Human Defensins Inhibit SARS-CoV-2 Infection by Blocking Viral Entry.

Innate immunity during acute infection plays a critical role in the disease severity of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), and is likely to contribute to COVID-19 disease outcomes. Defensins are highly abundant innate immune factors in neutrophils and epithelial cells, including intestinal Paneth cells, and exhibit antimicrobial and immune-modulatory activities. In this study, we investigated the effects of human α- and ß-defensins and RC101, a θ-defensin analog, on SARS-CoV-2 infection. We found that human neutrophil peptides (HNPs) 1-3, human defensin (HD) 5 and RC101 exhibited potent antiviral activity against pseudotyped viruses expressing SARS-CoV-2 spike proteins. HNP4 and HD6 had weak anti-SARS-CoV-2 activity, whereas human ß-defensins (HBD2, HBD5 and HBD6) had no effect. HNP1, HD5 and RC101 also inhibited infection by replication-competent SARS-CoV-2 viruses and SARS-CoV-2 variants. Pretreatment of cells with HNP1, HD5 or RC101 provided some protection against viral infection. These defensins did not have an effect when provided post-infection, indicating their effect was directed towards viral entry. Indeed, HNP1 inhibited viral fusion but not the binding of the spike receptor-binding domain to hACE2. The anti-SARS-CoV-2 effect of defensins was influenced by the structure of the peptides, as linear unstructured forms of HNP1 and HD5 lost their antiviral function. Pro-HD5, the precursor of HD5, did not block infection by SARS-CoV-2. High virus titers overcame the effect of low levels of HNP1, indicating that defensins act on the virion. HNP1, HD5 and RC101 also blocked viral infection of intestinal and lung epithelial cells. The protective effects of defensins reported here suggest that they may be useful additives to the antivirus arsenal and should be thoroughly studied.

Structural and biological features of a novel plant defensin from Brugmansia x candida.

Data from both the laboratory and clinic in the last decade indicate that antimicrobial peptides (AMPs) are widely regarded as potential sources of future antibiotics owing to their broad-spectrum activities, rapid killing, potentially low-resistance rate and multidirectional mechanisms of action compared to conventional antibiotics. Defensins, a prominent family of AMPs, have been found in a wide range of organisms including plants. Thailand is a rich source of plants including medicinal plants used therapeutically, however there is no report of defensin from among these plants. In this study, a novel plant defensin gene, BcDef, was successfully cloned from Brugmansia x candida (Bc). BcDef cDNA was 237 bp in length, encoding 78 amino acids with a putative 31-amino acid residue signal peptide at the N-terminal followed by the mature sequence. BcDef shared high sequence identity (78-85%) with Solanaceae defensins and belonged to the class I plant defensins. From homology modeling, BcDef shared a conserved triple stranded ß-sheet (ß1-ß3) and one α-helix (α1) connected by a loop (L1-L3). BcDef1 peptide, designed from the γ-core motifs of BcDef located in loop 3, showed antibacterial activity against both Gram-positive and Gram-negative pathogens with the lowest MIC (15.70 µM) against Staphylococcus epidermidis. This peptide affected cell membrane potential and permeability, and caused cell membrane disruption. Moreover, BcDef1 also exhibited antioxidant activity and showed low cytotoxicity against mouse fibroblast L929 cells. These findings may provide an opportunity for developing a promising antibacterial agent for medical application in the future.

Functions of Antimicrobial Peptides in Vertebrates.

OBJECTIVE: The aim of this review is to examine the multiple activities of antimicrobial peptides (AMPs) in vertebrates. CONTENT: The largest AMP families are the cathelicidins and defensins, but several peptides derived from bigger proteins have also been reported. Cathelicidins are characterized by a conserved Nterminal pro-region and a variable region that encodes the C-terminal mature peptide. The ß-defensins comprise a large family of AMPs that have diversified their functions, apparently without losing their antimicrobial activity. Cathelicidins and ß-defensins are present in all vertebrates studied so far; α- defensins are present in mammals, while θ-defensins are only present in some non-human primates. The AMPs are regulated by posttranslational modifications that mainly include proteolysis, amidation, ADP-ribosylation, glycosylation and phosphorylation. In addition to their antimicrobial effects, AMPs show activity against viral particles and interfere in different steps of virus replication. Moreover, AMPs may both promote and inhibit cancer growth: several vertebrate AMPs kill cancer cells, and some tumors grow in an environment wherein the expression of ß-defensins is reduced; however, human cathelicidin and some ß-defensins are overexpressed in several types of cancer and are correlated with tumor growth. AMPs are part of the complex network of cells and molecules that forms the vertebrate innate defense system and they induce adaptive responses. In addition, they participate in sperm maturation and male reproduction. CONCLUSION: AMPs are multifunctional peptides that participate in immune responses, wound healing, angiogenesis, toxin neutralization, iron metabolism, male reproduction, among other functions. However, AMPs may also contribute to excessive inflammation and tumorigenesis.

iDPF-PseRAAAC: A Web-Server for Identifying the Defensin Peptide Family and Subfamily Using Pseudo Reduced Amino Acid Alphabet Composition.

Defensins as one of the most abundant classes of antimicrobial peptides are an essential part of the innate immunity that has evolved in most living organisms from lower organisms to humans. To identify specific defensins as interesting antifungal leads, in this study, we constructed a more rigorous benchmark dataset and the iDPF-PseRAAAC server was developed to predict the defensin family and subfamily. Using reduced dipeptide compositions were used, the overall accuracy of proposed method increased to 95.10% for the defensin family, and 98.39% for the vertebrate subfamily, which is higher than the accuracy from other methods. The jackknife test shows that more than 4% improvement was obtained comparing with the previous method. A free online server was further established for the convenience of most experimental scientists at http://wlxy.imu.edu.cn/college/biostation/fuwu/iDPF-PseRAAAC/index.asp. A friendly guide is provided to describe how to use the web server. We anticipate that iDPF-PseRAAAC may become a useful high-throughput tool for both basic research and drug design.

In silico identification, structural characterization, and phylogenetic analysis of MdesDEF-2: a novel defensin from the Hessian fly, Mayetiola destructor.

The CSαß defensins are one of the most ancient antimicrobial peptide classes and are distributed in plants, invertebrates, and fungi. In the insect immunity, the defensins play a crucial role in protection against pathogens. The discovery of novel insect defensins could be a vital tool in developing novel antimicrobial agents, which are urgently needed because of growing resistance in pathogenic bacteria and the resulting reduction in the effectiveness of conventional antibiotics over the years. In this context, novel insect defensins could be identified from the potential resource of model insects. Here, a novel defensin, MdesDEF-2, was identified from the model insect Mayetiola destructor, the most destructive insect pest of wheat worldwide. The in silico identification of MdesDEF-2 was done through searching by regular expression in M. destructor's protein sequences available at NCBI. MdesDEF-2 has 36 amino acid residues and its model was composed of two ß-strands and one α-helix showing three disulfide bridges. According to the classification of CSαß defensins, MdesDEF-2 belongs to the group of ancient insect-type defensins. The molecular dynamics simulation revealed that MdesDEF-2 has a very flexible N-terminal loop. Moreover, phylogenetic analysis together with functional predictions indicated that MdesDEF-2 could have antibacterial activity without causing membrane disruption. However, while the actual activity of MdesDEF-2 is still unclear, it is evident that its role in the biology of M. destructor is similar to that of its paralogue, MdesDEF-1, protecting the insect against microbial invasion.

The genome of Mesobuthus martensii reveals a unique adaptation model of arthropods.

Representing a basal branch of arachnids, scorpions are known as 'living fossils' that maintain an ancient anatomy and are adapted to have survived extreme climate changes. Here we report the genome sequence of Mesobuthus martensii, containing 32,016 protein-coding genes, the most among sequenced arthropods. Although M. martensii appears to evolve conservatively, it has a greater gene family turnover than the insects that have undergone diverse morphological and physiological changes, suggesting the decoupling of the molecular and morphological evolution in scorpions. Underlying the long-term adaptation of scorpions is the expansion of the gene families enriched in basic metabolic pathways, signalling pathways, neurotoxins and cytochrome P450, and the different dynamics of expansion between the shared and the scorpion lineage-specific gene families. Genomic and transcriptomic analyses further illustrate the important genetic features associated with prey, nocturnal behaviour, feeding and detoxification. The M. martensii genome reveals a unique adaptation model of arthropods, offering new insights into the genetic bases of the living fossils.

Avian host defense peptides.

Host defense peptides (HDPs) are important effector molecules of the innate immune system of vertebrates. These antimicrobial peptides are also present in invertebrates, plants and fungi. HDPs display broad-spectrum antimicrobial activities and fulfill an important role in the first line of defense of many organisms. It is becoming increasingly clear that in the animal kingdom the functions of HDPs are not confined to direct antimicrobial actions. Research in mammals has indicated that HDPs have many immunomodulatory functions and are also involved in other physiological processes ranging from development to wound healing. During the past five years our knowledge about avian HDPs has increased considerably. This review addresses our current knowledge on the evolution, regulation and biological functions of HDPs of birds.

Molecular characterization of antimicrobial peptide genes of the carpenter ant Camponotus floridanus.

The production of antimicrobial peptides (AMPs) is a major defense mechanism against pathogen infestation and of particular importance for insects relying exclusively on an innate immune system. Here, we report on the characterization of three AMPs from the carpenter ant Camponotus floridanus. Due to sequence similarities and amino acid composition these peptides can be classified into the cysteine-rich (e.g. defensin) and glycine-rich (e.g. hymenoptaecin) AMP groups, respectively. The gene and cDNA sequences of these AMPs were established and their expression was shown to be induced by microbial challenge. We characterized two different defensin genes. The defensin-2 gene has a single intron, whereas the defensin-1 gene has two introns. The deduced amino acid sequence of the C. floridanus defensins is very similar to other known ant defensins with the exception of a short C-terminal extension of defensin-1. The hymenoptaecin gene has a single intron and a very peculiar domain structure. The corresponding precursor protein consists of a signal- and a pro-sequence followed by a hymenoptaecin-like domain and six directly repeated hymenoptaecin domains. Each of the hymenoptaecin domains is flanked by an EAEP-spacer sequence and a RR-site known to be a proteolytic processing site. Thus, proteolytic processing of the multipeptide precursor may generate several mature AMPs leading to an amplification of the immune response. Bioinformatical analyses revealed the presence of hymenoptaecin genes with similar multipeptide precursor structure in genomes of other ant species suggesting an evolutionary conserved important role of this gene in ant immunity.

Defensinpred: defensin and defensin types prediction server.

Defensins are considered to play an important role in the innate immune system of virtually all life forms, from insects and plants to amphibians and mammals. They are classified into alpha, beta and theta-defensins. Fast and accurate computational prediction of defensin and defensin types will help in annotating unidentified defensin novel peptides. Identified defensins, owing to their small length and potent antimicrobial activity can be used effectively for development of new clinically applicable antibiotics. Thus predicting the defensin candidates will aid in accurate identification of novel peptide drugs. Support vector machines prediction model accuracy was 99% for defensin and defensin types. The results indicate that it is most accurate and efficient prediction method for defensin peptides. User friendly defensin web server is provided at www.defensinpred.cdac.in for the benefit of scientific community.

Properties of the bubble protein, a defensin and an abundant component of a fungal exudate.

Colonies of the ascomycete fungus Penicillium brevicompactum Dierckx produce bright yellow-green fluorescent exudate bubbles on its surface when grown on standard plant cell culture medium. According to SDS-PAGE analysis, the exudate is enriched in one protein, named bubble protein (BP). Detailed characteristics of BP are described, and also its corresponding genomic promoter and terminator sequences that flank sequences encoding signal peptide and a precursor sequence upstream of that of the mature protein. Following on previous work, the protein is now biochemically characterized. BP, the structure of which mainly consists of beta sheets, has four very stable disulfide bridges that resist standard procedures for reduction. With such traits, BP can now be categorized as a new member of the ever growing class of defensins. Indeed, the protein revealed anti-fungal effects as it inhibits growth of the yeast Saccharomyces cerevisiae in a dose-dependent manner. Structural classification places BP into the group of proteins with a knottin fold, founding the BP superfamily. Based on genomic alignments that revealed very high homology to four proteins of related fungi, a 3D structure prediction of the corresponding proteins was made. In addition, it was discovered that the closely related fungus Penicillium chrysogenum encodes a BP homolog - in addition to its PAF protein, which also is similar to BP - further suggesting that fungi may possess more than one defensin.

Human defensins and LL-37 in mucosal immunity.

Defensins are widespread in nature and have activity against a broad range of pathogens. Defensins have direct antimicrobial effects and also modulate innate and adaptive immune responses. We consider the role of human defensins and the cathelicidin LL-37 in defense of respiratory, gastrointestinal, and genitourinary tracts and the oral cavity, skin, and eye. Human beta-defensins (hBDs) and human defensins 5 and 6 (HD5 and -6) are involved most obviously in mucosal responses, as they are produced principally by epithelial cells. Human alpha-defensins 1-4 (or HNPs 1-4) are produced principally by neutrophils recruited to the mucosa. Understanding the biology of defensins and LL-37 is the beginning to clarify the pathophysiology of mucosal inflammatory and infectious diseases (e.g., Crohn's disease, atopic dermatitis, lung or urinary infections). Challenges for these studies are the redundancy of innate defense mechanisms and the presence and interactions of many innate defense proteins in mucosal secretions.

Purification and molecular cloning of antimicrobial peptides from Scots pine seedlings.

A novel protocol for rapid and efficient purification of antimicrobial peptides from plant seedlings has been developed. Two peptides with antimicrobial activity, designated p1 and p2, were purified nearly to homogeneity from Scots pine seedlings by a combination of sulfuric acid extraction, ammonium sulfate precipitation, heat-inactivation and ion-exchange chromatography on phosphocellulose. Purified proteins had molecular masses of 11 kDa (p1) and 5.8 kDa (p2) and were identified by mass spectrometry as defensin and lipid-transfer protein, respectively. We demonstrated their growth inhibitory effects against a group of phytopathogenic fungi. Furthermore, we report for the first time molecular cloning and characterization of defensin 1 cDNA from Scots pine. A cDNA expression library from 7 days Scots pine seedlings was generated and used to isolate a cDNA clone corresponding to Scots pine defensin, termed PsDef1. The full-length coding sequence of PsDef1 is 252 bp in length and has an open reading frame capable to encode a protein of 83 amino residues. The deduced sequence has the typical features of plant defensins, including an endoplasmic reticulum signal sequence of 33aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5601.6 Da, which correlates well with the results of SDS-PAGE analysis. Finally, the antimicrobial properties of PsDef1 against a panel of fungi and bacteria define it as a member of the morphogenic group of plant defensins.

Expression of BrD1, a plant defensin from Brassica rapa, confers resistance against brown planthopper (Nilaparvata lugens) in transgenic rices.

Plant defensins are small (5-10 kDa) basic peptides thought to be an important component of the defense pathway against fungal and/or bacterial pathogens. To understand the role of plant defensins in protecting plants against the brown planthopper, a type of insect herbivore, we isolated the Brassica rapa Defensin 1 (BrD1) gene and introduced it into rice (Oryza sativa L.) to produce stable transgenic plants. The BrD1 protein is homologous to other plant defensins and contains both an N-terminal endoplasmic reticulum signal sequence and a defensin domain, which are highly conserved in all plant defensins. Based on a phylogenetic analysis of the defensin domain of various plant defensins, we established that BrD1 belongs to a distinct subgroup of plant defensins. Relative to the wild type, transgenic rices expressing BrD1 exhibit strong resistance to brown planthopper nymphs and female adults. These results suggest that BrD1 exhibits insecticidal activity, and might be useful for developing cereal crop plants resistant to sap-sucking insects, such as the brown planthopper.

Characterization of two isoforms of Japanese spiny lobster Panulirus japonicus defensin cDNA.

Antimicrobial peptides (AMPs) are components of the innate immune responses that form the first line of host defense against pathogens. In this study, cDNAs of two new isoforms of defensin (designated PJD1 and PJD2) from a Japanese spiny lobster Panulirus japonicus haemocytes cDNA library were cloned and sequenced. PJD1 and PJD2 consist of 656 and 673 nucleotides encoding putative proteins of 66 and 64 amino acids, respectively. The isoforms share 74.2% amino acid identity. In a phylogenetic analysis, the peptides clustered with vertebrate defensins and were closely mostly related to chicken beta-defensin. PJD1 and PJD2 were detected in all tissues examined including heart, nerves, intestine, haemocytes, gills and hepatopancreas.

Sequence structure and expression pattern of a novel anionic defensin-like gene from silkworm (Bombyx mori).

A defensin-like gene, BmdefA, was rediscovered in the silkworm genome and expressed sequence tags databases. The open reading frame of BmdefA encodes a prepropeptide consisting of a 22-residue signal peptide, a 34-residue propeptide, and a 36-residue mature peptide with a molecular mass of 4.0 kDa. The mature peptide possesses the characteristic six-cysteine motif of insect defensins, and its predicted isoelectric point is 4.12, indicating it is a novel anionic defensin. An intron is present in BmdefA and several cis-regulatory elements are in the regulating region. It is transcribed constitutively at a high level in the hemocyte, silk gland, head, and ovary of the silkworm larvae, and in the fat body of early-stage pupae and moth. BmdefA is also strongly induced by immune challenge. These results suggest that BmdefA plays an important role in both immunity and metamorphosis.

[Defensins: an important innate element of the immune system in mammals]. / Defensyny - wazny wrodzony element ukladu odpornosciowego u ssaków.

In this paper a group of proteins, defensins, is characterized as they seem to be a very important element of immunity, especially innate immunity. Twelve alpha-defensins (HNP1-4, HD5-6, NP1 and 5 theta, criptidin 3 and 4, RMAD3-4), 25 beta-defensins (HBD1-4, SBD1-2, TAP, LAP, EBD, BNBD1-13, PBD1-2, mBD-1), and 5 q-defensins (retrocyklin 1,2, RTD1-3) are described. They display biological roles which are not only antibacterial, but also antiviral, antiparasitic, and antifungal.

Analysis of Triticum boeoticum and Triticum urartu seed defensins: To the problem of the origin of polyploid wheat genomes.

The origin of polyploid wheat genomes has been the subject of numerous studies and is the key problem in wheat phylogeny. Different diploid species have been supposed to donate genomes to tetraploid and hexaploid wheat species. To shed light on phylogenetic relationships between the presumable A genome donors and hexaploid wheat species we have applied a new approach: the comparison of defensins from diploid Triticum species, Triticum boeoticum Boiss. and Triticum urartu Thum. ex Gandil., with previously characterized Triticum kiharae defensins [T.I. Odintsova et al., Biochimie 89 (2007) 605-612]. Defensins were isolated by acidic extraction of seeds followed by three-step chromatographic separation. Isolated defensins were identified by molecular masses using MALDI-TOF mass spectrometry and N-terminal sequencing. For the first time, we have shown that T. urartu defensins are more similar to those of the hexaploid wheat than T. boeoticum defensins, although variation among samples collected in different regions of the world was revealed. Our results clearly demonstrate that T. urartu of the Asian origin contributed the A genome to polyploid wheat species.

Human defensins: synthesis and structural properties.

Defensins are small, beta-sheet-rich, cationic peptides found in many organisms. All defensins have amphiphilic properties, which are central for antimicrobial activities of the proteins. The human genome encodes many defensin-line molecules, of which 10 have been characterized. Molecules of all known human defensins are stabilized by three intramolecular disulfide bonds arranged in a conserved pattern. To date, studies of human defensins indicate that these proteins are involved in various biological processes associated primarily with defensive and regulatory responses to infections by pathological agents. A comprehensive understanding of the multiple roles played by defensins within the immune system is greatly increased by reviewing the results of detailed structural studies. Emerging structural data, derived by the X-ray crystallography and the NMR spectroscopy in solution combined with functional studies; allow a rational understanding of the different activities of defensins and the mechanisms controlling these activities. Due to their well-established antimicrobial properties, defensins are also being investigated for their potential as therapeutics agents. Recently, increased effort has been focused on studies of the immunoregulatory properties of defensins, associated with their ability to bind and activate the G(i)-protein-coupled seven-transmembrane receptors. Comprehensive studies of defensins require development of simple, efficient, and inexpensive methods to generate these proteins and their derivatives in correctly folded form. This review highlights the current status of the sample generation methods, structural studies, and the structure-function relationships for human defensins.

Structural and functional studies of defensin-inspired peptides.

Mammals have evolved complex self-defence mechanisms to protect themselves from infection. This innate immune system comprises a large family of hundreds of peptides and proteins which have potent antibiotic activity at nanomolar concentrations. The defensins are a group of small cationic peptides which contain a high proportion of positively charged and hydrophobic amino acids. Their exact mechanism of antimicrobial action is unclear, but it is thought that the defensins bind to and disrupt the outer cell membrane which ultimately causes lysis and cell death. They are characterized by six conserved cysteine residues which oxidize to form three intramolecular disulphide (S-S) bonds. The human and mouse defensins have been subdivided into classes based on their sequence, site of expression and the S-S bond connectivity of the cysteine residues. Alpha-defensins are connected by cysteines 1 and 6, 2 and 4, and 3 and 5, whereas beta-defensins have a 1-5, 2-4 and 3-6 cysteine S-S connectivity. We present our structural and functional studies of a novel mouse beta-defensin-related peptide (Defr1) which contains only five cysteine residues. Synthetic Defr1 was more active than its six-cysteine analogue against a large panel of pathogens. High-resolution MS techniques revealed that Defr1 contains an unusual defensin structure. These studies have guided the design of novel peptides to explore the roles of defensins as antibiotics and as stimulants of the immune response.

[Antimicrobial peptides]. / Peptydy antydrobnoustrojowe.

For many years, attention has been focused on adaptive immunity as the main antimicrobial defense system. The discovery of antimicrobial peptides turned this point of view. In general, these peptides act on microorganisms directly by disrupting the structure of the microbial cytoplasmatic membranes and indirectly acting on immune system. This review provides an overview of some natural antimicrobial peptides and probability of their clinical usage.