid Sequence , Animals , Blood Proteins/chemistry , Cell Membrane Permeability , Crystallography , Defensins , Guinea Pigs , Humans , Macromolecular Substances , Membrane Proteins/chemistry , Membrane Proteins/ultrastructure , Models, Molecular , Molecular Sequence Data , Molecular Structure , Protein Conformation , Rabbits , Rats , Structure-Activity Relationship , X-Ray DiffractionABSTRACT
Analysis of rhesus macaque leukocytes disclosed the presence of an 18-residue macrocyclic, tridisulfide antibiotic peptide in granules of neutrophils and monocytes. The peptide, termed rhesus theta defensin-1 (RTD-1), is microbicidal for bacteria and fungi at low micromolar concentrations. Antibacterial activity of the cyclic peptide was threefold greater than that of an open-chain analog, and the cyclic conformation was required for antimicrobial activity in the presence of 150 millimolar sodium chloride. Biosynthesis of RTD-1 involves the head-to-tail ligation of two alpha-defensin-related nonapeptides, requiring the formation of two new peptide bonds. Thus, host defense cells possess mechanisms for synthesis and granular packaging of macrocyclic antibiotic peptides that are components of the phagocyte antimicrobial armamentarium.
Subject(s)
Anti-Infective Agents/metabolism , Monocytes/metabolism , Neutrophils/metabolism , Peptides, Cyclic/biosynthesis , Protein Biosynthesis , Amino Acid Sequence , Animals , Anti-Bacterial Agents , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Bacteria/drug effects , Cloning, Molecular , Defensins , Disulfides/chemistry , Fungi/drug effects , Humans , Leukopoiesis , Macaca mulatta , Molecular Sequence Data , Oligopeptides/chemistry , Oligopeptides/genetics , Oligopeptides/metabolism , Osmolar Concentration , Peptides, Cyclic/chemistry , Peptides, Cyclic/genetics , Peptides, Cyclic/pharmacology , Protein Conformation , Protein Precursors/chemistry , Protein Precursors/genetics , Protein Precursors/metabolism , Protein Processing, Post-Translational , Proteins/chemistry , Proteins/genetics , Proteins/pharmacologyABSTRACT
We tested the in vitro susceptibility of Candida albicans to three defensins from human neutrophilic granulocytes (HNP-1, 2, and 3), a homologous defensin from rabbit leukocytes (NP-1), and four unrelated cationic peptides. Although the primary amino acid sequences of HNP-1, 2, and 3 are identical except for a single amino-terminal amino acid alteration, HNP-1 and HNP-2 killed C. albicans but HNP-3 did not. C. albicans blastoconidia were protected from HNP-1 when incubations were performed in the absence of oxygen or in the presence of inhibitors that blocked both of its mitochondrial respiratory pathways. Neither anaerobiosis nor mitochondrial inhibitors substantially protected C. albicans exposed to NP-1, poly-L-arginine, poly-L-lysine, or mellitin. Human neutrophilic granulocyte defensin-mediated candidacidal activity was inhibited by both Mg2+ and Ca2+, and was unaffected by Fe2+. In contrast, Fe2+ inhibited the candidacidal activity of NP-1 and all of the model cationic peptides, whereas Mg2+ inhibited none of them. These data demonstrate that susceptibility of C. albicans to human defensins depends both on the ionic environment and on the metabolic state of the target cell. The latter finding suggests that leukocyte-mediated microbicidal mechanisms may manifest oxygen dependence for reasons unrelated to the production of reactive oxygen intermediates by the leukocyte.
Subject(s)
Blood Proteins/immunology , Candida albicans/immunology , Cations, Divalent/pharmacology , Neutrophils/immunology , alpha-Defensins , Anaerobiosis , Animals , Antifungal Agents/pharmacology , Antimycin A/pharmacology , Azides/pharmacology , Calcium/pharmacology , Candida albicans/metabolism , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Humans , Iron/pharmacology , Leukocytes/immunology , Magnesium/pharmacology , Mitochondria/drug effects , Mitochondria/metabolism , Peptides/immunology , Rabbits , Salicylamides/pharmacology , Sodium Azide , Sodium Chloride/pharmacology , Sodium Cyanide/pharmacologyABSTRACT
We investigated the monocyte-chemotactic activity of fractionated extracts of human neutrophil granules. Monocyte-chemotactic activity was found predominantly in the defensin-containing fraction of the neutrophil granules. Purified preparations of each of the three human defensins (HNP-1, HNP-2, HNP-3) were then tested. HNP-1 demonstrated significant chemotactic activity for monocytes: Peak activity was seen at HNP-1 concentrations of 5 X 10(-9) M and was 49 +/- 20% (mean +/- SE, n = 9) of that elicited by 10(-8) M FMLP. HNP-2 (peak activity at 5 X 10(-9) M) was somewhat less active, yielding 19 +/- 10% (n = 11). HNP-3 failed to demonstrate chemotactic activity. Checkerboard analysis of monocyte response to HNP-1 and HNP-2 confirmed that their activity was chemotactic rather than chemokinetic. Neutrophils demonstrated a low level of response to defensins but this reaction was primarily chemokinetic. Defensins may play a role in the recruitment of monocytes by neutrophils into inflammatory sites.
Subject(s)
Blood Proteins/pharmacology , Chemotaxis, Leukocyte/drug effects , Cytoplasmic Granules/analysis , Monocytes/physiology , Neutrophils/ultrastructure , alpha-Defensins , Blood Proteins/isolation & purification , Humans , N-Formylmethionine Leucyl-Phenylalanine/pharmacologyABSTRACT
The primary structures of three human neutrophil antimicrobial peptides (HNP) were determined. The peptides, HNP-1, HNP-2, and HNP-3, which we have termed defensins, were rich in cystine, arginine, and aromatic residues, but were devoid of free sulfhydryl groups and carbohydrate moieties. They were 29-30 residues in length and identical in sequence in all but their amino terminal residues. The defensins were homologous in sequence to peptides of similar size and biological activity previously purified from rabbit polymorphonuclear leukocytes, but unrelated to other neutrophil proteins of known sequence. 11 amino acid residues of the human defensins, including all six cysteinyl residues, were invariantly conserved in the six rabbit members of this multigene peptide family. That similarly structured antimicrobial peptides are present in both rabbit and human leukocytes supports their purported role as cidal agents in phagocyte-mediated host defense.
Subject(s)
Blood Proteins/analysis , Neutrophils/analysis , alpha-Defensins , Amino Acid Sequence , Animals , Cytoplasmic Granules/analysis , Humans , Protein Conformation , Rabbits , Sequence Homology, Nucleic Acid , Species SpecificityABSTRACT
Defensins are small, cysteine-rich antimicrobial peptides that are abundant in human, rabbit, and guinea pig neutrophils (PMN). Three defensins (human neutrophil peptide defensin [HNP]-1, HNP-2, and HNP-3) constitute between 30 and 50% of the total protein in azurophil granules of human PMN. We examined the mechanism of HNP-mediated bactericidal activity against Escherichia coli ML-35 (i-, y-, z+) and its pBR322-transformed derivative, E. coli ML-35p. Under conditions that supported bactericidal activity, HNP-1 sequentially permeabilized the outer membrane (OM) and inner membrane (IM) of E. coli. Coincident with these events, bacterial synthesis of DNA, RNA, and protein ceased and the colony count fell. Although these events were closely coupled under standard assay conditions, OM permeabilization was partially dissociated from IM permeabilization when experiments were performed with E. coli that had been plasmolyzed by mannitol. Under such conditions, the rate and extent of bacterial death more closely paralled loss of IM integrity than OM permeabilization. Electron microscopy of E. coli that had been killed by defensins revealed the presence of striking electron-dense deposits in the periplasmic space and affixed to the OM. Overall, these studies show that HNP-mediated bactericidal activity against E. coli ML-35 is associated with sequential permeabilization of the OM and IM, and that inner membrane permeabilization appears to be the lethal event.
Subject(s)
Blood Bactericidal Activity , Blood Proteins/pharmacology , Escherichia coli/drug effects , Neutrophils/physiology , alpha-Defensins , Cathepsin G , Cathepsins/pharmacology , Cell Membrane Permeability/drug effects , Escherichia coli/metabolism , Escherichia coli/ultrastructure , Humans , Nucleic Acids/biosynthesis , Protein Biosynthesis , Serine EndopeptidasesABSTRACT
We extracted a granule-rich sediment from normal human neutrophils and subjected it to chromatographic, electrophoretic, and functional analysis. The extract contained three small (molecular weight less than 3,500) antibiotic peptides that were named human neutrophil peptide (HNP)-1, HNP-2, and HNP-3, and which will be referred to as "defensins." HNP 1-3, a mixture of the three defensins, killed Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli effectively in vitro when tested in 10 mM phosphate buffer containing certain nutrients, but it had little or no bactericidal activity in nutrient-free buffer. In contrast, the nutrient-free buffer supported a high degree of activity by HNP 1-3 against Cryptococcus neoformans. In addition to its antibacterial and antifungal properties, HNP 1-3 directly inactivated herpes simplex virus, Type 1. Two of the individual purified defensins, HNP-1 and HNP-2, were as microbicidal as the mixture HNP 1-3. HNP-3 was less active than the other defensins against most but not all of the microbes tested. Immunoperoxidase stains revealed HNP 1-3 to have a granular localization in the neutrophil's cytoplasm by light microscopy. Frozen thin section immunogold transmission electron microscopy showed HNP 1-3 to be localized in azurophil granules. These studies define a broad-spectrum antimicrobial system in human neutrophils. The defensin system may operate in conjunction with or independently from oxygen-dependent microbicidal processes to enable human neutrophils to inactivate and destroy potential pathogens.
Subject(s)
Blood Proteins/isolation & purification , Neutrophils/analysis , alpha-Defensins , Amino Acids/analysis , Bacteria/drug effects , Blood Proteins/pharmacology , Blood Proteins/physiology , Chromatography , Cryptococcus neoformans/drug effects , Cytoplasmic Granules/analysis , Electrophoresis, Polyacrylamide Gel , Humans , Microbial Sensitivity Tests , Neutrophils/physiology , Simplexvirus/drug effectsABSTRACT
AML-1B is a hematopoietic transcription factor that is functionally inactivated by multiple chromosomal translocations in human acute myeloblastic and B-cell lymphocytic leukemias. The t(8;21)(q22;q22) translocation replaces the C terminus, including the transactivation domain of AML-1B, with ETO, a nuclear protein of unknown function. We previously showed that AML-1-ETO is a dominant inhibitor of AML-1B-dependent transcriptional activation. Here we demonstrate that AML-1-ETO also inhibits C/EBP-alpha-dependent activation of the myeloid cell-specific, rat defensin NP-3 promoter. AML-1B bound the core enhancer motifs present in the NP-3 promoter and activated transcription approximately sixfold. Similarly, C/EBP-alpha bound NP-3 promoter sequences and activated transcription approximately sixfold. Coexpression of C/EBP-alpha with AML-1B or its family members, AML-2 and murine AML-3, synergistically activated the NP-3 promoter up to 60-fold. The t(8;21) product, AML-1-ETO, repressed AML-1B-dependent activation of NP-3 and completely inhibited C/EBP-alpha-dependent activity as well as the synergistic activation. In contrast, the inv(16) product, which indirectly targets AML family members by fusing their heterodimeric DNA binding partner, CBF-beta, to the myosin heavy chain, inhibited AML-1B but not C/EBP-alpha activation or the synergistic activation. AML-1-ETO and C/EBP-alpha were coimmunoprecipitated and thus physically interact in vivo. Deletion mutants demonstrated that the C terminus of ETO was required for AML-1-ETO-mediated repression of the synergistic activation but not for association with C/EBP-alpha. Finally, overexpression of AML-1-ETO in myeloid progenitor cells prevented granulocyte colony-stimulating factor-induced differentiation. Thus, AML-1-ETO may contribute to leukemogenesis by specifically inhibiting C/EBP-alpha- and AML-1B-dependent activation of myeloid promoters and blocking differentiation.
Subject(s)
Chromosomes, Human, Pair 21 , Chromosomes, Human, Pair 8 , DNA-Binding Proteins/genetics , Granulocytes/metabolism , Nuclear Proteins/genetics , Oncogene Proteins, Fusion , Transcription Factors/genetics , Transcription, Genetic , Translocation, Genetic , Animals , CCAAT-Enhancer-Binding Proteins , COS Cells , Cell Differentiation/genetics , Core Binding Factor Alpha 2 Subunit , Gene Expression Regulation, Neoplastic , Granulocytes/cytology , Hematopoiesis/genetics , Humans , Leukemia/genetics , Promoter Regions, Genetic/genetics , RUNX1 Translocation Partner 1 Protein , Rats , Recombinant Fusion Proteins/geneticsABSTRACT
Defensins comprise a structural class of small cationic peptides that exert broad-spectrum antimicrobial activities through membrane permeabilization. Their predominantly beta-sheet structure, stabilized by three disulfide bonds, distinguishes them from other antimicrobial peptides which typically form amphiphilic helices. Defensins bind to membranes electrostatically and subsequently form apparently multimeric pores. Recent structural and biophysical studies are beginning to provide insights into the process of permeabilization.
Subject(s)
Blood Proteins/chemistry , Blood Proteins/physiology , Amino Acid Sequence , Animals , Blood Bactericidal Activity , Blood Proteins/genetics , Cell Membrane/metabolism , Defensins , Humans , Micrococcus luteus , Molecular Sequence Data , Structure-Activity RelationshipABSTRACT
Indolicidin, a cationic tridecapeptide amide isolated from the granules of bovine neutrophils, has been found to possess potent antimicrobial activity in vitro but its nonselective toxicity could restrict its therapeutic utility. We found that the concentration at which indolicidin disrupts washed human red blood cell membranes coincided with the concentration at which indolicidin self associates. Because of a preponderance of hydrophobic residues, we believed that indolicidin would partition into liposomes which would restrict its exchange with biological tissues and consequently reduce its toxicity. Fluorescence spectroscopy of indolicidin added to 100 nm liposomes comprised of POPC, POPC/cholesterol (60:40 mol%), DPPC, or DPPC/cholesterol (60:40) revealed a large blue-shift and an increase in intensity of the emission profile indicating insertion into the bilayer. Of the lipids tested, POPC exhibited the highest degree of indolicidin binding as determined by fluorescence and encapsulation efficiency. By sequestering indolicidin within the lipid bilayer of 100 nm POPC liposomes we significantly reduced its toxicity to CHO/K1 cells. Likewise, the systemic toxicity of liposomal indolicidin in Balb/c mice was decreased dramatically relative to aqueous solutions; the maximum dose at which no deaths occurred was 0.4 mg/kg for free indolicidin versus 40 mg/kg for indolicidin-POPC. Because of this decrease in toxicity, we were able to administer liposomally encapsulated material at significantly higher concentrations than unencapsulated aqueous material and achieve efficacy in treating animals systemically infected with Aspergillus fumigatus. Liposomal but not free indolicidin was found to be effective in obtaining cures. This report is the first description of the in vivo therapeutic activity of a neutrophil-derived antimicrobial peptide and suggests that liposomal treatment modalities will provide effective strategies for endowing this class of compounds with pharmacological utility.
Subject(s)
Antifungal Agents/pharmacology , Antimicrobial Cationic Peptides , Liposomes , Neutrophils/chemistry , Peptides/pharmacology , Amino Acid Sequence , Animals , Antifungal Agents/administration & dosage , Aspergillosis/drug therapy , Aspergillus fumigatus/drug effects , CHO Cells , Cattle , Cricetinae , Drug Carriers , Humans , Male , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Peptides/administration & dosageABSTRACT
A hexagonal crystal form (P6(3)22, a = b = 34.0 A, c = 113.5 A) and a monoclinic form (P2(1), a = 37.1 A, b = 32.2 A, c = 32.4 A, beta = 110 degrees) of neutrophil cationic protein NP2, isolated from rabbit leukocytes, have been characterized. The monoclinic form, containing two promoters (Mr = 3844) per asymmetric unit, diffracts to at least 1.8 A and is suitable for high-resolution structural studies.
Subject(s)
Anti-Infective Agents , Blood Proteins , Granulocytes/analysis , Animals , Antimicrobial Cationic Peptides , Crystallization , Rabbits , X-Ray DiffractionABSTRACT
Solution structures of the rabbit neutrophil defensin NP-5 have been determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and distance geometry techniques. This 33 amino acid peptide is part of the oxygen-independent mammalian defense system against microbial infection. The structures were generated from 107 n.m.r. derived inter-residue proton-proton distance constraints. A distance geometry algorithm was then used to determine the range of structures consistent with these distance constraints. These distance geometry calculations employed an improved algorithm that allowed the chirality constraints to be relaxed on prochiral centers when it was not possible to make stereo-specific assignments of protons on these centers. This procedure gave superior results compared with standard distance geometry methods and also produced structures that were more consistent with the original n.m.r. data. Analysis of the NP-5 structures shows that the overall folding of the peptide backbone is well defined by the n.m.r. distance information but that the side-chain group conformations are generally less well defined.
Subject(s)
Blood Proteins , Neutrophils/analysis , Animals , Defensins , Magnetic Resonance Spectroscopy , Protein Conformation , Protons , RabbitsABSTRACT
Indolicidin is a tridecapeptide amide isolated from the cytoplasmic granules of bovine neutrophils. It has potent, broad spectrum microbicidal activities in vitro that are thought to be related to the membrane-disruptive properties of the peptide. Based on the putative membrane-targeted mode of action, we postulated that indolicidin would be active against HIV-1, an enveloped virus. Indolicidin was reproducibly virucidal against HIV-1 at a concentration of 333 microg/mL (174 microM) with a 50% inhibitory dose between 67 and 100 microg/mL. At 37 degrees C, killing was rapid with >50% killing of HIV occurring within 5 min, and nearly 100% viral inactivation achieved by 60 min. The anti-HIV activity of indolicidin was temperature-sensitive, a finding consistent with a membrane-mediated antiviral mechanism. Parallel experiments revealed that indolicidin lysed cultured lymphoblastoid cells at concentrations similar to those required for antiviral activity. However, a des-R13-amide indolicidin analog (R12-OH), previously shown to have less antibacterial activity than indolicidin, was significantly less active against HIV and was non-toxic to lymphoid target cells at concentrations up to 333 microg/mL, the highest level tested.
Subject(s)
Anti-HIV Agents/pharmacology , Antimicrobial Cationic Peptides , HIV Infections/drug therapy , Neutrophils/physiology , Peptides/pharmacology , Animals , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/toxicity , CD4-Positive T-Lymphocytes/drug effects , Cattle , Cell Survival/drug effects , Cells, Cultured , Humans , Peptides/chemical synthesis , Peptides/toxicity , Structure-Activity Relationship , TemperatureABSTRACT
Defensins, a family of cationic peptides isolated from mammalian granulocytes and believed to permeabilize membranes, were tested for their ability to cause fusion and lysis of liposomes. Unlike alpha-helical peptides whose lytic effects have been extensively studied, the defensins consist primarily of beta-sheet. Defensins fuse and lyse negatively charged liposomes but display reduced activity with neutral liposomes. These and other experiments suggest that fusion and lysis is mediated primarily by electrostatic forces and to a lesser extent, by hydrophobic interactions. Circular dichroism and fluorescence spectroscopy of native defensins indicate that the amphiphilic beta-sheet structure is maintained throughout the fusion process. Taken together, these results support the idea that protein-mediated membrane fusion depends not only on hydrophobic and electrostatic forces but also on the spatial arrangement of the amino acid residues to form a three-dimensional amphiphilic structure, which promotes the efficient mixing of the lipids between membranes. A molecular model for membrane fusion by defensins is presented, which takes into account the contributions of electrostatic forces, hydrophobic interactions, and structural amphiphilicity.
Subject(s)
Blood Proteins/chemistry , Liposomes , Membrane Fusion , Protein Structure, Secondary , 1,2-Dipalmitoylphosphatidylcholine , Amino Acid Sequence , Animals , Blood Proteins/isolation & purification , Cholesterol , Circular Dichroism , Defensins , Humans , Kinetics , Macromolecular Substances , Models, Structural , Molecular Sequence Data , Neutrophils/physiology , Phosphatidylserines , Rabbits , Rats , Sequence Homology, Amino Acid , Spectrometry, FluorescenceABSTRACT
Defensins comprise a family of broad-spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP-2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP-2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP-2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions. Because vesicle aggregation and fusion can confound studies of the interaction of HNP-2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP-2, and the POPG:POPC ratio. Vesicles (300 microM POPG) readily aggregated at HNP-2 concentrations above 1 microM, but no mixing of vesicle contents could be detected for concentrations as high as 2 microM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi-fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 microM POPG vesicles, 50% of entrapped solute is released by 0.7 microM HNP-2. We introduce a simple method for determining whether leakage from vesicles is graded or all-or-none. We show by means of this fluorescence "requenching" method that native HNP-2 induces vesicle leakage in an all-or-none manner, whereas reduced HNP-2 induces partial, or graded, leakage of vesicle contents. At HNP-2 concentrations that release 100% of small (approximately 400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900-Da dextran is mostly retained. These results suggest that HNP-2 can form pores that have a maximum diameter of approximately 25 A. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.
Subject(s)
Anti-Infective Agents/chemistry , Blood Proteins/chemistry , Lipid Bilayers/chemistry , Neutrophils/chemistry , alpha-Defensins , Amino Acid Sequence , Anti-Infective Agents/isolation & purification , Anti-Infective Agents/metabolism , Blood Proteins/isolation & purification , Blood Proteins/metabolism , Defensins , Humans , Membrane Fusion , Models, Molecular , Molecular Sequence Data , Naphthalenes/metabolism , Permeability , Phosphatidylcholines/chemistry , Phosphatidylglycerols/chemistry , Protein Binding , Sequence Homology, Amino AcidABSTRACT
We have purified and determined the amino acid sequence of cryptdin-1, a murine Paneth cell defensin. The peptide corresponds to a previously characterized mRNA that accumulates to high abundance during postnatal ontogeny of the small bowel. Acid-extracted intestinal protein was fractionated by cation-exchange chromatography and fractions were assayed for antimicrobial activity. One peak of anti-Salmonella activity contained a putative defensin, based on its predicted electrophoretic migration in acid-urea PAGE. The peptide was purified to homogeneity by RP-HPLC and sequenced. These studies demonstrate defensin expression in non-myeloid tissue. The N-terminal extension of cryptdin-1 is a unique structural feature of this novel epithelial defensin.
Subject(s)
Ileum/chemistry , Intestinal Mucosa/chemistry , Jejunum/chemistry , Protein Precursors/chemistry , Proteins/chemistry , Amino Acid Sequence , Animals , Consensus Sequence , Male , Mice , Molecular Sequence Data , Protein Precursors/isolation & purification , Proteins/isolation & purification , Sequence Homology, Nucleic AcidABSTRACT
Defensins, human neutrophil peptide (HNP) antibiotics, potently inhibited phospholipid/Ca2+ protein kinase (protein kinase C, PKC) and phosphorylation of endogenous proteins from rat brains catalyzed by the enzyme. Of the three defensin peptides, HNP-2 appeared to be more potent than HNP-1 and HNP-3. Kinetic studies indicated that defensins inhibited PKC noncompetitively with respect to phosphatidylserine (a phospholipid cofactor), Ca2+ (an activator), ATP (a phosphoryl donor) and histone H1 (a substrate protein) with Ki values ranging from 1.2 to 1.7 microM. Defensins, unlike polymyxin B (another peptide inhibitor of PKC), did not inhibit the binding of [3H]phorbol 12,13-dibutyrate to PKC; however, defensins, like polymyxin B, inhibited the PKC activity stimulated by 12-O-tetradecanoylphorbol-13-acetate. Defensins had little or no effect on myosin light chain kinase (a calmodulin/Ca2+-dependent protein kinase) and the holoenzyme or catalytic subunit of cyclic AMP-dependent protein kinase, indicating a specificity of action of defensins. It is suggested that defensins, among the most potent peptide inhibitors of PKC so far identified, may have profound effects on functions of neutrophils and other mammalian cells, in addition to their well-recognized antimicrobial activities.
Subject(s)
Blood Bactericidal Activity , Blood Proteins/pharmacology , Neutrophils/analysis , Protein Kinase C/antagonists & inhibitors , alpha-Defensins , Animals , Brain Chemistry , Cytoplasmic Granules/analysis , Humans , Kinetics , Phorbol Esters/metabolism , Phosphorylation , Polymyxin B/pharmacology , RatsABSTRACT
New approaches to antimicrobial therapy for ocular pathogens must overcome organisms that are resistant to current therapeutic modalities. This investigation examined the antimicrobial activity of novel antimicrobial neutrophil peptides (defensins NP-1 and NP-5) against isolates from clinical ocular microbial infections in humans and horses. The test panel of human clinical isolates included Candida albicans, an alpha-hemolytic Streptococcus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Morganella morganii. The test panel of equine pathogens included three clinical isolates of P aeruginosa and two clinical isolates of Staphylococcus aureus. The equine isolates were chosen for their relative resistance to commonly employed antimicrobial therapy. The two defensins differed markedly in their bactericidal activity. Defensin NP-5, at a 50-micrograms/mL concentration, exhibited minimal bactericidal activity against the majority of isolates of the test panel. The inferior microbicidal activity of NP-5 is consistent with previously published results. However, at this concentration, NP-5 did exhibit appreciable bacteriostatic activity against human ocular pathogens M morganii (74%), alpha-hemolytic Streptococcus (57%), and P aeruginosa (93%) during the 2-hour incubation period. In contrast, defensin NP-1 at 10 micrograms/mL exerted potent microbicidal activity against all isolates, effecting a 2 to 3 log10 decrease in colony-forming units within a 60-minute incubation period. Under the assay conditions employed, these findings demonstrate: (1) two distinct mechanisms by which defensins exert their antimicrobial activity against microbial pathogens associated with clinical ocular disease in humans and horses, and (2) that rabbit defensin NP-1 is a potent antimicrobial agent against a wide array of ocular pathogens.