1. Evaluation of susceptibility of gram-positive and -negative bacteria to human defensins by using radial diffusion assay
H Takemura, M Kaku, S Kohno, Y Hirakata, H Tanaka, R Yoshida, K Tomono, H Koga, A Wada, T Hirayama, S Kamihira Antimicrob Agents Chemother. 1996 Oct;40(10):2280-4. doi: 10.1128/AAC.40.10.2280.
Defensins are small cationic bactericidal peptides present abundantly in the granules of polymorphonuclear neutrophils (PMNs). Human PMNs contain four defensins termed HNP-1 to HNP-4. We used a new assay system in agar plates, the radial diffusion assay, to evaluate the effects of human defensins against gram-positive and -negative bacteria. A crude mixture of HNP-1, -2, and -3 (crude HNPs) was purified from human PMN extracts by reversed-phase high-pressure liquid chromatography (RP-HPLC). The different components were later separated by RP-HPLC and gel permeation chromatography. We compared the antibacterial activities of purified HNP-1, -2, and -3 against Escherichia coli, Pseudomonas aeruginosa, methicillin-susceptible Staphylococcus aureus, and methicillin-resistant S. aureus strains using the radial diffusion assay. The antibacterial activities of HNP-1 and HNP-2 against all strains tested were similar to those of the crude HNPs, but the activity of HNP-3 was less than those of the other defensins. To quantitate the activities of HNPs against different bacteria, we defined the minimal dose of crude HNPs forming a detectable clear zone around the bacteria as the minimal inhibitory dose (MID) and determined the MIDs for 10 strains of E. coli, 12 strains of P. aeruginosa, 10 strains of methicillin-susceptible S. aureus, and 12 strains of methicillin-resistant S. aureus isolates, including clinical isolates. In general, the MIDs of the HNPs were similar against similar bacterial species. However, the MIDs for P. aeruginosa were higher than those for the other organisms tested. The radial diffusion assay is suitable as a screening test for measuring the susceptibilities of isolates to defensins, because it is sensitive and simple and has good reproducibility.
2. Purification, primary structures, and antibacterial activities of beta-defensins, a new family of antimicrobial peptides from bovine neutrophils
M E Selsted, Y Q Tang, W L Morris, P A McGuire, M J Novotny, W Smith, A H Henschen, J S Cullor J Biol Chem. 1993 Mar 25;268(9):6641-8.
A new family of cysteine-rich antimicrobial peptides from bovine neutrophils was isolated and characterized. Thirteen structurally homologous peptides were purified to homogeneity from a granule-rich cytoplasmic fraction of purified blood neutrophils. The complete sequences of the peptides were determined by a combination of enzymatic digestion, Edman degradation, and additional biochemical characterization of the carboxyl termini. The peptides are characterized by a highly cationic 38-42-residue chain which includes 6 invariantly spaced cysteines which form three disulfides. They share a highly conserved consensus sequence which is also found in a recently described epithelial antimicrobial peptide from bovine trachea. The in vitro antibacterial activities of the 13 neutrophil peptides, determined in assays using Staphylococcus aureus and Escherichia coli as test organisms, demonstrated that each peptide possessed antimicrobial activity, and that several were as active as the most potent neutrophil defensin, rabbit NP-1. Though the structural and functional attributes of the bovine neutrophil peptides are similar to those of defensins, the two peptide families are distinguished by their unique consensus sequences and additionally by differing tridisulfide motifs. We therefore propose that this new defensin-like antimicrobial peptide family be named beta-defensins.
3. A novel big defensin identified in horseshoe crab hemocytes: isolation, amino acid sequence, and antibacterial activity
T Saito, S Kawabata, T Shigenaga, Y Takayenoki, J Cho, H Nakajima, M Hirata, S Iwanaga J Biochem. 1995 May;117(5):1131-7. doi: 10.1093/oxfordjournals.jbchem.a124818.
Hemocytes of the horseshoe crab (limulus) contain a family of arthropodous peptide antibiotics, termed the tachyplesin family, and antibacterial protein, called anti-LPS factor, of which the former is located in the small (S) granules and the latter in the large (L) granules of the hemocytes. In our ongoing studies on granular components, we have identified here a novel defensin-like substance present in both L- and S-granules. This substance strongly inhibits the growth of Gram-negative and -positive bacteria, and fungi, such as Candida albicans. The isolated substance, tentatively termed "big defensin," consists of 79 amino acid residues, of which the COOH-terminal 37 residues have a sequence similar to those of mammalian neutrophil-derived defensins, especially rat defensin. Characterization of the disulfide motif in big defensin indicated that the disulfide array is identical to that of beta-defensins from bovine neutrophils. One clear structural difference is that the limulus hemocyte-derived big defensin has an extension of the NH2-terminal hydrophobic sequence with 35 amino acid residues followed by the COOH-terminal cationic defensin portion. This amphipathic nature of big defensin seems likely to be associated with its potent antibacterial activity. Furthermore, antibacterial activities of the NH2-terminal hydrophobic region and the COOH-terminal defensin portion separated by tryptic digestion are significantly different: the former displays a more potent activity against Gram-positive bacteria, whereas the latter is more potent against Gram-negative bacteria. Big defensin, therefore, may prove to represent a new class of defensin family possessing two functional domains with different antimicrobial activities.
