protegrin-1 and Pseudomonas-Infections

Antibiotics with new mechanisms of action are urgently required to combat the growing health threat posed by resistant pathogenic microorganisms. We synthesized a family of peptidomimetic antibiotics based on the antimicrobial peptide protegrin I. Several rounds of optimization gave a lead compound that was active in the nanomolar range against Gram-negative Pseudomonas spp., but was largely inactive against other Gram-negative and Gram-positive bacteria. Biochemical and genetic studies showed that the peptidomimetics had a non-membrane-lytic mechanism of action and identified a homolog of the beta-barrel protein LptD (Imp/OstA), which functions in outer-membrane biogenesis, as a cellular target. The peptidomimetic showed potent antimicrobial activity in a mouse septicemia infection model. Drug-resistant strains of Pseudomonas are a serious health problem, so this family of antibiotics may have important therapeutic applications.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacterial Outer Membrane Proteins; Cell Membrane; Drug Design; Drug Resistance, Bacterial; Genes, Bacterial; Lipopolysaccharides; Mice; Microbial Sensitivity Tests; Molecular Mimicry; Mutation; Peptide Library; Peptides; Protein Structure, Tertiary; Pseudomonas aeruginosa; Pseudomonas Infections; Sepsis

Antimicrobial peptides are naturally occurring cationic peptides. The first-line of defense in infected burns is the innate immune system, of which antimicrobial peptides are essential parts. To facilitate their topical use in infected partial-thickness burns, the efficacy of a mixture with fibrin glue in vitro and in vivo was tested.. After in vitro tests, 15 male Sprague-Dawley rats received partial-thickness burns. Afterwards, the wounds were infected with multiresistant Pseudomonas aeruginosa. The animals received PG-1 (100 microg/ml, n=5), fibrin glue (n=5), or a mixture of both (n=5) topically. The efficacy of the materials was previously proven by radial diffusion assay. After 24 h, the infected and burned skin was harvested and quantitative bacterial counts per gram of skin performed.. The biologic effect of the peptides was confirmed in vitro. The PG-1 and fibrin glue groups did not show significant differences in bacterial numbers, whereas the mixture group showed significant reduction in Pseudomonas in vivo (P<0.04 and P<0.01).. A mixture of an antimicrobial peptide and commercially available fibrin glue is capable of significantly reducing bacteria in infected partial thickness burns in vivo compared to controls.

Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Burns; Colony Count, Microbial; Dose-Response Relationship, Drug; Drug Combinations; Drug Resistance, Multiple; Escherichia coli; Fibrin Tissue Adhesive; Male; Microbial Sensitivity Tests; Proteins; Pseudomonas Infections; Rats; Rats, Sprague-Dawley; Wound Infection

Infections with multidrug-resistant microorganisms (e.g. Pseudomonas aeruginosa and Staphylococcus aureus) cause immense complications in wound care and in the treatment of immunosuppressed patients. Like most antimicrobial peptides, histones are relatively small polycationic proteins located in each eukaryotic nucleus, which naturally supercoil DNA. The aim of this study was to investigate the in vitro and in vivo activity of histone H1.2 in infected burn wounds and its potential toxicity.. To characterize the antimicrobial properties of histone H1.2 against potential causative organisms of burn wound infections, the in vitro radial diffusion assay and modified NCCLS microbroth dilution MIC assay were carried out. Haemolytic and cytotoxic properties were determined in human red blood cells and primary human keratinocytes. In vivo antimicrobial activity was tested in an infected rat burn model with P. aeruginosa (ATCC 27853). All results were compared with the naturally occurring broad-spectrum antimicrobial peptide protegrin-1 and with antibiotics clinically used against the corresponding bacteria.. Human histone H1.2 exerted good antimicrobial activity against all tested microorganisms without significant haemolytic activity. Surprisingly, histone H1.2 showed cytotoxicity with an LD50 of 7.91 mg/L in primary human keratinocytes. The in vivo burn model data revealed a significant three-fold higher reduction in bacterial counts within 4 h compared with carrier control.. These findings indicate that histone H1.2 is a potential candidate for use as a local and, because of its low haemolytic activity, systemic antimicrobial agent. However, further investigations are needed to specify the cytotoxicity and the dose-response relationship for histone H1.2.

Topics: Animals; Antimicrobial Cationic Peptides; Bacteria; Burns; Cells, Cultured; Disease Models, Animal; Erythrocytes; Hemolysis; Histones; Humans; Keratinocytes; Microbial Sensitivity Tests; Proteins; Pseudomonas aeruginosa; Pseudomonas Infections; Rats; Skin; Toxicity Tests; Wound Infection

We investigated the therapeutic potential of the pig-derived antimicrobial peptide protegrin-1 (PG-1) against porcine skin wounds infected with Pseudomonas aeruginosa.. Using a porcine skin wound model, PG-1 was added to the wound fluid either at the time of P. aeruginosa inoculation, four hours after inoculation or 24 hours after inoculation. Wound fluids were analyzed 20-24 hours later by use of colony-forming unit (CFU) assays, semiquantitative immunoblot analysis for PG-1, and radial diffusion assays (RDA) for residual in vitro activity.. Results of the CFU assays indicated a 10,000-fold decrease in the number of bacteria when PG-1 was added at the time of inoculation, a 120-fold decrease when added 4 hours after inoculation and a 10-fold decrease when added 24 hours after inoculation. Results of immunoblot analysis and RDA indicated that PG-1 concentrations for each of the three conditions remained increased in wound fluid 20 to 24 hours after treatment, and correlated with increased residual in vitro antimicrobial activity.. These results document that the endogenous antibiotic PG-1 significantly prevented the colonization of P. aeruginosa in wounds and reduced the in vivo bacterial concentration in established wound infections. Therapeutics used in the same animal species from which they were derived are a promising means for preventing and treating localized infections.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Female; In Vitro Techniques; Proteins; Pseudomonas aeruginosa; Pseudomonas Infections; Skin; Swine; Swine Diseases; Wound Infection