Defensin-neurotoxin dyad in a basally branching metazoan sea anemone.

Recent studies suggest that vertebrate and invertebrate defensins have evolved from two independent ancestors, and that both defensins could share origins with animal toxins. Here, we purified novel sea anemone neurotoxin (BDS)-like antimicrobial peptides (AMPs)-Crassicorin-I and its putative homolog (Crassicorin-II)-from the pharynx extract of an anthozoan sea anemone (Urticina crassicornis). Based on structural analyses and cDNA cloning, mature Crassicorin-I represents a cationic AMP likely generated from a precursor and comprising 40 amino acid residues, including six cysteines forming three intramolecular disulfide bonds. Recombinant Crassicorin-I produced in a heterologous bacterial-expression system displayed antimicrobial activity against both a gram-positive bacterium (Bacillus subtilis) and gram-negative bacteria (Escherichia coli and Salmonella enterica). The Crassicorin-I transcript was upregulated by immune challenge, suggesting its involvement in defense mechanisms against infectious pathogens in sea anemone. Sequence alignment and three-dimensional molecular modeling revealed that Crassicorin-I exhibits high degrees of structural similarity to sea anemone neurotoxins that share ß-defensin fold which is found in vertebrate defensins and invertebrate big-defensins. Consistent with its structural similarity to neurotoxins, Crassicorin-I exhibited paralytic activity toward a crustacean. These findings motivated our investigation and subsequent discovery of antimicrobial activity from other known sea anemone neurotoxins, such as APETx1 and ShK. Collectively, our work signified that Crassicorin-I is the first AMP identified from a sea anemone and provided evidence of a functional linkage between AMPs and neurotoxins in a basally branching metazoan.

Toxicity assessment of (99m)technetium-labeled human beta-defensin-3 in CD1 mice.

OBJECTIVE: Human beta-defensin-3 (HBD-3) is an antimicrobial peptide which is up-regulated during inflammation. Based on the previously demonstrated capacity of technetium-99m ((99m)Tc) labelled HBD-3 of distinguishing infection from inflammation in rats, we have decided to collect information on the potential toxicity of the tracer in view of its possible use for imaging in humans. MATERIALS AND METHODS: Recombinant HBD-3 underwent labeling with (99m)Tc. The CD1 mice were selected as standard rodent species. Ten mice, 5 male and 5 female, were subjected to physical examination and housed in a dedicated room in 5 per cage. After 9 days pre-test period, all mice were weighted for dose adjustment and received intravenously 6mcg/mouse of (99m)Tc-HBD-3. Mortality was recorded daily, while body weight was registered once a week. Clinical observation of animals was performed daily for sickness symptoms due to the drug treatment. At day 19 a second dose of 6mcg/mouse (99m)Tc-HBD-3, was administered. Twenty-four hours after the second dose (day 20) the animals were euthanized. A piece of liver, kidneys, heart and lungs was collected for histopathological analysis. RESULTS: Our results showed that the labelled-HBD-3 dose did not induce significant toxicity in mice. Of course these parameters were not sufficient to authorize use in humans. This non-toxic dose of HBD-3 when translated from animals to humans resulted in an equivalent dose of approximately 25 times higher than that needed for imaging. CONCLUSION: Our non toxicity data of using (99m)Tc-beta-defensin-3 in mice offer a further indication in favour of the clinical use of this radiopharmaceutical in all cases where discrimination between infection and inflammation is needed.

Cytotoxicity of HBD3 for dendritic cells, normal human epidermal keratinocytes, hTERT keratinocytes, and primary oral gingival epithelial keratinocytes in cell culture conditions.

Human ß-defensin 3 (HBD3) is a prominent host defense peptide. In our recent work, we observed that HBD3 modulates pro-inflammatory agonist-induced chemokine and cytokine responses in human myeloid dendritic cells (DCs), often at 20.0 µM concentrations. Since HBD3 can be cytotoxic in some circumstances, it is necessary to assess its cytotoxicity for DCs, normal human epidermal keratinocytes (NHEKs), human telomerase reverse transcriptase (hTERT) keratinocytes, and primary oral gingival epithelial (GE) keratinocytes in different cell culture conditions. Cells, in serum free media with resazurin and in complete media with 10% fetal bovine serum and resazurin, were incubated with 5, 10, 20, and 40 µM HBD3. Cytotoxicity was determined by measuring metabolic conversion of resazurin to resorufin. The lethal dose 50 (LD50, mean µM±Std Err) values were determined from the median fluorescent intensities of test concentrations compared to live and killed cell controls. The LD50 value range of HBD3 was 18.2-35.9 µM in serum-free media for DCs, NHEKs, hTERT keratinocytes, and GE keratinocytes, and >40.0 µM in complete media. Thus, HBD3 was cytotoxic at higher concentrations, which must be considered in future studies of HBD3-modulated chemokine and cytokine responses in vitro.

Antimicrobial activities of chicken ß-defensin (4 and 10) peptides against pathogenic bacteria and fungi.

Host Defense Peptides (HDPs) are small cationic peptides found in several organisms. They play a vital role in innate immunity response and immunomodulatory stimulation. This investigation was designed to study the antimicrobial activities of ß-defensin peptide-4 (sAvBD-4) and 10 (sAvBD-4) derived from chickens against pathogenic organisms including bacteria and fungi. Ten bacterial strains and three fungal species were used in investigation. The results showed that the sAvBD-10 displayed a higher bactericidal potency against all the tested bacterial strains than that of sAvBD-4. The exhibited bactericidal activity was significant against almost the different bacterial strains at different peptide concentrations except for that of Pseudomonas aeruginosa (P. aeruginosa) and Streptococcus bovis (Str. bovis) strains where a moderate effect was noted. Both peptides were effective in the inactivation of fungal species tested yielding a killing rate of up to 95%. The results revealed that the synthetic peptides were resistant to salt at a concentration of 50 mM NaCl. However, they lost antimicrobial potency when applied in the presence of high salt concentrations. Based on blood hemolysis studies, a little hemolytic effect was showed in the case of both peptides even when applied at high concentrations. The data obtained from this study indicated that synthetic avian peptides exhibit strong antibacterial and antifungal activity. In conclusion, future work and research should be tailored to a better understanding of the mechanisms of action of those peptides and their potential use in the pharmaceutical industry to help reduce the incidence and impact of infectious agent and be marketed as a naturally occurring antibiotic.

Antimicrobial characterization of site-directed mutagenesis of porcine beta defensin 2.

Porcine ß defensin 2 (pBD2) is a small, cationic and amphiphilic antimicrobial peptide. It has broad antimicrobial activities against bacteria and plays an important role in host defense. In order to enhance its antimicrobial activity and better understand the effect of positively charged residues on its activity, we substituted eight amino acid residues with arginine or lysine respectively. All mutants were cloned and expressed in BL21 (DE3) plysS and the mutant proteins were then purified. These mutant versions had higher positive charges but similar structural configurations compared to the wild-type pBD2. Moreover, these mutant proteins showed different antimicrobial activities against E. coli and S. aureus. The mutant I4R of pBD2 had the highest antimicrobial activity. In addition, all the mutants showed low hemolytic activities. Our results indicated that the positively charged residues were not the only factor that influenced antimicrobial activity, but other factors such as distribution of these residues on the surface of defensins might also contribute to their antimicrobial potency.

Antimicrobial peptide coating of dental implants: biocompatibility assessment of recombinant human beta defensin-2 for human cells.

PURPOSE: Artificial materials such as dental implants are at risk of bacterial contamination in the oral cavity. Human beta defensins (HBDs), small cationic antimicrobial peptides that exert a broad-spectrum antibacterial function at epithelial surfaces and within some mesenchymal tissues, could probably help to reduce such contamination. HBDs also have protective immunomodulatory effects and have been reported to promote bone remodeling. The aim of this study, therefore, was to investigate the influence of recombinant HBD-2 on the proliferation and survival of cells in culture. MATERIALS AND METHODS: Human mesenchymal stem cells (hMSCs), human osteoblasts, human keratinocytes (control), and the HeLa cancer cell line (control) were incubated with recombinant HBD-2 (1, 5, 10, or 20 µg/mL). Cell proliferation and cytotoxicity were evaluated via a water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase assays, respectively. RESULTS: HBD-2 was not toxic in any tested concentration to hMSCs, osteoblasts, keratinocytes, or HeLa cells. Furthermore, proliferation of hMSCs and osteoblasts increased after treatment with HBD-2 at all tested concentrations, and keratinocyte proliferation increased when treated at 20 µg/mL. In contrast, HeLa cancer cells were not affected by HBD-2 as tested. CONCLUSIONS: HBD-2 is not only biocompatible but also promotes proliferation of hMSCs, osteoblasts, and keratinocytes in culture. Further investigation of HBD-2 functional surface coating of artificial materials is recommended.

Antiviral activity of recombinant mouse ß-defensin 3 against influenza A virus in vitro and in vivo.

BACKGROUND: Influenza causes significant morbidity and mortality. Mammalian ß-defensins are small peptides of about 4.5-6 kDa in mass and are effectors of the innate immune response with potent antimicrobial activity. In this paper, we focused on the anti-influenza A activity of the recombinant mouse ß-defensin 3 (rMBD-3) in vivo and in vitro. METHODS: The rMBD-3 peptide was added to Madin-Darby canine kidney (MDCK) cells at different stages of influenza A virus (IAV) A/PR/8/34 (H1N1) infection and its virus inhibitory properties were determined. Mice were infected with IAV and treated with rMBD-3 peptide from 12 h post-infection. The effect of rMBD-3 peptide was determined by pulmonary viral load, pathology and mortality. In addition, the expression of interleukin (IL)-12, interferon (IFN)-γ and tumour necrosis factor (TNF)-α genes in mice with or without rMBD-3 treatment was determined by semi-quantitative reverse transcriptase PCR. RESULTS: rMBD-3 was shown to protect MDCK cells against IAV infection and had a major role in inhibition of adsorption and uptake by cells infected with IAV. Following the addition of 100 µg/ml rMBD-3 to MDCK cells medium, approximately 80% of cells were protected from infection in vitro. rMBD-3 given by tail vein injection (10 mg/kg/day) was the most effective method to improve the survival rate of the mice. Treatment with rMBD-3 was found to up-regulate IFN-γ and IL-12 gene expression, but reduced expression of the TNF-α gene. CONCLUSIONS: These results demonstrate that rMBD-3 possesses anti-influenza virus activity both in vivo and in vitro that might be of therapeutic use.

Human beta-defensin-3 up-regulates cyclooxygenase-2 expression and prostaglandin E2 synthesis in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Oral epithelial cells express three antimicrobial peptide human beta-defensins (hBDs) that have previously been demonstrated to exert proinflammatory effects on various immune cells. We wanted to examine whether hBDs could induce cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) synthesis in non-immune cells, such as human gingival fibroblasts. MATERIAL AND METHODS: Cultured fibroblasts were treated with different concentrations of hBD-1, -2, -3 or interleukin-1 beta, as a positive control, for various times, in the presence or absence of NS-398, a specific COX-2 inhibitor. The levels of COX-1 and COX-2 mRNA expression were analyzed using RT-PCR and real-time PCR. Whole cell lysates were analyzed for COX-1 and COX-2 protein expression by western blotting. Cell-free culture supernatants were assayed for PGE(2) levels by ELISA. The lactate dehydrogenase assay was performed to determine the cytotoxicity of hBDs. RESULTS: Ten and 40 microg/mL of hBD-3 up-regulated COX-2 mRNA and protein expression, consistent with COX-2 up-regulation by interleukin-1 beta, whereas hBD-1 and hBD-2 did not. However, COX-1 mRNA and protein were constitutively expressed. The time-course study revealed that hBD-3 up-regulated COX-2 mRNA and protein expression at 6 and 12 h, respectively. Consistent with COX-2 up-regulation, 10 and 40 microg/mL of hBD-3 significantly increased PGE(2) levels in cell-free culture supernatants (p < 0.05), and this was inhibited by NS-398 in a dose-dependent manner. Neither of the hBD concentrations tested in this study was toxic to the cells. CONCLUSION: These findings indicate that epithelial human beta-defensin-3 functions as a proinflammatory mediator in controlling arachidonic acid metabolism in underlying fibroblasts.

Selective reciprocity in antimicrobial activity versus cytotoxicity of hBD-2 and crotamine.

Recent discoveries suggest cysteine-stabilized toxins and antimicrobial peptides have structure-activity parallels derived by common ancestry. Here, human antimicrobial peptide hBD-2 and rattlesnake venom-toxin crotamine were compared in phylogeny, 3D structure, target cell specificity, and mechanisms of action. Results indicate a striking degree of structural and phylogenetic congruence. Importantly, these polypeptides also exhibited functional reciprocity: (i) they exerted highly similar antimicrobial pH optima and spectra; (ii) both altered membrane potential consistent with ion channel-perturbing activities; and (iii) both peptides induced phosphatidylserine accessibility in eukaryotic cells. However, the Na(v) channel-inhibitor tetrodotoxin antagonized hBD-2 mechanisms, but not those of crotamine. As crotamine targets eukaryotic ion channels, computational docking was used to compare hBD-2 versus crotamine interactions with prototypic bacterial, fungal, or mammalian Kv channels. Models support direct interactions of each peptide with Kv channels. However, while crotamine localized to occlude Kv channels in eukaryotic but not prokaryotic cells, hBD-2 interacted with prokaryotic and eukaryotic Kv channels but did not occlude either. Together, these results support the hypothesis that antimicrobial and cytotoxic polypeptides have ancestral structure-function homology, but evolved to preferentially target respective microbial versus mammalian ion channels via residue-specific interactions. These insights may accelerate development of anti-infective or therapeutic peptides that selectively target microbial or abnormal host cells.

Linear analogues of human beta-defensin 3: concepts for design of antimicrobial peptides with reduced cytotoxicity to mammalian cells.

A series of engineered linear analogues [coded as F6, W6, Y6, A6, S6 and C(Acm)6] were modeled, designed, synthesized and structurally characterized by mass spectra, circular dichroism, hydrophobicity analysis and molecular modeling. We have screened antimicrobial activity, hemolysis to rabbit erythrocytes, and cytotoxicity to human conjunctival epithelial cells. No significant hemolytic effect was observed for hBD3 or from five of the six analogues [F6, Y6, A6, S6 and C(Acm)6] over the range of 3-100 microg mL(-1). The six linear analogues have reduced cytotoxicity to human conjunctival epithelial cells over the range of 6-100 microg mL(-1) compared to hBD3. By tuning the overall hydrophobicity of linear hBD3 analogues, reduced cytotoxicity and hemolysis were obtained while preserving the antimicrobial properties. The decreased cytotoxicity of the linear analogues is suggested to be structurally related to the removal of disulfide bridges, and the flexible structure of the linear forms, which seem to be associated with loss of secondary structure. These results suggest a new approach for guiding the design of new linear analogues of defensin peptides with strong antibiotic properties and reduced cytotoxicity to mammalian cells.

Structure-activity relation of human beta-defensin 3: influence of disulfide bonds and cysteine substitution on antimicrobial activity and cytotoxicity.

Human beta-defensins form a group of cysteine-rich antimicrobial peptides which have been found in epithelial tissue and, more recently, in the male genital tract. They play a role in the defense against microbial pathogens in innate immunity and display additional chemotactic functions in the adaptive immune system. An important characteristic of antimicrobial peptides is that they also exhibit toxic potential on eukaryotic cells. Very little is known about the structure dependence of antimicrobial and cytotoxic effects. We investigated human beta-defensin 3 (hBD-3), a potent broad-spectrum antimicrobial effector peptide, regarding the influence of structural parameters on the antimicrobial and cytotoxic activity. We have established a structure-activity relation of the hBD-3 using synthetic derivatives differing in length, charge, disulfide connectivity, and overall hydrophobicity. The antimicrobial activity of the peptides was compared to the cyctotoxic effects on monocytic THP-1 cells and the hemolytic activity on human erythrocytes. We found that it is not important for antimicrobial and cytotoxic activity whether and how cysteine residues are arranged to form disulfide bonds. Substitution of half-cystinyl residues by tryptophan resulted in increased activities, while other substitutions did not change activity. Correlation of activities with the structural changes demonstrates that the activity on eukaryotic cells appears to depend strongly on the overall hydrophobicity. In contrast, the antimicrobial potency of hBD-3 peptides is determined by the distribution of positively charged amino acid residues and hydrophobic side chains. The results facilitate the understanding of beta-defensin interaction with different cell types and guide the design of antimicrobially active peptides.