Range of activity and metabolic stability of synthetic antibacterial glycopeptides from insects.
Antibacterial glycopeptides isolated from insects are exciting bio-oligomers because they represent a family of compounds in which the structural and functional effects of incorporating short O-linked sugars to protein fragments can be studied. Additionally, their high activity in vitro warrants detailed further drug development efforts. Due to the limited availability of the isolated material, we used synthetic glycopeptides and some analogs to investigate the range of activity of drosocin and pyrrhocoricin. While addition of the Gal-GalNAc disaccharide to the natural mid-chain position generally increased the antibacterial activity of drosocin, pyrrhocoricin lacking sugar appeared to be more potent, with an IC50 against Escherichia coli D22 of 150 nM. Although glycosylated drosocin was active against E. coli in the low microM range in vitro, this peptide was completely inactive when injected into mice. The lack of in vivo activity of drosocin could be explained by the unusually high degradation rate of the peptides in mammalian sera. The early degradation products were inactive in vitro. In contrast, the peptides were considerably more stable in insect hemolymph, where their natural activity is manifested. (+info)
Biological properties of structurally related alpha-helical cationic antimicrobial peptides.
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. (+info)
Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides.
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. (+info)
Recombinant bactericidal/permeability-increasing protein (rBPI21) in combination with sulfadiazine is active against Toxoplasma gondii.
The activity of recombinant bactericidal/permeability-increasing protein (rBPI21), alone or in combination with sulfadiazine, on the intracellular replication of Toxoplasma gondii was assessed in vitro and in mice with acute toxoplasmosis. rBPI21 markedly inhibited the intracellular growth of T. gondii in human foreskin fibroblasts (HFFs). Following 72 h of exposure, the 50% inhibitory concentration of rBPI21 for T. gondii was 2.6 micrograms/ml, whereas only slight cytotoxicity for HFF cells was observed at the concentrations tested. Subsequent mathematical analyses revealed that the combination of rBPI21 with sulfadiazine yielded slight to moderate synergistic effects against T. gondii in vitro. Infection of mice orally with C56 cysts or intraperitoneally (i.p.) with RH tachyzoites resulted in 100% mortality, whereas prolongation of the time to death or significant survival (P = 0.002) was noted for those animals treated with 5 to 20 mg of rBPI21 per kg of body weight per day. Treatment with rBPI21 in combination with sulfadiazine resulted in significant (P = 0.0001) survival of mice infected i.p. with tachyzoites but not of mice infected orally with T. gondii cysts. These results indicate that rBPI21 is active in vitro and in vivo against T. gondii and that its activity is significantly enhanced when it is used in combination with sulfadiazine. To our knowledge, this is the first report of the activity of rBPI21 against a protozoan parasite. (+info)
In vitro antibacterial properties of pexiganan, an analog of magainin.
Pexiganan, a 22-amino-acid antimicrobial peptide, is an analog of the magainin peptides isolated from the skin of the African clawed frog. Pexiganan exhibited in vitro broad-spectrum antibacterial activity when it was tested against 3,109 clinical isolates of gram-positive and gram-negative, anaerobic and aerobic bacteria. The pexiganan MIC at which 90% of isolates are inhibited (MIC90) was 32 micrograms/ml or less for Staphylococcus spp., Streptococcus spp., Enterococcus faecium, Corynebacterium spp., Pseudomonas spp., Acinetobacter spp., Stenotrophomonas spp., certain species of the family Enterobacteriaceae, Bacteroides spp., Peptostreptococcus spp., and Propionibacterium spp. Comparison of the MICs and minimum bactericidal concentrations (MBCs) of pexiganan for 143 isolates representing 32 species demonstrated that for 92% of the isolates tested, MBCs were the same or within 1 twofold difference of the MICs, consistent with a bactericidal mechanism of action. Killing curve analysis showed that pexiganan killed Pseudomonas aeruginosa rapidly, with 10(6) organisms/ml eliminated within 20 min of treatment with 16 micrograms of pexiganan per ml. No evidence of cross-resistance to a number of other antibiotic classes was observed, as determined by the equivalence of the MIC50s and the MIC90s of pexiganan for strains resistant to oxacillin, cefazolin, cefoxitin, imipenem, ofloxacin, ciprofloxacin, gentamicin, and clindamicin versus those for strains susceptible to these antimicrobial agents. Attempts to generate resistance in several bacterial species through repeated passage with subinhibitory concentrations of pexiganan were unsuccessful. In conclusion, pexiganan exhibits properties in vitro which make it an attractive candidate for development as a topical antimicrobial agent. (+info)
A gene encoding a hevein-like protein from elderberry fruits is homologous to PR-4 and class V chitinase genes.
We isolated SN-HLPf (Sambucus nigra hevein-like fruit protein), a hevein-like chitin-binding protein, from mature elderberry fruits. Cloning of the corresponding gene demonstrated that SN-HLPf is synthesized as a chimeric precursor consisting of an N-terminal chitin-binding domain corresponding to the mature elderberry protein and an unrelated C-terminal domain. Sequence comparisons indicated that the N-terminal domain of this precursor has high sequence similarity with the N-terminal domain of class I PR-4 (pathogenesis-related) proteins, whereas the C terminus is most closely related to that of class V chitinases. On the basis of these sequence homologies the gene encoding SN-HLPf can be considered a hybrid between a PR-4 and a class V chitinase gene. (+info)
Endocytosis of heparin-binding protein (CAP37) is essential for the enhancement of lipopolysaccharide-induced TNF-alpha production in human monocytes.
Heparin-binding protein (HBP), also known as CAP37, is a proteolytically inactive serine protease homologue that is released from activated granulocytes. However, HBP is not a biologically inactive molecule but rather a multifunctional protein with properties that include the enhancement of LPS-induced TNF-alpha production from monocytes. We have previously demonstrated that HBP is internalized in monocytes. In the current study, we hypothesize that HBP is internalized in monocytes via endocytosis, and this internalization is an important mechanism by which HBP enhances LPS-induced TNF-alpha release. Using whole blood from healthy donors and flow cytometry, we found that colchicine (0.1-10 mM), cytochalasin D (1000 microM), NH4Cl (10-50 mM), and bafilomycin A1 (0.1-3 microM) significantly reduced the affinity of FITC-HBP for CD14-positive monocytes. Using isolated human monocytes and ELISA, we found that colchicine (0.1 mM), cytochalasin D (30 and 300 microM), NH4Cl (30 mM), and bafilomycin A1 (1 microM) significantly reduced the effect of HBP (10 microg/ml) to enhance LPS (10 ng/ml)-induced TNF-alpha release after 24 h. These findings demonstrate that internalization of HBP in monocytes is essential for the enhancement of LPS-induced TNF-alpha release. Transport of HBP to an activating compartment depends on intact F-actin polymerization and endosomal acidification, an important mechanism for endosomal protein sorting and trafficking. (+info)
Transfer of a cathelicidin peptide antibiotic gene restores bacterial killing in a cystic fibrosis xenograft model.
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. (+info)