The best offense is a good beta-defensin.

The full range of receptors through which antimicrobial peptides exert their immunologic effects remains incompletely explored. Dong and colleagues identify Mgrpra2 as a G-coupled protein receptor on neutrophils, for which keratinocyte-derived Beta-defensins serve as key ligands. Binding of Mgrpra2 leads to release of neutrophil granules and Il-1ß, which helps shape skin microbiome composition and augments cutaneous defense against bacterial infection.

Expression, purification and investigation of antibacterial activity of a novel hybrid peptide LL37/hBD-129 by applied comprehensive computational and experimental approaches.

Antibiotic-resistant pathogens have become a great universal health concern. Antimicrobial peptides (AMPs) are small amphipathic and cationic polypeptides with high therapeutic potential against various microorganisms containing drug-resistant strains. Two major groups of these peptides, which have antibacterial activity against Gram-positive and Gram-negative bacteria, antiviral activity, and even antifungal activity, are defensins and cathelicidins. Hybridization of various AMPs is an appropriate approach to achieving new fusion AMPs with high antibacterial activity but low cellular toxicity. In the current research, the amino-acid sequence of human cathelicidin LL-37 (2-31) and Human beta-defensin (hBD)-129 were combined, and the fusion protein was evaluated by bioinformatics tool. The designed AMP gene sequence was commercially synthesized and cloned in the pET-28a expression vector. The LL-37/hBD-129 fusion protein was expressed in E.coli BL21-gold (DE3). The expression of the recombinant protein was evaluated using the SDS-PAGE method. The LL37/hBD-129 was successfully expressed as a recombinant hybrid AMP in E.coli BL21-gold (DE3) strain. Purification of the expressed AMP was performed by Ni-NTA column affinity chromatography, and the purified AMP was validated using the Western blot technic. Finally, the antimicrobial activity of the fusion AMP against Staphylococcus aureus and Escherichia coli bacteria was assessed. Based on the in silico analysis and experimental evaluations, the fusion AMP showed a significant antimicrobial effect on E. coli and Staphylococcus aureus bacteria.

A novel smaller ß-defensin-derived peptide is active against multidrug-resistant bacterial strains.

Antibiotic resistance is becoming a severe obstacle in the fight against acute and chronic infectious diseases that accompany most degenerative illnesses from neoplasia to osteo-arthritis and obesity. Currently, the race is on to identify pharmaceutical molecules or combinations of molecules able to prevent or reduce the insurgence and/or progression of infectivity. Attempts to substitute antibiotics with antimicrobial peptides have, thus far, met with little success against multidrug-resistant (MDR) bacterial strains. During the last decade, we designed and studied the activity and features of human ß-defensin analogs, which are salt-resistant, and hence active also under high salt concentrations as, for instance, in cystic fibrosis. Herein, we describe the design, synthesis, and major features of a new 21 aa long molecule, peptide γ2. The latter derives from the γ-core of the ß-defensin natural molecules, a small fragment of these molecules still bearing high antibacterial activity. We found that peptide γ2, which contains only one disulphide bond, recapitulates most of the biological properties of natural human ß-defensins and can also counteract both Gram-positive and Gram-negative MDR bacterial strains and biofilm formation. Moreover, it has great stability in human serum thereby enhancing its antibacterial presence and activity without cytotoxicity in human cells. In conclusion, peptide γ2 is a promising new weapon also in the battle against intractable infectious diseases.

Investigation of human ß-defensins 1, 2 and 3 in human saliva by molecular dynamics.

Human ß-defensins present in saliva have a broad spectrum of antimicrobial activities that work against infections in oral cavity. To provide a better understanding of these molecules' properties and functions at the molecular level, we have investigated and compared the important structural properties of human ß-defensin-1, -2 and -3 using molecular dynamics simulations. Our results have shown that human ß-defensin-3 has a more flexible structure in water than the other two because of its high hydrophilicity, low ß-sheet content and high repulsive forces between its charged residues. Moreover, we found that the location of the salt bridges is important in protein's stability in water. Molecular dynamics simulations of human ß-defensins 1, 2 and 3 revealed that the hbd-3 is more flexible in water than hbd-1 and hbd-2.

Real-Time Fluorescence Microscopy on Living E. coli Sheds New Light on the Antibacterial Effects of the King Penguin ß-Defensin AvBD103b.

(1) Antimicrobial peptides (AMPs) are a promising alternative to conventional antibiotics. Among AMPs, the disulfide-rich ß-defensin AvBD103b, whose antibacterial activities are not inhibited by salts contrary to most other ß-defensins, is particularly appealing. Information about the mechanisms of action is mandatory for the development and approval of new drugs. However, data for non-membrane-disruptive AMPs such as ß-defensins are scarce, thus they still remain poorly understood. (2) We used single-cell fluorescence imaging to monitor the effects of a ß-defensin (namely AvBD103b) in real time, on living E. coli, and at the physiological concentration of salts. (3) We obtained key parameters to dissect the mechanism of action. The cascade of events, inferred from our precise timing of membrane permeabilization effects, associated with the timing of bacterial growth arrest, differs significantly from the other antimicrobial compounds that we previously studied in the same physiological conditions. Moreover, the AvBD103b mechanism does not involve significant stereo-selective interaction with any chiral partner, at any step of the process. (4) The results are consistent with the suggestion that after penetrating the outer membrane and the cytoplasmic membrane, AvBD103b interacts non-specifically with a variety of polyanionic targets, leading indirectly to cell death.

Molecular and functional identification of a ß-defensin homolog in large yellow croaker (Larimichthys crocea).

ß-defensin (BD) is a cysteine-rich cationic antibacterial peptide that is active against a wide range of bacteria. Here, a ß-defensin homolog (LcBD2) was identified in large yellow croaker (Larimichthys crocea). The open reading frame of LcBD2 contains 195 nucleotides, encoding a protein of 64 amino acids that possesses a typical arrangement of six conserved cysteine residues (C31 , C37 , C41 , C53 , C59 and C60 ). LcBD2 transcripts were constitutively expressed in all examined tissues and significantly increased in head kidney, spleen and gills by Vibrio alginolyticus. The synthetic LcBD2 peptide imparted antimicrobial effects on both Gram-negative bacteria (V. campbellii, V. parahaemolyticus, V. alginolyticus, V. harveyi and Pseudomonas plecoglossicida) and Gram-positive bacteria (Bacillus subtilis). We also observed that after treatment with synthetic LcBD2 peptide, numerous blisters appeared on the membrane of P. plecoglossicida, which in turn may result in cell membrane breakage and bacterial death. Moreover, the synthetic LcBD2 peptide significantly upregulated the expression levels of TNF-α2, IL-1ß and CXCL8_L1 in monocytes/macrophages, while downregulated expression level of IL-10. The LcBD2 peptide also remarkedly enhanced the phagocytosis of monocytes/macrophages. These results indicate that LcBD2 not only protects large yellow croaker against multiple bacterial pathogens but also plays a role in activation of monocytes/macrophages.

Antimicrobial activity and structure of a consensus human ß-defensin and its comparison to a novel putative hBD10.

The spread of multidrug resistant bacteria owing to the intensive use of antibiotics is challenging current antibiotic therapies, and making the discovery and evaluation of new antimicrobial agents a high priority. The evaluation of novel peptide sequences of predicted antimicrobial peptides from different sources is valuable approach to identify alternative antibiotic leads. Two strategies were pursued in this study to evaluate novel antimicrobial peptides from the human ß-defensin family (hBD). In the first, a 32-residue peptide was designed based on the alignment of all available hBD primary structures, while in the second a putative 35-residue peptide, hBD10, was mined from the gene DEFB110. Both hBDconsensus and hBD10 were chemically synthesized, folded and purified. They showed antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Mycobacterium tuberculosis, but were not hemolytic on human red blood cells. The NMR-based solution structure of hBDconsensus revealed that it adopts a classical ß-defensin fold and disulfide connectivities. Even though the mass spectrum of hBD10 confirmed the formation of three disulfide bonds, it showed limited dispersion in 1 H NMR spectra and structural studies were not pursued. The evaluation of different ß-defensin structures may identify new antimicrobial agents effective against multidrug-resistant bacterial strains.

Unusual interplay of contrasting selective pressures on ß-defensin genes implicated in male fertility of the Buffalo (Bubalus bubalis).

BACKGROUND: The buffalo, despite its superior milk-producing ability, suffers from reproductive limitations that constrain its lifetime productivity. Male sub-fertility, manifested as low conception rates (CRs), is a major concern in buffaloes. The epididymal sperm surface-binding proteins which participate in the sperm surface remodelling (SSR) events affect the survival and performance of the spermatozoa in the female reproductive tract (FRT). A mutation in an epididymal secreted protein, beta-defensin 126 (DEFB-126/BD-126), a class-A beta-defensin (CA-BD), resulted in decreased CRs in human cohorts across the globe. To better understand the role of CA-BDs in buffalo reproduction, this study aimed to identify the BD genes for characterization of the selection pressure(s) acting on them, and to identify the most abundant CA-BD transcript in the buffalo male reproductive tract (MRT) for predicting its reproductive functional significance. RESULTS: Despite the low protein sequence homology with their orthologs, the CA-BDs have maintained the molecular framework and the structural core vital to their biological functions. Their coding-sequences in ruminants revealed evidence of pervasive purifying and episodic diversifying selection pressures. The buffalo CA-BD genes were expressed in the major reproductive and non-reproductive tissues exhibiting spatial variations. The Buffalo BD-129 (BuBD-129) was the most abundant and the longest CA-BD in the distal-MRT segments and was predicted to be heavily O-glycosylated. CONCLUSIONS: The maintenance of the structural core, despite the sequence divergence, indicated the conservation of the molecular functions of the CA-BDs. The expression of the buffalo CA-BDs in both the distal-MRT segments and non-reproductive tissues indicate the retention the primordial microbicidal activity, which was also predicted by in silico sequence analyses. However, the observed spatial variations in their expression across the MRT hint at their region-specific roles. Their comparison across mammalian species revealed a pattern in which the various CA-BDs appeared to follow dissimilar evolutionary paths. This pattern appears to maintain only the highly efficacious CA-BD alleles and diversify their functional repertoire in the ruminants. Our preliminary results and analyses indicated that BuBD-129 could be the functional ortholog of the primate DEFB-126. Further studies are warranted to assess its molecular functions to elucidate its role in immunity, reproduction and fertility.

The Komodo dragon (Varanus komodoensis) genome and identification of innate immunity genes and clusters.

BACKGROUND: We report the sequencing, assembly and analysis of the genome of the Komodo dragon (Varanus komodoensis), the largest extant lizard, with a focus on antimicrobial host-defense peptides. The Komodo dragon diet includes carrion, and a complex milieu of bacteria, including potentially pathogenic strains, has been detected in the saliva of wild dragons. They appear to be unaffected, suggesting that dragons have robust defenses against infection. While little information is available regarding the molecular biology of reptile immunity, it is believed that innate immunity, which employs antimicrobial host-defense peptides including defensins and cathelicidins, plays a more prominent role in reptile immunity than it does in mammals. . RESULTS: High molecular weight genomic DNA was extracted from Komodo dragon blood cells. Subsequent sequencing and assembly of the genome from the collected DNA yielded a genome size of 1.6 Gb with 45x coverage, and the identification of 17,213 predicted genes. Through further analyses of the genome, we identified genes and gene-clusters corresponding to antimicrobial host-defense peptide genes. Multiple ß-defensin-related gene clusters were identified, as well as a cluster of potential Komodo dragon ovodefensin genes located in close proximity to a cluster of Komodo dragon ß-defensin genes. In addition to these defensins, multiple cathelicidin-like genes were also identified in the genome. Overall, 66 ß-defensin genes, six ovodefensin genes and three cathelicidin genes were identified in the Komodo dragon genome. CONCLUSIONS: Genes with important roles in host-defense and innate immunity were identified in this newly sequenced Komodo dragon genome, suggesting that these organisms have a robust innate immune system. Specifically, multiple Komodo antimicrobial peptide genes were identified. Importantly, many of the antimicrobial peptide genes were found in gene clusters. We found that these innate immunity genes are conserved among reptiles, and the organization is similar to that seen in other avian and reptilian species. Having the genome of this important squamate will allow researchers to learn more about reptilian gene families and will be a valuable resource for researchers studying the evolution and biology of the endangered Komodo dragon.

Characterization of grass carp (Ctenopharyngodon idella) beta-defensin 1: implications for its role in inflammation control.

Considering that fish grows in a complex aquatic environment, there is an increasing interest in fish ß-defensins, which is an important group of antimicrobial peptides (AMPs). In this study, grass carp (Ctenopharyngodon idella) ß-defensin 1 (gcdefb1) was isolated using homology cloning technology. Tissue distribution assay showed that gcdefb1 transcripts were expressed with the highest levels in brain and liver, followed by some mucous tissues. To examine gcDefb1 bioactivities, the recombinant gcDefb1 proteins fused with thioredoxin tag protein (Trx) (Trx-Defb1) were induced for production in Escherichia coli Rosetta-gami2(DE3)pLysS under optimal expression conditions. The antibacterial activity of Trx-Defb1 against Aeromonas hydrophila was assessed and its minimum inhibitory concentration (MIC) was 36 µM. Interestingly, Trx-Defb1 significantly inhibited LPS-induced Tnf-α (gcTnf-α) secretion and nitric oxide production in grass carp head kidney monocytes/macrophages (HKM), although Trx-Defb1 alone had no effect. Our studies provide the first evidence of fish ß-defensin 1 engaging in both antimicrobial and inflammation suppression process.

The Designer Antimicrobial Peptide A-hBD-2 Facilitates Skin Wound Healing by Stimulating Keratinocyte Migration and Proliferation.

BACKGROUND/AIMS: Antimicrobial peptides are effective promoters of wound healing but are susceptible to degradation. In this study, we replaced the GIGDP unit on the N-terminal of the endogenous human antimicrobial peptide hBD-2 with APKAM to produce A-hBD-2 and analyzed the effect on wound healing both in vitro and in vivo. METHODS: The effects of A-hBD-2 and hBD-2 on cytotoxicity and proliferation in keratinocytes were assessed by Cell Counting Kit-8 assay. The structural stability and antimicrobial activity of hBD-2 and A-hBD-2 were evaluated against Staphylococcus aureus. RNA and proteins levels were evaluated by real-time PCR and western blotting, respectively. Cell migration was evaluated using a transwell assay. Cell cycle analysis was performed by flow cytometry. Wound healing was assessed in Sprague-Dawley rats. Epidermal thickness was evaluated by hematoxylin and eosin staining. RESULTS: We found that hBD-2 exhibited cytotoxicity at high concentrations and decreased the structural stability in the presence of high sodium chloride concentrations. A-hBD-2 exhibited increased structural stability and antimicrobial activity, and had lower cytotoxicity in keratinocytes. A-hBD-2 increased the migration and proliferation of keratinocytes via phosphorylation of EGFR and STAT3 and suppressed terminal differentiation of keratinocytes. We also found that A-hBD-2 elicited mobilization of intracellular Ca2+ and stimulated keratinocytes to produce pro- and anti-inflammatory cytokines and chemokines via phospholipase C activation. Furthermore, A-hBD-2 promoted wound healing in vivo. CONCLUSION: Our data suggest that A-hBD-2 may be a promising candidate therapy for wound healing.

Beta-defensin derived cationic antimicrobial peptides with potent killing activity against gram negative and gram positive bacteria.

BACKGROUND: Avian ß-defensins (AvBD) are cationic antimicrobial peptides (CAMP) with broad-spectrum antimicrobial activity, chemotactic property, and low host cytotoxicity. However, their bactericidal activity is greatly compromised under physiological salt concentrations which limits the use of these peptides as therapeutic agents. The length and the complex structure involving three conserved disulfide bridges are additional drawbacks associated with high production cost. In the present study, short linear CAMPs (11 to 25 a.a. residues) were developed based on the key functional components of AvBDs with additional modifications. Their biological functions were characterized. RESULTS: CAMP-t1 contained the CCR2 binding domain (N-terminal loop and adjacent α-helix) of AvBD-12 whereas CAMP-t2 comprised the key a.a. residues responsible for the concentrated positive surface charge and hydrophobicity of AvBD-6. Both CAMP-t1 and CAMP-t2 demonstrated strong antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus pseudintermedius. However, CAMP-t1 failed to show chemotactic activity and CAMP-t2, although superior in killing Staphylococcus spp., remained sensitive to salts. Using an integrated design approach, CAMP-t2 was further modified to yield CAMP-A and CAMP-B which possessed the following characteristics: α-helical structure with positively and negatively charged residues aligned on the opposite side of the helix, lack of protease cutting sites, C-terminal poly-Trp tail, N-terminal acetylation, and C-terminal amidation. Both CAMP-A and CAMP-B demonstrated strong antimicrobial activity against multidrug-resistant P. aeruginosa and methicillin-resistant S. pseudintermedius (MRSP) strains. These peptides were resistant to major proteases and fully active at physiological concentrations of NaCl and CaCl2. The peptides were minimally cytotoxic to avian and murine cells and their therapeutic index was moderate (≥ 4.5). CONCLUSIONS: An integrated design approach can be used to develop short and potent antimicrobial peptides, such as CAMP-A and CAMP-B. The advantageous characteristics, including structural simplicity, resistance to salts and proteases, potent antimicrobial activity, rapid membrane attacking mode, and moderate therapeutic index, suggest that CAMP-A and CAMP-B are excellent candidates for development as therapeutic agents against multidrug-resistant P. aeruginosa and methicillin-resistant staphylococci.

Human ß-defensin 2 plays a regulatory role in innate antiviral immunity and is capable of potentiating the induction of antigen-specific immunity.

BACKGROUND: Antimicrobial peptides (AMPs) are primarily known for their innate immune defense against invading microorganisms, including viruses. In addition, recent research has suggested their modulatory activity in immune induction. Given that most subunit vaccines require an adjuvant to achieve effective immune induction through the activation of innate immunity, AMPs are plausible candidate molecules for stimulating not only innate immune but also adaptive immune responses. RESULTS: In this study, we investigated the ability of human ß-defensin (HBD) 2 to promote antiviral immunity in vitro and in vivo using a receptor-binding domain (RBD) of Middle East respiratory syndrome-coronavirus (MERS-CoV) spike protein (S RBD) as a model antigen (Ag). When HBD 2-conjugated S RBD was used to treat THP-1 human monocytic cells, the expression levels of antiviral (IFN-ß, IFN-γ, MxA, PKR, and RNaseL) and primary immune-inducing (NOD2, TNF-α, IL-1ß, and IL-6) molecules were enhanced compared to those expressed after treatment with S RBD only. The expression of chemokines capable of recruiting leukocytes, including monocytes/macrophages, natural killer cells, granulocytes, T cells, and dendritic cells, was also increased following HBD 2-conjugated S RBD treatment. More important, immunization of mice with HBD 2-conjugated S RBD enhanced the immunogenicity of the S RBD and elicited a higher S RBD-specific neutralizing antibody response than S RBD alone. CONCLUSIONS: We conclude that HBD 2 activates the primary antiviral innate immune response and may also mediate the induction of an effective adaptive immune response against a conjugated Ag.

Different dynamics and pathway of disulfide bonds reduction of two human defensins, a molecular dynamics simulation study.

Human defensins are a class of antimicrobial peptides that are crucial components of the innate immune system. Both human α defensin type 5 (HD5) and human ß defensin type 3 (hBD-3) have 6 cysteine residues which form 3 pairs of disulfide bonds in oxidizing condition. Disulfide bond linking is important to the protein structure stabilization, and the disulfide bond linking and breaking order have been shown to influence protein function. In this project, microsecond long molecular dynamics simulations were performed to study the structure and dynamics of HD5 and hBD-3 wildtype and analogs which have all 3 disulfide bonds released in reducing condition. The structure of hBD-3 was found to be more dynamic and flexible than HD5, based on RMSD, RMSF, and radius of gyration calculations. The disulfide bridge breaking order of HD5 and hBD-3 in reducing condition was predicted by two kinds of methods, which gave consistent results. It was found that the disulfide bonds breaking pathways for HD5 and hBD-3 are very different. The breaking of disulfide bonds can influence the dimer interface by making the dimer structure less stable for both kinds of defensin. In order to understand the difference in dynamics and disulfide bond breaking pathway, hydrophilic and hydrophobic accessible surface areas (ASA), buried surface area between cysteine pairs, entropy of cysteine pairs, and internal energy were calculated. Comparing to the wildtype, hBD-3 analog is more hydrophobic, while HD5 is more hydrophilic. For hBD-3, the disulfide breaking is mainly entropy driven, while other factors such as the solvation effects may take the major role in controlling HD5 disulfide breaking pathway. Proteins 2017; 85:665-681. © 2016 Wiley Periodicals, Inc.

Comparative genomic identification and validation of ß-defensin genes in the Ovis aries genome.

BACKGROUND: ß-defensins are small, cationic, antimicrobial peptides found in species across the plant and animal kingdoms. In addition to microbiocidal activity, roles in immunity as well as reproduction have more recently been documented. ß-defensin genes in Ovis aries (domestic sheep) have been poorly annotated, having been identified only by automatic gene prediction algorithms. The objective of this study was to use a comparative genomics approach to identify and characterise the ß-defensin gene repertoire in sheep using the bovine genome as the primary reference. RESULTS: All 57 currently predicted bovine ß-defensin genes were used to find orthologous sequences in the most recent version of the sheep genome (OAR v4.0). Forty three genes were found to have close genomic matches (>70% similarity) between sheep and cattle. The orthologous genes were located in four clusters across the genome, with 4 genes on chromosome 2, 19 genes on chromosome 13, 5 genes on chromosome 20 and 15 genes on chromosome 26. Conserved gene order for the ß-defensin genes was apparent in the two smaller clusters, although gene order was reversed on chromosome 2, suggesting an inversion between sheep and cattle. Complete conservation of gene order was also observed for chromosome 13 ß-defensin orthologs. More structural differences were apparent between chromosome 26 genes and the orthologous region in the bovine reference genome, which is known to be copy-number variable. In this cluster, the Defensin-beta 1 (DEFB1) gene matched to eleven Bovine Neutrophil beta-Defensin (BNBD) genes on chromosome 27 with almost uniform similarity, as well as to tracheal, enteric and lingual anti-microbial peptides (TAP, EAP and LAP), suggesting that annotation of the bovine reference sequence is still incomplete. qPCR was used to profile the expression of 34 ß-defensin genes, representing each of the four clusters, in the ram reproductive tract. Distinct site-specific and differential expression profiles were detected across the reproductive tract of mature rams with preferential ß-defensin gene expression in the epididymis, recapitulating observations for orthologous genes in other species. CONCLUSIONS: This is the first comprehensive analysis of ß-defensin genes encoded by the ovine reference sequence, and the first report of an expanded repertoire of ß-defensin genes in this species. The preferential expression of these genes in the epididymis suggests a role in fertility, possibly providing immunoprotection for sperm within the female reproductive tract.

AvBD1 nucleotide polymorphisms, peptide antimicrobial activities and microbial colonisation of the broiler chicken gut.

BACKGROUND: The importance of poultry as a global source of protein underpins the chicken genome and associated SNP data as key tools in selecting and breeding healthy robust birds with improved disease resistance. SNPs affecting host peptides involved in the innate defences tend to be rare, but three non-synonymous SNPs in the avian ß-defensin (AvBD1) gene encoding the variant peptides NYH, SSY and NYY were identified that segregated specifically to three lines of commercial broiler chickens Line X (LX), Line Y(LY) and Line Z. The impacts of such amino acid changes on peptide antimicrobial properties were analysed in vitro and described in relation to the caecal microbiota and gut health of LX and LY birds. RESULTS: Time-kill and radial immune diffusion assays indicated all three peptides to have antimicrobial properties against gram negative and positive bacteria with a hierarchy of NYH > SSY > NYY. Calcein leakage assays supported AvBD1 NYH as the most potent membrane permeabilising agent although no significant differences in secondary structure were identified to explain this. However, distinct claw regions, identified by 3D modelling and proposed to play a key role in microbial membrane attachment, and permeation, were more distinct in the NYH model. In vivo AvBD1 synthesis was detected in the bird gut epithelia. Analyses of the caecal gut microbiota of young day 4 birds suggested trends in Lactobacilli sp. colonisation at days 4 (9% LX vs × 30% LY) and 28 (20% LX vs 12% LY) respectively, but these were not statistically significant (P > 0.05). CONCLUSION: Amino acid changes altering the killing capacity of the AvBD1 peptide were associated with two different bird lines, but such changes did not impact significantly on caecal gut microbiota.

Two arginine residues in the COOH-terminal of human ß-defensin-3 constitute an essential motif for antimicrobial activity and IL-6 production.

Human ß-defensin-3 (HBD-3) possesses antimicrobial activities and the potential to induce proinflammatory cytokines. HBD-3 contains a unique motif of two arginine residues (Arg or R) in the COOH-terminal region. To understand the bioactive properties of the Arg residues of HBD-3, we examined antimicrobial activities against Staphylococcus aureus and Pseudomonas aeruginosa using synthetic HBD-2, HBD-3 and two variant peptides of HBD-3: the Arg-truncated variant designated desR HBD-3 and NRR HBD-3, in which both Arg residues were shifted to the N-terminal region. IL-6 production from keratinocytes was studied using the peptides. HBD-3 possessed approximately five-fold more potent antimicrobial activities, evaluated as the minimum inhibitory concentration (MIC), against S. aureus compared with desR and NRR HBD-3, while no significant activity was observed in HBD-2. The antimicrobial activity of HBD-3 against S. aureus was well preserved even at high sodium chloride concentrations, but was attenuated in desR and NRR HBD-3. All the peptides exhibited similar antimicrobial activities against P. aeruginosa, but HBD-2 and desR HBD-3 showed diminished antimicrobial activities against P. aeruginosa at high salt concentrations. IL-6 production was significantly induced in keratinocytes with HBD-3, but not remarkably with stimulation by other peptide. These Arg residues are essential for the antimicrobial and biological properties of HBD-3.

Chimeric analogs of human ß-defensin 1 and θ-defensin disrupt pre-established bacterial biofilms.

Antibiofilm activity of several human defensin analogs that have the ability to kill planktonic bacteria, against pre-established biofilms of Escherichia coli MG1655 and Staphylococcus aureus NCTC 8530 were examined. Linear and linear fatty acylated analogs did not show any activity while disulfide constrained analogs disrupted pre-established S. aureus biofilms. Chimeric analogs of human ß-defensin 1 and θ-defensin, hBTD-1 and [d]hBTD-1 were highly active against S. aureus biofilms. Among the analogs tested, only the d-enantiomer [d]hBTD-1 showed activity against E. coli biofilm. Our study provides insights into the structural requirements for the eradication of pre-established biofilms in defensin analogs.

Determining the cleavage site for the mature antimicrobial peptide of Nile tilapia ß-defensin using 2D electrophoresis, western blot, and mass spectrometry analysis.

Several proteomic techniques were used to determine the cleavage site of the mature antimicrobial peptide of Nile tilapia ß-defensin. The computer-predicted Nile tilapia ß-defensin (25ASFPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL66) composed of 42 amino acids was chemically synthesized and prepared to produce an antibody for Western blotting. Total proteins from the skin of the Nile tilapia were separated on two-dimensional electrophoresis, and the spot of Nile tilapia ß-defensin was recognized using Western blot analysis. It was then excised and extracted from the gel. The precise molecular mass of this spot was determined by LC-MS/MS spectrometry. Four major peptides were discovered, with molecular weights of 4293.2 Da, 4306.5 Da, 4678.9 Da, and 4715.0 Da. The calculated mass of the 40-amino-acid sequence (27FPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL66) of Nile tilapia ß-defensin starting from Phe27 and ending with Leu66 was 4293.18 Da, which completely matched the 4293.2 Da peptide that was obtained from the mass spectrometry analysis. This result confirmed that the cleavage site for the mature C-terminal Nile tilapia ß-defensin is at residue Ser26-Phe27, not at Ala24-25 as predicted by computer analysis. This study provides a simple but reliable model to determine the cleavage site for a mature antimicrobial peptide.

Host defense peptide-derived privileged scaffolds for anti-infective drug discovery.

'Privileged scaffolds' are molecular frameworks which have been successfully exploited for small molecule drug discovery. Peptide privileged scaffolds, featuring a strictly conserved multiple-disulfide framework and high variability in the rest of the sequence, display a broad range of biological effects, including antimicrobial and antiviral activity. Unlike small molecules, however, the cost of manufacturing these peptides is high, and their synthesis challenging. We previously described a simplified privileged scaffold corresponding to the γ-core of human ß-defensin-3 (HBD3). The γ-core is a common structural signature found in virtually all host defense peptides (HDPs) stabilized by multiple disulfides, and we showed that for HBD3, it represents the evolutionary starting point of the full-length molecule and, thus, is itself a primordial HDP. Accordingly, we showed that the peptide folded rapidly and was stable in human serum, and displayed many of the biological activities of HBD3. We report here that in addition to the previously reported antibacterial activity on planktonic bacteria, the γ-core peptide is active against biofilm formation and maturation. We also show that it is readily cell penetrant, like HBD3, although with a different mechanism, which is independent from CD98. Overall, the potency of the single-disulfide, 23-amino acid γ-core is comparable with the full-length peptide across the whole spectrum of examined properties, and the peptide is not toxic to human cells. The HBD3 γ-core peptide may therefore represent the first example of an economically viable lead peptide derived from a HDP privileged scaffold. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.