protegrin-1 and protegrin-5

Protegrins are broad spectrum antibiotic peptides isolated from porcine leukocytes. In this study, we (i) examine the sensitivity of Gram-negative, anaerobic periodontal pathogens to synthetic protegrins; (ii) determine the relative potencies of protegrin congeners against these bacteria; and (iii) compare the potency of protegrins with other antibiotic peptides, including magainin MSI-78, tachyplesin I, cecropin P1, human defensins HNP-1-3, and clavanin A. Synthetic L- and D-enantiomers of protegrin 1 (PG-1 and D-PG-1, respectively), and L-enantiomers of protegrins 2, 3 and 5 (PG-2, PG-3 and PG-5) were tested against Fusobacterium nucleatum, and black-pigmented organisms including Porphyromonas gingivalis and Prevotella intermedia. Strains of both F. nucleatum and the black-pigmented organisms were sensitive to PG-1, and exhibited mean ED99 of 2.2-2.3 micrograms/ml and 3.4-9.9 micrograms/ml, respectively. The D-form was statistically more potent than the L-form against these oral anaerobes, and although this difference in potency is unlikely to be of decisive therapeutic significance, the D-form may be of value given ability to resist microbial and host-derived proteases. PG-1 was more potent than magainin, tachyplesin, cecropin, defensins and clavanin under test conditions. Hypertonic salt concentrations and heat-inactivated serum were found to be inhibitory to the bactericidal activity of PG-1. PG-1 was found to induce morphologic alterations in the ultrastructural appearance of F. nucleatum consistent with damage to the bacterial membranes. We conclude that protegrins may be useful antimicrobial agents in therapy against Gram-negative anaerobic bacteria believed to be involved in chronic, adult forms of periodontal infections.

Topics: alpha-Defensins; Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Blood Proteins; Cell Membrane; Colony Count, Microbial; Defensins; DNA-Binding Proteins; Fusobacterium nucleatum; Gram-Negative Anaerobic Bacteria; Magainins; Microbial Sensitivity Tests; Molecular Sequence Data; Peptides; Peptides, Cyclic; Porphyromonas gingivalis; Prevotella intermedia; Proteins; Swine; Toxicity Tests; Xenopus Proteins

We used a two-stage radial diffusion assay to perform a structure-activity study of the antifungal effects of protegrin-1 (PG-1) on yeast-phase Candida albicans. While doing so, we computed MICs from the radial diffusion assay data by three methods and compared the respective values with results from colony count and broth microdilution assays. This allowed us to identify several technical modifications that improved the sensitivity and accuracy of radial diffusion assays. We found that both PG-1 and enantiomeric PG-1 (composed exclusively of D-amino acids) were potently fungicidal for yeast-phase C. albicans. The protegrins PG-2, -3, and -5, but not PG-4, were as effective as PG-1. At least one intramolecular disulfide bond was required to retain optimal candidacidal activity at physiological NaCl concentrations. Truncated variants of PG-1 that lacked its first four residues showed decreased candidacidal activity, although their activity against bacteria was substantially intact. Altering the beta-turn region (residues 9 to 12) of PG-1 or its variants further decreased candidacidal activity. These studies suggest that only 12 residues are needed to endow protegrin molecules with strong antibacterial activity and that at least 4 additional residues are needed to add potent antifungal properties. Thus, the 16-residue protegrin PG-2 likely represents the minimal structure needed for broad-spectrum antimicrobial activity encompassing bacteria and fungi.

Topics: Amino Acid Sequence; Antifungal Agents; Antimicrobial Cationic Peptides; Candida; Candida albicans; Cations, Divalent; Colony Count, Microbial; Diffusion; Disulfides; Drug Interactions; Microbial Sensitivity Tests; Molecular Sequence Data; Peptides; Protein Structure, Secondary; Proteins; Species Specificity; Structure-Activity Relationship

Protegrins, small peptides (1900 to 2160 daltons) isolated from porcine leukocytes, are bactericidal against a broad range of medical pathogens in vitro under conditions which reflect the extracellular milieu. The purpose of this study was to determine whether Gram-negative, facultative periodontal pathogens were sensitive to the protegrins. Synthetic L- and D-enantiomers of protegrin 1 (PG-1 and D-PG-1, respectively), and L-enantiomers of protegrins 2, 3, and 5 (PG-2, PG-3, and PG-5) were tested against Actinobacillus actinomycetemcomitans (three strains) and Capnocytophaga spp. (three strains). Strains of both A. actinomycetemcomitans and Capnocytophaga spp. were sensitive to PG-1, and exhibited ED99 (dose at which 99% killing was observed after 1 hr at 37 degrees C) of 0.5 to 3 microg/mL and 4 to 19 microg/mL, respectively. The D-form and the L-form were equally effective. Serum (above 5% v/v) inhibited the bactericidal effects of 10 microg/mL PG-1, but the inhibitory effect was overcome by concentrations of PG-1 at 100 microg/mL. Different patterns of sensitivity were observed when the effects of PG-1, D-PG-1, PG-2, PG-3, and PG-5 were compared against A. actinomycetemcomitans and the Capnocytophaga. We conclude that protegrins may be useful antimicrobial agents in therapy against periodontal infections.

Topics: Aggregatibacter actinomycetemcomitans; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Blood Physiological Phenomena; Capnocytophaga; Colony Count, Microbial; Humans; Leukocytes; Microbial Sensitivity Tests; Periodontal Diseases; Proteins; Swine

We tested 20 protegrins against Chlamydia trachomatis serovar L2 (L2/434/Bu). Five of the protegrins had native structures; the others included nonamidated, enantiomeric, and truncated variants and peptides with <2 disulfide bonds. Antichlamydial activity resided principally in residues 5 to 15 of native protegrin PG-1, and optimal activity required both intramolecular disulfide bonds.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Cell Line; Chlamydia trachomatis; Mice; Molecular Sequence Data; Proteins

We cloned the genes of three protegrins, a family of cathelin-associated antimicrobial peptides originally isolated from porcine leukocytes. Each gene comprised 4 exons and 3 introns, wherein Exon I encoded the signal sequence and the first 37 amino acids of cathelin, Exons II and III contained 36 and 24 additional cathelin residues and Exon IV contained the final two cathelin residues followed by the protegrin sequence. This quadripartite gene structure helps explain how structurally diverse antimicrobial peptides can be expressed on common, cathelin-containing precursors. Southern blot probed with an oligonucleotide specific for protegrin genes suggested that several identical or nearly identical protegrin genes were densely clustered in the pig chromosome.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Base Sequence; Cloning, Molecular; Consensus Sequence; Exons; Genes; Introns; Molecular Sequence Data; Multigene Family; Proteins; Sequence Analysis, DNA; Swine