Showing papers on "Antimicrobial peptides published in 1999"

Antimicrobial peptides in insects; structure and function

Abstract: Antimicrobial peptides appear to be ubiquitous and multipotent components of the innate immune defense arsenal used by both prokaryotic and eukaryotic organisms. During the past 15 years a multitude of these peptides have been isolated largely from insects. In spite of great differences in size, amino acid composition and structure, most of the antimicrobial peptides from insects can be grouped into one of three categories. The largest category in number contains peptides with intramolecular disulfide bonds forming hairpin-like β-sheets or α-helical–β-sheet mixed structures. The second most important group is composed of peptides forming amphipathic α-helices. The third group comprises peptides with an overrepresentation in proline and/or glycine residues. In general, the insect antimicrobial peptides have a broad range of activity and are not cytotoxic. Despite a wealth of information on structural requirements for their antimicrobial activity, the mode of action of these peptides is not yet fully understood. However, some data suggest the existence of two types of mode of action: 1. through peptide–lipid interaction or 2. through receptor-mediated recognition processes. This review presents the main results obtained during the last four years in the field of antimicrobial peptides from insects with a special focus on the proline-rich and cysteine-rich peptides.

Mechanism of Interaction of Different Classes of Cationic Antimicrobial Peptides with Planar Bilayers and with the Cytoplasmic Membrane of Escherichia coli

Abstract: Antimicrobial cationic peptides are prevalent throughout nature as part of the intrinsic defenses of most organisms, and have been proposed as a blueprint for the design of novel antimicrobial agents. They are known to interact with membranes, and it has been frequently proposed that this represents their antibacterial target. To see if this was a general mechanism of action, we studied the interaction, with model membranes and the cytoplasmic membrane of Escherichia coli, of 12 peptides representing all 4 structural classes of antimicrobial peptides. Planar lipid bilayer studies indicated that there was considerable variance in the interactions of the peptides with model phospholipid membranes, but generally both high concentrations of peptide and high transmembrane voltages (usually −180 mV) were required to observe conductance events (channels). The channels observed for most peptides varied widely in magnitude and duration. An assay was developed to measure the interaction with the Escherichia coli cy...

Expression and Regulation of the Human β-Defensins hBD-1 and hBD-2 in Intestinal Epithelium

Abstract: The intestinal epithelium forms a physical barrier to limit access of enteric microbes to the host and contributes to innate host defense by producing effector molecules against luminal microbes. To further define the role of the intestinal epithelium in antimicrobial host defense, we analyzed the expression, regulation, and production of two antimicrobial peptides, human defensins hBD-1 and hBD-2, by human intestinal epithelial cells in vitro and in vivo. The human colon epithelial cell lines HT-29 and Caco-2 constitutively express hBD-1 mRNA and protein but not hBD-2. However, hBD-2 expression is rapidly induced by IL-1α stimulation or infection of those cells with enteroinvasive bacteria. Moreover, hBD-2 functions as a NF-κB target gene in the intestinal epithelium as blocking NF-κB activation inhibits the up-regulated expression of hBD-2 in response to IL-1α stimulation or bacterial infection. Caco-2 cells produce two hBD-1 isoforms and a hBD-2 peptide larger in size than previously described hBD-2 isoforms. Paralleling the in vit ro findings, human fetal intestinal xenografts constitutively express hBD-1, but not hBD-2, and hBD-2 expression, but not hBD-1, is up-regulated in xenografts infected intraluminally with Salmonella . hBD-1 is expressed by the epithelium of normal human colon and small intestine, with a similar pattern of expression in inflamed colon. In contrast, there is little hBD-2 expression by the epithelium of normal colon, but abundant hBD-2 expression by the epithelium of inflamed colon. hBD-1 and hBD-2 may be integral components of epithelial innate immunity in the intestine, with each occupying a distinct functional niche in intestinal mucosal defense.

Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity.

Abstract: The antimicrobial peptide LL-37 belongs to the cathelicidin family and is the first amphipathic alpha-helical peptide isolated from human. LL-37 is considered to play an important role in the first line of defence against local infection and systemic invasion of pathogens at sites of inflammation and wounds. Understanding its mode of action may assist in the development of antimicrobial agents mimicking those of the human immune system. In vitro studies revealed that LL-37 is cytotoxic to both bacterial and normal eukaryotic cells. To gain insight into the mechanism of its non-cell-selective cytotoxicity, we synthesized and structurally and functionally characterized LL-37, its N-terminal truncated form FF-33, and their fluorescent derivatives (which retained structure and activity). The results showed several differences, between LL-37 and other native antimicrobial peptides, that may shed light on its in vivo activities. Most interestingly, LL-37 exists in equilibrium between monomers and oligomers in solution at very low concentrations. Also, it is significantly resistant to proteolytic degradation in solution, and when bound to both zwitterionic (mimicking mammalian membranes) and negatively charged membranes (mimicking bacterial membranes). The results also showed a role for the N-terminus in proteolytic resistance and haemolytic activity, but not in antimicrobial activity. The LL-37 mode of action with negatively charged membranes suggests a detergent-like effect via a 'carpet-like' mechanism. However, the ability of LL-37 to oligomerize in zwitterionic membranes might suggest the formation of a transmembrane pore in normal eukaryotic cells. To examine this possibility we used polarized attenuated total reflectance Fourier-transform infrared spectroscopy and found that the peptide is predominantly alpha-helical and oriented nearly parallel with the surface of zwitterionic-lipid membranes. This result does not support the channel-forming hypothesis, but rather it supports the detergent-like effect.

An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides

Abstract: Four macrocyclic cystine-knot peptides of 29–31 residues, kalata, circulin A and B (CirA and CirB), and cyclopsychotride, have been isolated from coffee plants but have undetermined physiological functions. These macrocycles and 10 of their analogs prepared by chemical synthesis were tested against nine strains of microbes. Kalata and CirA were specific for the Gram-positive Staphylococcus aureus with a minimum inhibition concentration of ≈0.2 μM. They were relatively ineffective against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. However, CirB and cyclopsychotride were active against both Gram-positive and Gram-negative bacteria. In particular, CirB showed potent activity against E. coli with a minimum inhibitory concentration of 0.41 μM. All four cyclic peptides were moderately active against two strains of fungi, Candida kefyr and Candida tropicalis, but were inactive against Candida albicans. These macrocycles are cytotoxic and lysed human red blood cell with a lethal dose 50% of 400 μM. Modifying the Arg residue in kalata with a keto aldehyde significantly reduced its activity against S. aureus whereas blocking the arg in CirA produced no significant effect. The two-disulfide variants and their scrambled disulfide isomers exhibited antimicrobial profiles and potency similar to their native peptides. However, in high-salt assays (100 mM NaCl), few of these macrocyclic peptides, natives or analogs, retained antimicrobial activity. These results show that the macrocyclic peptides possess specific and potent antimicrobial activity that is salt-dependent and that their initial interactions with the microbial surfaces may be electrostatic, an effect commonly found in defensin antimicrobial peptides. Furthermore, their end-to-end cyclic structure with a cystine-knot motif represents a molecular structure of antimicrobials and may provide a useful template for the design of novel peptide antibiotics.

Production of β-Defensin Antimicrobial Peptides by the Oral Mucosa and Salivary Glands

Abstract: β-Defensins are cationic peptides with broad-spectrum antimicrobial activity that are produced by epithelia at mucosal surfaces. Two human β-defensins, HBD-1 and HBD-2, were discovered in 1995 and 1997, respectively. However, little is known about the expression of HBD-1 or HBD-2 in tissues of the oral cavity and whether these proteins are secreted. In this study, we characterized the expression of HBD-1 and HBD-2 mRNAs within the major salivary glands, tongue, gingiva, and buccal mucosa and detected β-defensin peptides in salivary secretions. Defensin mRNA expression was quantitated by RNase protection assays. HBD-1 mRNA expression was detected in the gingiva, parotid gland, buccal mucosa, and tongue. Expression of HBD-2 mRNA was detected only in the gingival mucosa and was most abundant in tissues with associated inflammation. To test whether β-defensin expression was inducible, gingival keratinocyte cell cultures were treated with interleukin-1β (IL-1β) or bacterial lipopolysaccharide (LPS) for 24 h. HBD-2 expression increased ∼16-fold with IL-1β treatment and ∼5-fold in the presence of LPS. Western immunoblotting, liquid chromatography, and mass spectrometry were used to identify the HBD-1 and HBD-2 peptides in human saliva. Human β-defensins are expressed in oral tissues, and the proteins are secreted in saliva; HBD-1 expression was constitutive, while HBD-2 expression was induced by IL-1β and LPS. Human β-defensins may play an important role in the innate defenses against oral microorganisms.

Defensins and Host Defense

Abstract: Defensins are antimicrobial peptides produced by cells as part of the host9s immune defense system against invading pathogens. A Perspective by Tomas Ganz highlights three papers that provide new insights into the structure and function of vertebrate defensins. In mice, a-defensins produced by the Paneth cells of the small intestine help defend the host against invasion by intestinal pathogens such as Escherichia coli and Salmonella ( Wilson et al.). Two human b-defensins have been shown to act as chemoattractants for dendritic cells and memory T cells ( Yang et al.), and a new cyclic q-defensin identified in monkeys has powerful antimicrobial activity and may be a molecular template for designing new antibiotics ( Tang et al.).

Augmentation of Innate Host Defense by Expression of a Cathelicidin Antimicrobial Peptide

Abstract: Antimicrobial peptides, such as defensins or cathelicidins, are effector substances of the innate immune system and are thought to have antimicrobial properties that contribute to host defense. The evidence that vertebrate antimicrobial peptides contribute to innate immunity in vivo is based on their expression pattern and in vitro activity against microorganisms. The goal of this study was to investigate whether the overexpression of an antimicrobial peptide results in augmented protection against bacterial infection. C57BL/6 mice were given an adenovirus vector containing the cDNA for LL-37/hCAP-18, a human cathelicidin antimicrobial peptide. Mice treated with intratracheal LL-37/hCAP-18 vector had a lower bacterial load and a smaller inflammatory response than did untreated mice following pulmonary challenge with Pseudomonas aeruginosa PAO1. Systemic expression of LL-37/hCAP-18 after intravenous injection of recombinant adenovirus resulted in improved survival rates following intravenous injection of lipopolysaccharide with galactosamine or Escherichia coli CP9. In conclusion, the data demonstrate that expression of an antimicrobial peptide by gene transfer results in augmentation of the innate immune response, providing support for the hypothesis that vertebrate antimicrobial peptides protect against microorganisms in vivo.

Bioactive peptides encrypted in milk proteins: proteolytic activation and thropho-functional properties.

Abstract: The bioactivities of peptides encrypted in major milk proteins are latent until released and activated by enzymatic proteolysis, e.g. during gastrointestinal digestion or food processing. The proteolytic system of lactic acid bacteria can contribute to the liberation of bioactive peptides. In vitro, the purified cell wall proteinase of Lactococcus lactis was shown to liberate oligopeptides from β- and α-caseins which contain amino acid sequences present in casomorphins, casokinines, and immunopeptides. The further degradation of these peptides by endopeptidases and exopeptidases of lactic acid bacteria could lead to the liberation of bioactive peptides in fermented milk products. However, the sequences of practically all known biologically active peptides can also be cleaved by peptidases from lactic acid bacteria. Activated peptides are potential modulators of various regulatory processes in the body: Opioid peptides are opioid receptor ligands which can modulate absorption processes in the intestinal tract, angiotensinI-converting enzyme (ACE)-inhibitory peptides are hemodynamic regulators and exert an antihypertensive effect, immunomodulating casein peptides stimulate the activities of cells of the immune system, antimicrobial peptides kill sensitive microorganisms, antithrombotic peptides inhibit aggregation of platelets and caseinophosphopeptides may function as carriers for different minerals, especially calcium. Bioactive peptides can interact with target sites at the luminal side of the intestinal tract. Furthermore, they can be absorbed and then reach peripheral organs. Food-derived bioactive peptides are claimed to be health enhancing components which can be used for functional food and pharmaceutical preparations.

Antimicrobial peptides as mediators of epithelial host defense.

Abstract: Mammalian epithelial surfaces are remarkable for their ability to provide critical physiologic functions in the face of frequent microbial challenges. The fact that these mucosal surfaces remain infection-free in the normal host suggests that highly effective mechanisms of host defense have evolved to protect these environmentally exposed tissues. Throughout the animal and plant kingdoms, endogenous genetically encoded antimicrobial peptides have been shown to be key elements in the response to epithelial compromise and microbial invasion. In mammals, a variety of such peptides have been identified, including the well-characterized defensins and cathelicidins. A major source of these host defense molecules is circulating phagocytic leukocytes. However, more recently, it has been shown that resident epithelial cells of the skin and respiratory, alimentary, and genitourinary tracts also synthesize and release antimicrobial peptides. Both in vitro and in vivo data support the hypothesis that these molecules are important contributors to intrinsic mucosal immunity. Alterations in their level of expression or biologic activity can predispose the organism to microbial infection. The regulatory and developmental aspects of antimicrobial peptide synthesis are discussed from a perspective that emphasizes the possible relevance to pediatric medicine.

Myticin, a novel cysteine-rich antimicrobial peptide isolated from haemocytes and plasma of the mussel Mytilus galloprovincialis.

Abstract: We report here the isolation of two isoforms of a novel cysteine-rich peptide from haemocytes (isoform A of 4.438 Da and B of 4.562 Da) and plasma (isoform A) of the mussel, Mytilus galloprovincialis. The two molecules display antibacterial activity against gram-positive bacteria, whereas only isoform B is active against the fungus Fusarium oxysporum and a gram-negative bacteria Escherichia coli D31. Complete peptide sequences were determined by a combination of Edman degradation, mass spectrometry and cDNA cloning using a haemocyte cDNA library. The mature molecules, named myticins, comprise 40 residues with four intramolecular disulfide bridges and a cysteine array in the primary structure different to that of the previously characterized cysteine-rich antimicrobial peptides. Sequence analysis of the cloned cDNAs revealed that myticin precursors consist of 96 amino acids with a putative signal peptide of 20 amino acids, the antimicrobial peptide sequence and a 36-residue C-terminal extension. This structure suggests that myticins are synthesized as preproproteins and then processed by various proteolytic events before storage of the active peptide in the haemocytes. Myticin precursors are expressed mainly in the haemocytes as revealed by Northern blot analysis.

Salt-Resistant Alpha-Helical Cationic Antimicrobial Peptides

Abstract: Analogues based on the insect cecropin-bee melittin hybrid peptide (CEME) were studied and analyzed for activity and salt resistance. The new variants were designed to have an increase in amphipathic alpha-helical content (CP29 and CP26) and in overall positive charge (CP26). The alpha-helicity of these peptides was demonstrated by circular dichroism spectroscopy in the presence of liposomes. CP29 was shown to have activity against gram-negative bacteria that was similar to or better than those of the parent peptides, and CP26 had similar activity. CP29 had cytoplasmic membrane permeabilization activity, as assessed by the unmasking of cytoplasmic beta-galactosidase, similar to that of CEME and its more positively charged derivative named CEMA, whereas CP26 was substantially less effective. The activity of the peptides was not greatly attenuated by an uncoupler of membrane potential, carbonyl cyanide-m-chlorophenylhydrazone. The tryptophan residue in position 2 was shown to be necessary for interaction with cell membranes, as demonstrated by a complete lack of activity in the peptide CP208. Peptides CP29, CEME, and CEMA were resistant to antagonism by 0.1 to 0.3 M NaCl; however, CP26 was resistant to antagonism only by up to 160 mM NaCl. The peptides were generally more antagonized by 3 and 5 mM Mg2+ and by the polyanion alginate. It appeared that the positively charged C terminus in CP26 altered its ability to permeabilize the cytoplasmic membrane of Escherichia coli, although CP26 maintained its ability to kill gram-negative bacteria. These peptides are potential candidates for future therapeutic drugs.

Transfer of a cathelicidin peptide antibiotic gene restores bacterial killing in a cystic fibrosis xenograft model

Abstract: Recent studies suggest that the gene defect in cystic fibrosis (CF) leads to a breach in innate immunity. We describe a novel genetic strategy for reversing the CF-specific defect of antimicrobial activity by transferring a gene encoding a secreted cathelicidin peptide antibiotic into the airway epithelium grown in a human bronchial xenograft model. The airway surface fluid (ASF) from CF xenografts failed to kill Pseudomonas aeruginosa or Staphylococcus aureus. Partial reconstitution of CF transmembrane conductance regulator expression after adenovirus-mediated gene transfer restored the antimicrobial activity of ASF from CF xenografts to normal levels. Exposure of CF xenografts to an adenovirus expressing the human cathelicidin LL-37/hCAP-18 increased levels of this peptide in the ASF three- to fourfold above the normal concentrations, which were equivalent in ASF from CF and normal xenografts before gene transfer. The increase of LL-37 was sufficient to restore bacterial killing to normal levels. The data presented describe an alternative genetic approach to the treatment of CF based on enhanced expression of an endogenous antimicrobial peptide and provide strong evidence that expression of antimicrobial peptides indeed protects against bacterial infection.

Recombinant expression and range of activity of penaeidins, antimicrobial peptides from penaeid shrimp

Abstract: Penaeidins are 5.5- to 6.6-kDa antimicrobial peptides recently isolated from the plasma and haemocytes of the tropical shrimp Penaeus vannamei. These molecules differ from the other classes of antimicrobial peptides in that they are composed of a proline-rich N-terminus and of a C-terminus containing six cysteine residues engaged in three disulfide bridges. In order to gain information on their antimicrobial activity, two penaeidins (Pen-2 and Pen-3a) were expressed in Saccharomyces cerevisiae. The recombinant Pen-2 and -3a were characterized in terms of primary structure by Edman degradation, mass spectrometry and gas chromatography. A protocol was then established to purify the amount of penaeidins required for the determination of their activity spectrum. We demonstrate in this study that expression in yeast is appropriate for the large-scale production of functional penaeidins, whose activities are almost indistinguishable from those of the native molecules. Data on Pen-2 and -3a activity demonstrate that penaeidins have a broad spectrum of antifungal properties associated with a fungicidal activity, and that their antibacterial activities are essentially directed against Gram-positive bacteria, with a strain-specific inhibition mechanism. Despite a better efficiency of Pen-3a on most of the tested strains, similar activity spectra and inhibition mechanisms were observed for both Pen-2 and -3a. Finally, no synergistic effect could be observed between the two molecules.

Biological Properties of Structurally Related α-Helical Cationic Antimicrobial Peptides

Abstract: A series of alpha-helical cationic antimicrobial peptide variants with small amino acid changes was designed. Alterations in the charge, hydrophobicity, or length of the variant peptides did not improve the antimicrobial activity, and there was no statistically significant correlation between any of these factors and the MIC for Pseudomonas aeruginosa, Escherichia coli, or Salmonella typhimurium. Individual peptides demonstrated synergy with conventional antibiotics against antibiotic-resistant strains of P. aeruginosa. The peptides varied considerably in the ability to bind E. coli O111:B4 lipopolysaccharide (LPS), and this correlated significantly with their antimicrobial activity and ability to block LPS-stimulated tumor necrosis factor and interleukin-6 production. In general, the peptides studied here demonstrated a broad range of activities, including antimicrobial, antiendotoxin, and enhancer activities.