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1.
Antimicrob Agents Chemother ; 55(9): 4238-50, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21768520

ABSTRACT

Retrocyclins are humanized versions of the -defensin peptides expressed by the leukocytes of several nonhuman primates. Previous studies, performed in serum-free media, determined that retrocyclins 1 (RC1) and RC2 could prevent successful germination of Bacillus anthracis spores, kill vegetative B. anthracis cells, and inactivate anthrax lethal factor. We now report that retrocyclins are extensively bound by components of native mouse, human, and fetal calf sera, that heat-inactivated sera show greatly enhanced retrocyclin binding, and that native and (especially) heat-inactivated sera greatly reduce the direct activities of retrocyclins against spores and vegetative cells of B. anthracis. Nevertheless, we also found that retrocyclins protected mice challenged in vivo by subcutaneous, intraperitoneal, or intranasal instillation of B. anthracis spores. Retrocyclin 1 bound extensively to B. anthracis spores and enhanced their phagocytosis and killing by murine RAW264.7 cells. Based on the assumption that spore-bound RC1 enters phagosomes by "piggyback phagocytosis," model calculations showed that the intraphagosomal concentration of RC1 would greatly exceed its extracellular concentration. Murine alveolar macrophages took up fluorescently labeled retrocyclin, suggesting that macrophages may also acquire extracellular RC1 directly. Overall, these data demonstrate that retrocyclins are effective in vivo against experimental murine anthrax infections and suggest that enhanced macrophage function contributes to this property.


Subject(s)
Anthrax/prevention & control , Bacillus anthracis/pathogenicity , Defensins/therapeutic use , Macrophages/drug effects , Animals , Anthrax/immunology , Bacillus anthracis/drug effects , Cell Line , Female , Mice , Mice, Inbred BALB C , Phagocytosis/drug effects
2.
Biochemistry (Mosc) ; 71(8): 879-83, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16978151

ABSTRACT

Three antimicrobial peptides named PHD1-3 (Papio hamadryas defensin) have been isolated from hamadryas baboon blood leukocytes using preparative electrophoresis and reverse-phase HPLC. The primary structures of these peptides have been determined by automated Edman degradation and mass-spectrometry. The results suggest that the peptides belong to the alpha-defensin family. Structural homology analysis reveals that among alpha-defensins from other animal species, PHD3 is the most closely related to RMAD5 (rhesus macaque alpha-defensin) (90% homology) from rhesus macaque leukocytes and also highly similar to human alpha-defensin HD5 (60% homology), which is produced by intestinal Paneth cells. The homology of PHD3 with human neutrophil alpha-defensin HNP1 (human natural peptide) was 30%. The primary structures of PHD1 and PHD2 are most similar to RED1 (rhesus enteral defensin), one of six enteral alpha-defensins of rhesus monkeys. PHD1-3 have been shown to be active against the Gram-positive bacteria Listeria monocytogenes and Staphylococcus aureus, the Gram-negative bacterium Escherichia coli, and the fungus Candida albicans, similarly to the human HNP1 defensin.


Subject(s)
Papio hamadryas/blood , alpha-Defensins/blood , Amino Acid Sequence , Animals , Candida albicans/drug effects , Chromatography, High Pressure Liquid , Escherichia coli/drug effects , Humans , In Vitro Techniques , Leukocytes/chemistry , Listeria monocytogenes/drug effects , Macaca mulatta , Molecular Sequence Data , Papio hamadryas/genetics , Phylogeny , Sequence Homology, Amino Acid , Species Specificity , Staphylococcus aureus/drug effects , alpha-Defensins/genetics , alpha-Defensins/isolation & purification , alpha-Defensins/pharmacology
3.
J Pept Res ; 63(6): 469-76, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15175019

ABSTRACT

The ability of certain theta-defensins, including retrocyclin-1, to protect human cells from infection by HIV-1 marks them as potentially useful molecules. Theta-defensins composed of L-amino acids are likely to be unstable in environments that contain host and microbial proteases. This study compared the properties of two enantiomeric theta-defensins, retrocyclin-1, and RC-112. Although these peptides have identical sequences, RC-112 is composed exclusively of D-amino acids, whereas retrocyclin-1 contains only L-amino acids. We compared the ability of these peptides to protect JC53-BL human cells from infection by 30 primary HIV-1 isolates. JC53-BL cells are modified HeLa cells that express surface CD4, CXCR4, and CCR5. They also contain reporter cassettes that are driven by the HIV-1 LTR, and express beta-galactosidase and luciferase. The HIV-1 isolates varied in co-receptor specificity and included subtypes A, B, C, D, CRF01-AE, and G. RC-112 was several fold more potent than retrocyclin-1 across the entire HIV-1 panel. Although RC-112 bound immobilized gp120 and CD4 with lower affinity than did retrocyclin-1, surface plasmon resonance experiments performed with 1 microg/mL of RC-112 and retrocyclin-1 revealed that both glycoproteins were bound to a similar extent. The superior antiviral performance of RC-112 most likely reflected its resistance to degradation by surface-associated or secreted proteases of the JC53-BL target cells. Theta-defensins composed exclusively of D-amino acids merit consideration as starting points for designing microbicides for topical application to the vagina or rectum.


Subject(s)
Defensins/chemistry , Defensins/pharmacology , HIV-1/drug effects , Amino Acids/chemistry , Animals , Anti-HIV Agents/pharmacology , Defensins/metabolism , HIV Infections/drug therapy , Humans , Stereoisomerism
4.
Exp Mol Pathol ; 74(2): 190-5, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12710952

ABSTRACT

We tested the activity of Novispirin G-10, a novel antimicrobial alpha-helical octadecapeptide structurally related to cathelicidins and other innate immunity peptides, against Chlamydia trachomatis serovars L2, D, and E and three organisms associated with bacterial vaginosis (BV). The peptide's activity against C. trachomatis was measured in 48-h shell vial assays with McCoy cell targets. Exposure to 100 micro g/ml of Novispirin G-10 reduced the infectivity of serovars D and E by 99.4-100% and serovar L2 by 91.7-99.1%. At the same concentration of 100 micro g/ml, Novispirin G-10 rapidly killed >99% of Mobiluncus curtisii, Gardnerella vaginalis, and Prevotella bivia, in standard colony-forming unit (CFU) assays. Given its simple structure and relative lack of cytotoxic and hemolytic activity, Novispirin G-10 may be a useful component of microbicide preparations designed to prevent chlamydial infection and/or remediate the abnormal vaginal flora associated with BV.


Subject(s)
Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Antimicrobial Cationic Peptides/pharmacology , Antimicrobial Cationic Peptides/therapeutic use , Chlamydia Infections/drug therapy , Chlamydia trachomatis/drug effects , Peptides/pharmacology , Peptides/therapeutic use , Vaginosis, Bacterial/drug therapy , Vaginosis, Bacterial/microbiology , Antimicrobial Cationic Peptides/chemical synthesis , Antimicrobial Cationic Peptides/chemistry , Bacteroidaceae Infections/drug therapy , Cell Line , Chlamydia Infections/microbiology , Chlamydia trachomatis/classification , Colony-Forming Units Assay , Female , Gardnerella vaginalis/drug effects , Humans , Microbial Sensitivity Tests , Mobiluncus/drug effects , Peptides/chemistry , Peptides/urine , Prevotella/drug effects , Protein Structure, Secondary , Serotyping
5.
Antimicrob Agents Chemother ; 45(10): 2695-702, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11557457

ABSTRACT

Rhesus monkey bone marrow expresses a cathelicidin whose C-terminal domain comprises a 37-residue alpha-helical peptide (RL-37) that resembles human LL-37. Like its human counterpart, RL-37 rapidly permeabilized the membranes of Escherichia coli ML-35p and lysed liposomes that simulated bacterial membranes. When tested in media whose NaCl concentrations approximated those of extracellular fluids, RL-37 was considerably more active than LL-37 against staphylococci. Whereas human LL-37 contains five acidic residues and has a net charge of +6, rhesus RL-37 has only two acidic residues and a net charge of +8. Speculating that the multiple acidic residues of human LL-37 reduced its efficacy against staphylococci, we made a peptide (LL-37 pentamide) in which each aspartic acid of LL-37 was replaced by an asparagine and each glutamic acid was replaced by a glutamine. LL-37 pentamide's antistaphylococcal activity was substantially greater than that of LL-37. Thus, although the precursor of LL-37 is induced in human skin keratinocytes by injury or inflammation, its insufficiently cationic antimicrobial domain may contribute to the success of staphylococci in colonizing and infecting human skin.


Subject(s)
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Proteins/pharmacology , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemistry , Antimicrobial Cationic Peptides/chemistry , Base Sequence , Cathelicidins , Circular Dichroism , DNA, Complementary/analysis , Escherichia coli/drug effects , Lipopolysaccharides/metabolism , Liposomes/metabolism , Macaca mulatta , Microbial Sensitivity Tests , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Protein Structure, Secondary , Protein Structure, Tertiary , Proteins/chemistry , Sequence Homology, Amino Acid , Staphylococcus epidermidis/drug effects
6.
Biophys J ; 81(4): 2203-14, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11566791

ABSTRACT

The orientation and dynamics of an 18-residue antimicrobial peptide, ovispirin, has been investigated using solid-state NMR spectroscopy. Ovispirin is a cathelicidin-like model peptide (NH(2)-KNLRRIIRKIIHIIKKYG-COOH) with potent, broad-spectrum bactericidal activity. (15)N NMR spectra of oriented ovispirin reconstituted into synthetic phospholipids show that the helical peptide is predominantly oriented in the plane of the lipid bilayer, except for a small portion of the helix, possibly at the C-terminus, which deviates from the surface orientation. This suggests differential insertion of the peptide backbone into the lipid bilayer. (15)N spectra of both oriented and unoriented peptides show a reduced (15)N chemical shift anisotropy at room temperature compared with that of rigid proteins, indicating that the peptide undergoes uniaxial rotational diffusion around the bilayer normal with correlation times shorter than 10(-4) s. This motion is frozen below the gel-to-liquid crystalline transition temperature of the lipids. Ovispirin interacts strongly with the lipid bilayer, as manifested by the significantly reduced (2)H quadrupolar splittings of perdeuterated palmitoyloleoylphosphatidylcholine acyl chains upon peptide binding. Therefore, ovispirin is a curved helix residing in the membrane-water interface that executes rapid uniaxial rotation. These structural and dynamic features are important for understanding the antimicrobial function of this peptide.


Subject(s)
Antimicrobial Cationic Peptides/chemistry , Lipid Bilayers/chemistry , Models, Chemical , Phospholipids/chemistry , Binding Sites , Diffusion , Magnetic Resonance Spectroscopy , Membranes, Artificial , Rotation , Surface Properties , Thermodynamics , Water/chemistry
7.
J Leukoc Biol ; 70(3): 461-4, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11527997

ABSTRACT

We purified two new minidefensins (RTD-2 and RTD-3) from the bone marrow of rhesus monkeys. Both were circular octadecapeptides that contained three intramolecular disulfide bonds and were homologous to RTD-1, a circular (theta) defensin previously described by Tang et al. (Science, 286, 498-502, 1999). However, whereas the 18 residues of RTD-1 represent spliced nonapeptide fragments derived from two different demidefensin precursors, RTD-2 and -3 comprise tandem nonapeptide repeats derived from only one of the RTD-1 precursors. Thus, circular minidefensins are products of a novel posttranslational system that generates effector molecule diversity without commensurate genome expansion. A system wherein two demidefensin genes can produce three circular minidefensins might allow n such genes to produce (n/2)(n+1) peptides.


Subject(s)
Defensins/genetics , Defensins/metabolism , Macaca mulatta/metabolism , Amino Acid Sequence , Animals , Bone Marrow/metabolism , Cloning, Molecular , Defensins/chemistry , Disulfides/chemistry , Genetic Variation , Humans , Macaca mulatta/genetics , Molecular Sequence Data , Oxidation-Reduction , Peptides, Cyclic/chemical synthesis , Polymerase Chain Reaction , Protein Processing, Post-Translational , RNA, Messenger/genetics , Sequence Homology, Amino Acid
8.
Biochim Biophys Acta ; 1527(3): 141-8, 2001 Aug 15.
Article in English | MEDLINE | ID: mdl-11479030

ABSTRACT

We isolated a novel antimicrobial peptide, dicynthaurin, from hemocytes of a tunicate, Halocynthia aurantium. The native peptide had a mass of approximately 6.2 kDa and was composed of two 30-residue monomers without sequence homology to any previously identified peptides (ILQKAVLDCLKAAGSSLSKAAITAIYNKIT). Most cynthaurin molecules were C-terminally amidated and were linked covalently by a single cystine disulfide bond. When performed in membrane-mimetic environments, circular dichroism studies of dicynthaurin revealed largely alpha-helical conformations. Dicynthaurin's broad-spectrum activity encompassed Gram-positive (Micrococcus luteus, Staphylococcus aureus, Listeria monocytogenes) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), but not Candida albicans, a fungus. Although dicynthaurin was purified from a marine invertebrate, its antimicrobial activity was optimal at NaCl concentrations below 100 mM. This suggests that the antimicrobial actions of this molecule may take place intracellularly (e.g., within a phagosome) rather than extracellularly.


Subject(s)
Anti-Infective Agents/isolation & purification , Dipeptides/isolation & purification , Peptides/isolation & purification , Urochordata/metabolism , Amino Acid Sequence , Animals , Anti-Bacterial Agents , Anti-Infective Agents/chemistry , Circular Dichroism , Dimerization , Dipeptides/biosynthesis , Dipeptides/chemistry , Hemocytes/metabolism , Microbial Sensitivity Tests , Models, Molecular , Molecular Sequence Data , Molecular Weight , Peptides/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tissue Extracts/chemistry
10.
Infect Immun ; 69(4): 2684-91, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11254635

ABSTRACT

Gallinacin-3 and gallopavin-1 (GPV-1) are newly characterized, epithelial beta-defensins of the chicken (Gallus gallus) and turkey (Meleagris gallopavo), respectively. In normal chickens, the expression of gallinacin-3 was especially prominent in the tongue, bursa of Fabricius, and trachea. It also occurred in other organs, including the skin, esophagus, air sacs, large intestine, and kidney. Tracheal expression of gallinacin-3 increased significantly after experimental infection of chickens with Haemophilus paragallinarum, whereas its expression in the tongue, esophagus, and bursa of Fabricius was unaffected. The precursor of gallinacin-3 contained a long C-terminal extension not present in the prepropeptide. By comparing the cDNA sequences of gallinacin-3 and GPV-1, we concluded that a 2-nucleotide insertion into the gallinacin-3 gene had induced a frameshift that read through the original stop codon and allowed the chicken propeptide to lengthen. The striking structural resemblance of the precursors of beta-defensins to those of crotamines (highly toxic peptides found in rattlesnake venom) supports their homology, even though defensins are specialized to kill microorganisms and crotamines are specialized to kill much larger prey.


Subject(s)
Anti-Infective Agents/metabolism , Antimicrobial Cationic Peptides/genetics , Avian Proteins , Defensins , Amino Acid Sequence , Animals , Antimicrobial Cationic Peptides/biosynthesis , Antimicrobial Cationic Peptides/chemistry , Base Sequence , Chickens , DNA, Complementary/chemistry , Molecular Sequence Data , Turkeys
11.
Eur J Biochem ; 268(3): 703-12, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11168409

ABSTRACT

Four analogs of the antimicrobial peptide trichogin GA IV were studied. Their sequences are as follows: GT, n-octanoyl-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OMe; ST, n-octanoyl-Aib-Ser-Leu-Aib-Ser-Ser-Leu-Aib-Ser-Ile-Leu-OMe; BT, n-octanoyl-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-OMe; and DT, n-octanoyl-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-OMe. The trichogin GA IV differs from GT only in the nature of the C-terminal residue, being a 1,2 aminoalcohol (leucinol) in the case of the parent peptide. Compared with GT, ST has an increased amphiphilicity. In contrast, BT has little amphiphilicity being composed only of hydrophobic amino acids. DT is an octanoylated head-to-tail dimer of BT. We show that BT and DT lower the bilayer-to-hexagonal phase transition temperature (T(H)) of dipalmitoleoylphosphatidylethanolamine, indicating that the peptides promote negative curvature. These two peptides, composed of only hydrophobic amino acids, have their bulkier groups on one face of the helix, suggesting that they may penetrate membranes at an oblique angle. In contrast, GT and ST, like trichogin itself, increase TH, promoting positive curvature. These peptides have contrasting membrane lytic activities. Whereas DT and BT did not produce leakage of aqueous contents, GT and ST, like trichogin, did cause rapid leakage. The leakage activity with liposomes also correlates with the greater potency of GT and ST, compared with the hydrophobic analogs, in their hemolytic and bacteriostatic action. ST has greater lytic ability than GT in liposomal leakage as well as hemolysis. We also measured the rate of peptide-promoted lipid mixing as an indication of membrane fusion. BT produced lipid mixing only with large unilamellar vesicles enriched with dioleoylphosphatidylethanolamine; ST did not produce lipid mixing, as its apparent reduction of energy transfer proved to be artifactual. Quasi-elastic light scattering of large unilamellar vesicles was also carried out after adding ST and BT. Peptide BT, but not ST, was able to aggregate large unilamellar vesicles. Thus, one of the properties of BT that leads to the induction of lipid mixing is that it is able to aggregate vesicles, placing the bilayers in juxtaposition. Thus, the two pairs of peptides, BT and DT vs GT and ST, exhibit contrasting behaviour with respect to a number of membrane biophysical properties. This occurs despite the fact that the chemical structures of the peptides are rather similar. Such distinct behavior is also reflected in their hemolytic and bacteriostatic actions.


Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Cell Membrane/immunology , Peptides , Amino Acid Sequence , Calorimetry, Differential Scanning , Cell Membrane/drug effects , Cell Membrane/metabolism , Glycopeptides , Hemolysis/drug effects , Light , Lipid Metabolism , Lipopeptides , Liposomes/metabolism , Molecular Sequence Data , Scattering, Radiation , Staphylococcus aureus/metabolism , Temperature , Time Factors
12.
J Pept Res ; 58(6): 445-56, 2001 Dec.
Article in English | MEDLINE | ID: mdl-12005415

ABSTRACT

We cloned the precursor of a novel peptide from a cDNA library prepared from pharyngeal tissues of the tunicate, Styela clava. Its sequence predicted a histidine-rich, amidated 23-residue peptide (FLRF(IG)SVIHGIGHLVHHIGVAL-NH2) that we named clavaspirin. A synthetic clavaspirin was prepared and it was found that it killed Gram-positive and Gram-negative bacteria, permeabilized the outer and inner membranes of Escherichia coli, lysed phosphatidylglycerol (POPG) liposomes, and was potently haemolytic towards human and bovine erythrocytes. Each of these activities was performed more effectively at an acidic pH. Circular dichroism measurements of synthetic clavaspirin revealed a largely alpha-helical structure and polarized and residue-specific FTIR spectrometry showed that its association with phospholipid membranes was influenced by pH. Peptides such as clavaspirin may equip tunicate haemocytes to mediate cytotoxicity and participate in antimicrobial defence.


Subject(s)
Anti-Bacterial Agents/pharmacology , Peptides/genetics , Peptides/pharmacology , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemistry , Bacteria/drug effects , Base Sequence , Blood Proteins/chemistry , Blotting, Northern , Cell Membrane Permeability , Circular Dichroism , Cloning, Molecular , Hemolysis , Humans , Liposomes/metabolism , Microbial Sensitivity Tests/methods , Molecular Sequence Data , Peptides/chemistry , Sequence Homology, Amino Acid , Spectroscopy, Fourier Transform Infrared , Urochordata
13.
Expert Opin Investig Drugs ; 9(8): 1731-42, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11060772

ABSTRACT

Protegrins and their derivatives are a new class of peptide antibiotics based on mammalian antimicrobial peptides. Their pharmacological properties include an unusually broad spectrum of antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi and some enveloped viruses. Preclinical and clinical studies of the lead compound, IB-367, developed for topical applications, show promise for the prevention of chemotherapy- and radiation-induced oral mucositis.


Subject(s)
Anti-Bacterial Agents/pharmacology , Mammals/metabolism , Peptides , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/therapeutic use , Bacteria/drug effects , Bacterial Infections/drug therapy , Humans
14.
Biophys J ; 79(4): 2002-9, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11023904

ABSTRACT

Membrane pores spontaneously formed by antimicrobial peptides in membranes were crystallized for the first time by manipulating the sample hydration and temperature. Neutron diffraction shows that magainins and protegrins form stable pores in fully hydrated fluid membranes. At lower hydration levels or low temperature, the membrane multilayers crystallize. In one crystalline phase, the pores in each bilayer arrange in a regular hexagonal array and the bilayers are stacked into a hexagonal ABC lattice, corresponding to the cubic close-packed structure of spheres. In another crystalline phase, the bilayers are modulated into the rippled multilamellae, corresponding to a 2D monoclinic lattice. The phase diagrams are described. Crystallization of the membrane pores provides possibilities for diffraction studies that might provide useful information on the pore structures.


Subject(s)
Anti-Infective Agents/chemistry , Antimicrobial Cationic Peptides/chemistry , Proteins/chemistry , Xenopus Proteins , Amino Acid Sequence , Biophysical Phenomena , Biophysics , Crystallization , Crystallography, X-Ray , Lipid Bilayers/chemistry , Magainins , Membrane Proteins/chemistry , Molecular Sequence Data
15.
J Biol Chem ; 275(49): 38417-26, 2000 Dec 08.
Article in English | MEDLINE | ID: mdl-10978343

ABSTRACT

We isolated styelin D, a 32-residue, C-terminally amidated antimicrobial peptide, from the blood cells (hemocytes) of the solitary ascidian, Styela clava. Styelin D had remarkably extensive post-translational modifications, containing two novel amino acids, dihydroxyarginine and dihydroxylysine, and two distinctly unusual ones, 6-bromotryptophan and 3,4-dihydroxyphenylalanine. In addition, the peptide exhibited microheterogeneity because of differential mono- and dihydroxylation of several lysine residues. The primary sequence of one variant was: GW(*)LR(**)K(**)AAK(**)SVGK(**)FY(*)Y(*)K(**)HK(*)Y(*) Y(*)IK(*)AAWQIG KHAL-NH(2), where W(*) is 6-bromotryptophan, R(**) is dihydroxyarginine, Y(*) is 3,4-dihydroxyphenylalanine, K(*) is 5-hydroxylysine, and K(**) is dihydroxylysine. Styelin D exhibited activity against Gram-negative and Gram-positive bacteria, and this activity was retained in 200 mm NaCl. The role of the extensive modifications may be to preserve activity at low pH and/or high salinity because, under these conditions, the native peptide was considerably more active against the Gram-positive bacterial strains than its unmodified synthetic analogue. The peptide was also hemolytic and quite cytotoxic to eukaryotic cells. These broad ranging activities, combined with its relative abundance in ascidian hemocytes, suggest that styelin D plays a significant role in the innate immune mechanisms of S. clava.


Subject(s)
Anti-Bacterial Agents/chemistry , Hemocytes/chemistry , Proteins/chemistry , Urochordata , Amino Acid Sequence , Animals , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides , Cell Membrane Permeability/drug effects , Chromatography, High Pressure Liquid , Dihydroxyphenylalanine/analysis , Escherichia coli/drug effects , Escherichia coli/physiology , Gas Chromatography-Mass Spectrometry , Molecular Sequence Data , Peptide Fragments/chemistry , Proteins/isolation & purification , Proteins/pharmacology , Tryptophan/analogs & derivatives , Tryptophan/analysis
16.
Eur J Clin Microbiol Infect Dis ; 19(3): 187-94, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10795591

ABSTRACT

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide microplate assay was adapted to screen for the ability of 20 host-defense peptides to inactivate herpes simplex virus type 1 and type 2. The procedure required minimal amounts of material, was reproducible, and was confirmed with standard antiviral testing techniques. In screening tests, with the exception of melittin, a highly cytotoxic and hemolytic peptide found in bee venom, the alpha-helical peptides in our test panel (magainins, cecropins, clavanins, and LL-37) caused little viral inactivation. Several beta-sheet peptides (defensins, tachyplesin, and protegrins) inactivated one or both viruses, sometimes with remarkable selectivity. Two peptides were identified as having antiviral activity against both viruses, indolicidin (a tryptophan-rich peptide from bovine neutrophils) and brevinin-1 (a peptide found in frog skin). The antiviral activity of these two peptides was confirmed with standard antiviral assays. Interestingly, the antiviral activity of brevinin-1 was maintained after reduction and carboxamidomethylation, procedures that abolished its otherwise prominent hemolytic and cytotoxic effects.


Subject(s)
Amphibian Proteins , Antimicrobial Cationic Peptides , Antiviral Agents/pharmacology , Herpesvirus 1, Human/drug effects , Herpesvirus 2, Human/drug effects , Peptides/pharmacology , Amino Acid Sequence , Animals , Cattle , Chlorocebus aethiops , Dose-Response Relationship, Drug , Erythrocytes/drug effects , Hemolysis/drug effects , Herpesvirus 1, Human/physiology , Herpesvirus 2, Human/physiology , Humans , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/chemistry , Rabbits , Tumor Cells, Cultured , Vero Cells , Viral Plaque Assay
17.
Infect Immun ; 68(5): 2748-55, 2000 May.
Article in English | MEDLINE | ID: mdl-10768969

ABSTRACT

Endogenous antimicrobial peptides of the cathelicidin family contribute to innate immunity. The emergence of widespread antibiotic resistance in many commonly encountered bacteria requires the search for new bactericidal agents with therapeutic potential. Solid-phase synthesis was employed to prepare linear antimicrobial peptides found in cathelicidins of five mammals: human (FALL39/LL37), rabbit (CAP18), mouse (mCRAMP), rat (rCRAMP), and sheep (SMAP29 and SMAP34). These peptides were tested at ionic strengths of 25 and 175 mM against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. Each peptide manifested activity against P. aeruginosa irrespective of the NaCl concentration. CAP18 and SMAP29 were the most effective peptides of the group against all test organisms under both low- and high-salt conditions. Select peptides of 15 to 21 residues, modeled on CAP18 (37 residues), retained activity against the gram-negative bacteria and methicillin-sensitive S. aureus, although the bactericidal activity was reduced compared to that of the parent peptide. In accordance with the behavior of the parent molecule, the truncated peptides adopted an alpha-helical structure in the presence of trifluoroethanol or lipopolysaccharide. The relationship between the bactericidal activity and several physiochemical properties of the cathelicidins was examined. The activities of the full-length peptides correlated positively with a predicted gradient of hydrophobicity along the peptide backbone and with net positive charge; they correlated inversely with relative abundance of anionic residues. The salt-resistant, antimicrobial properties of CAP18 and SMAP29 suggest that these peptides or congeneric structures have potential for the treatment of bacterial infections in normal and immunocompromised persons and individuals with cystic fibrosis.


Subject(s)
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/pharmacology , Escherichia coli/drug effects , Pseudomonas aeruginosa/drug effects , Staphylococcus aureus/drug effects , Animals , Anti-Bacterial Agents/chemistry , Cathelicidins , Hemolysis , Humans , Luminescent Measurements , Mammals , Mice , Protein Conformation , Rabbits , Rats , Sheep
18.
J Biol Chem ; 275(6): 3943-9, 2000 Feb 11.
Article in English | MEDLINE | ID: mdl-10660548

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/chemical synthesis , Peptides, Cyclic/chemical synthesis , Proteins/chemical synthesis , Salts/pharmacology , alpha-Defensins , Amino Acid Sequence , Animals , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides , Defensins , Disulfides/chemistry , Escherichia coli/drug effects , Microbial Sensitivity Tests , Molecular Sequence Data , Protein Engineering , Proteins/chemistry , Rabbits , Salmonella typhimurium/drug effects , Structure-Activity Relationship
19.
Biochemistry ; 39(1): 139-45, 2000 Jan 11.
Article in English | MEDLINE | ID: mdl-10625488

ABSTRACT

Lipid bilayers containing the antimicrobial peptide protegrin-1 (PG-1) were studied by lamellar X-ray diffraction. Previously, we have shown that the peptide exists in two distinct states when associated with lipid bilayers depending on the peptide concentration [Heller, W. T., Waring, A. J., Lehrer, R. I., and Huang, H. W. (1998) Biochemistry 37, 17331-17338]. For concentrations below a lipid-dependent threshold, PG-1 exhibits a unique oriented circular dichroism spectrum called the S state. X-ray experiments show that in this state PG-1 decreases the thickness of the lipid bilayer in proportion to the peptide concentration, similar to alamethicin's membrane thinning effect. This indicates that the S state is adsorbed in the headgroup region of the lipid bilayer, where the peptide is in an inactive state. For PG-1 above the threshold concentration, X-ray diffraction shows that the interaction between the peptide and the bilayer changes significantly. These results suggest that PG-1 has the same concentration-gated mechanism of action as alamethicin.


Subject(s)
Anti-Infective Agents/chemistry , Lipid Bilayers/chemistry , Proteins/chemistry , Alamethicin/chemistry , Amino Acid Sequence , Antimicrobial Cationic Peptides , Molecular Sequence Data , Peptides/chemistry , Phosphatidylcholines/chemistry , Protein Structure, Secondary , X-Ray Diffraction
20.
Oral Microbiol Immunol ; 15(4): 226-31, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11154407

ABSTRACT

The bactericidal activity of synthetic LL-37, a cathelicidin, was assessed against Actinobacillus actinomycetemcomitans (three strains) and Capnocytophaga spp. (three strains). All strains were sensitive to LL-37, and exhibited 99% effective dose of 7.5-to-11.6 micrograms/ml. An amidated form of LL-37, pentamide-37, killed with about the same efficacy as LL-37. Partial inhibition of killing was noted at physiologic concentrations of NaCl, and complete inhibition was observed at 400 mM NaCl. At approximately the 99% effective dose--i.e., 10 micrograms/ml--LL-37 also lost activity against A. actinomycetemcomitans in the presence of native or heat-inactivated 10-15% normal human AB serum. Pentamide-37 was less sensitive to serum inhibition than LL-37. In conclusion, certain oral, gram-negative bacteria are sensitive to the bactericidal activity of LL-37 at low concentrations of serum and salt, a condition likely to be found within the membrane-delimited phagolysosome. Modified forms of LL-37, such as pentamide-37, may be more suitable for future therapeutic application in the presence of serum.


Subject(s)
Aggregatibacter actinomycetemcomitans/drug effects , Antimicrobial Cationic Peptides/pharmacology , Capnocytophaga/drug effects , Amino Acid Sequence , Antimicrobial Cationic Peptides/chemistry , Blood Proteins/pharmacology , Cathelicidins , Dose-Response Relationship, Drug , Epithelial Cells/chemistry , Humans , Leukocytes/chemistry , Microbial Sensitivity Tests , Molecular Sequence Data , Sodium Chloride/pharmacology
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