Defensins in granules of phagocytic and non-phagocytic cells

Antimicrobial proteins stored in lysosome-like granules of neutrophils and macrophages probably play an important role in killing phagocytosed microbes after delivery to the phagolysosome. Among the granules' antimicrobial armamentarium are defensins, peptides that kill a broad spectrum of microorganisms in vitro. Antimicrobial defensins were recently also isolated from non-phagocytic granulocytes of the mouse small intestinal epithelium, from where they are secreted into the lumen to function extracellularly. Clarification of the antimicrobial mechanisms of defensins in intracellular and extracellular environments will provide a key to understanding peptide-mediated host defence.     

Defensins in innate antiviral immunity

Defensins are small antimicrobial peptides that are produced by leukocytes and epithelial cells, and that have an important role in innate immunity. Recent advances in understanding the mechanisms of the antiviral action(s) of defensins indicate that they have a dual role in antiviral defence, acting directly on the virion and on the host cell. This Review focuses on the antiviral activities and mechanisms of action of mammalian defensins, and on the clinical relevance of these activities. Understanding the complex function of defensins in innate immunity against viral infection has implications for the prevention and treatment of viral disease.     

Primate defensins

Defensins are endogenous, cysteine-rich antimicrobial peptides that contribute to host defence against bacterial, fungal and viral infections. There are three subfamilies of defensins in primates: alpha-defensins are most common in neutrophils and Paneth cells of the small intestine; beta-defensins protect the skin and the mucous membranes of the respiratory, genitourinary and gastrointestinal tracts; and theta-defensins, which are expressed only in Old World monkeys, lesser apes and orangutans, are lectins with broad-spectrum antiviral efficacy. Here, their discovery and recent advances in understanding their properties and functions are described.     

Antimicrobial Peptides from Plants

Peptides with antimicrobial properties are present in most if not all plant species. All plant antimicrobial peptides isolated so far contain even numbers of cysteines (4, 6, or 8), which are all pairwise connected by disulfide bridges, thus providing high stability to the peptides. Based on homologies at the primary structure level, plant antimicrobial peptides can be classified into distinct families including thionins, plant defensins, lipid transfer proteins, and he vein- and knottin-type antimicrobial peptides. Detailed three-dimensional structure information has been obtained for one or more members of these peptide families. All antimicrobial peptides studied thus far appear to exert their antimicrobial effect at the level of the plasma membrane of the target microorganism, but the different peptide types are likely to act via different mechanisms. Antimicrobial peptides can occur in all plant organs. In unstressed organs, antimicrobial peptides are usually most abundant in the outer cell layer lining the organ, which is consistent with a role for the antimicrobial peptides in constitutive host defense against microbial invaders attacking from the outside. Thionins are predominantly located intracellularly but are also found in the extracellular space, whereas most plant defensins and lipid transfer proteins are deposited exclusively in the extracellular space. In a number of plant species, a strong induction of genes expressing either thionins, plant defensins, or lipid transfer proteins has been observed on infection of the leaves by microbial pathogens. Hence, antimicrobial peptides can also take part in the inducible defense response of plants. Constitutive expression in transgenic plants of heterologous antimicrobial peptide genes has been achieved, which in some cases has led to enhanced resistance to particular microbial plant pathogens.     

In-silico homology modeling of three isoforms of insect defensins from the dengue vector mosquito, Aedes aegypti (Linn., 1762)

Dengue is a serious public health problem in tropical and subtropical countries. It is caused by any of the four serologically distinct dengue viruses, namely DENV1–4. The viruses are transmitted by Aedes mosquitoes. Understanding various defence mechanisms of insects has become a prime area of research worldwide. In insects, the first line of defence against invading pathogens includes cellular mechanisms and a battery of antimicrobial peptides such as defensins, cecropins etc. Defensins—cationic, cysteine-rich peptides consisting of ∼40 amino acids with broad-spectrum activity against Gram-positive bacteria—have been reported from a wide range of organisms. In the dengue vector mosquito, Aedes aegypti, three isoforms of defensins are reported to be expressed in a spatial and temporal fashion. This report presents the three-dimensional structures of the three isoforms of Ae. aegypti defensins predicted by comparative modeling. Prediction was done with Modeller 9v1 and the structures validated through a series of tests. The best results of the prediction study are presented, and may help lead to the discovery of new synthetic peptides or derivatives of defensins that could be useful in the control of vector-borne diseases.     

Staphylococcal resistance to antimicrobial peptides of mammalian and bacterial origin

Antimicrobial host defense peptides, such as defensins, protegrins, and platelet microbicidal proteins are deployed by mammalian skin, epithelia, phagocytes, and platelets in response to Staphylococcus aureus infection. In addition, staphylococcal products with similar structures and activities, called bacteriocins, inhibit competing microorganisms. Staphylococci have developed resistance mechanisms, which are either highly specific for certain host defense peptides or bacteriocins or which broadly protect against a range of cationic antimicrobial peptides. Experimental infection models can be used to study the molecular mechanisms of antimicrobial peptides, the peptide resistance strategies of S. aureus, and the therapeutic potential of peptides in staphylococcal diseases.     

Function and therapeutic potential of host defence peptides.

Cationic host defence (antimicrobial) peptides are an important component of the innate immune systems of a wide variety of plants, animals, and bacteria. Although most of these compounds have direct antimicrobial activities under specific conditions, a greater appreciation for the diversity of functions of these molecules is beginning to develop in the field. In addition to their directly antimicrobial activities, they also have a broad spectrum of activity on the host immune system, with both pro-inflammatory and anti-inflammatory effects being invoked. Increasingly sophisticated approaches to understand the role of host defence peptides in modulating innate immunity are already serving to guide the development of novel therapeutics.     

Recent advances in the research and development of human defensins

Human defensins are a family of cationic antimicrobial peptides with molecular weights of 4-5 kDa, containing a conserved six disulphide-linked cysteine motif. During the last two decades a series of endogenous alpha- and beta-human defensins were discovered. They exhibit a broad range of antimicrobial properties and are thought to be ideal therapeutic agents because of their potential ability to circumvent the problems of acquired resistance often observed with other antimicrobial therapies. Because of their appealing medical and pharmaceutical potential there has been an emphasis on human defensins in medical and molecular biology research in recent years. This paper aims to present a comprehensive review of recent advances in the study of human defensins including their discovery, classification, molecular properties, expression, mechanisms of action and potential medical applications. In addition, the advances in producing human defensins via genetic engineered cells are summarized from research works in our group (besides host cells including E. coli, B. subtilis and yeast systems, the cell-free protein synthesis system was also employed to express human beta-defensin-2) along with other related published works. The present challenges and prospects for the potential application of human defensins are also discussed.     

Crystal structures of human alpha-defensins HNP4, HD5, and HD6

Six alpha-defensins have been found in humans. These small arginine-rich peptides play important roles in various processes related to host defense, being the effectors and regulators of innate immunity as well as enhancers of adoptive immune responses. Four defensins, called neutrophil peptides 1 through 4, are stored primarily in polymorphonuclear leukocytes. Major sites of expression of defensins 5 and 6 are Paneth cells of human small intestine. So far, only one structure of human alpha-defensin (HNP3) has been reported, and the properties of the intestine defensins 5 and 6 are particularly poorly understood. In this report, we present the high-resolution X-ray structures of three human defensins, 4 through 6, supplemented with studies of their antimicrobial and chemotactic properties. Despite only modest amino acid sequence identity, all three defensins share their tertiary structures with other known alpha- and beta-defensins. Like HNP3 but in contrast to murine or rabbit alpha-defensins, human defensins 4-6 form characteristic dimers. Whereas antimicrobial and chemotactic activity of HNP4 is somewhat comparable to that of other human neutrophil defensins, neither of the intestinal defensins appears to be chemotactic, and for HD6 also an antimicrobial activity has yet to be observed. The unusual biological inactivity of HD6 may be associated with its structural properties, somewhat standing out when compared with other human alpha-defensins. The strongest cationic properties and unique distribution of charged residues on the molecular surface of HD5 may be associated with its highest bactericidal activity among human alpha-defensins.     

Tool developments for structure-function studies of host defense peptides

Antimicrobial peptides, or host defense peptides, are universal signaling and effector molecules in host defense and innate immunity. This article highlights various tools developed for cathelicidins and defensins, ranging from peptide identification, production, and structural biology, including the eight databases for antimicrobial peptides. Novel peptides can be identified from natural sources at both gene and protein levels. Solid-phase synthesis and bacterial expression are the two important methods for peptide production. Three-dimensional structures of antimicrobial peptides, primarily determined by solution NMR techniques, are essential for an in-depth understanding of the mode of action. The introduction of octanoyl phosphatidylglycerol as a bacterial membrane-mimetic model provides new insights into peptide-lipid interactions. The incorporation of structure and activity data into the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html) will lead to an integrated understanding of these peptides via structural bioinformatics.     

Antimicrobial activity of three tick defensins and four mammalian cathelicidin-derived synthetic peptides against Lyme disease spirochetes and bacteria isolated from the midgut

In this study, chemically synthesized tick defensins and cathelicidin-derived mammalian peptides were used to investigate the activity spectrum against Borrelia garinii and symbiotic Stenotrophomonas maltophila. Synthetic tick defensins showed antimicrobial activity against Staphylococcus aureus but not B. garinii and S. maltophila. Mammalian peptides which have cationic property similar to tick defensins, showed antimicrobial activity similar to tick defensins. The antimicrobial peptides in ticks and mammalian hosts have common characteristics against microbial invasion in the innate immune system.     

Modes of antifungal action and in planta functions of plant defensins and defensin-like peptides

Plant defensins are small basic peptides that are inhibitory against a range of plant and human pathogens. Their in vitro antimicrobial activity and structural similarity with human and insect defensins indicated an important role for plant defensins in the innate immune system of plants. Regarding their mode of antimicrobial action, most plant defensins interact with a specific microbial surface receptor, resulting in microbial cell death via e.g. induction of apoptosis. However, accumulating evidence suggests additional in vivo functions of these plant defensins, and by extension of the more recently discovered defensin-like peptides, in general plant development. In this review we will discuss both, the functional roles of defensins in the plant and their modes of antimicrobial action.     

Beta-defensin antibiotic peptides in the innate immunity of the buffalo: in vivo and in vitro studies

Beta-defensin antimicrobial peptides are multifunctional biomolecules, which are a major component of the oxygen-independent microbicidal system of buffalo polymorphonuclear (PMN) cells. They have great potential for use as proteomic biomarkers of host cell responses in the presence of microbial agents. On purifying these peptides by RP-HPLC, four defensin peptides were revealed. The results from testing against Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Candida albicans, Rinderpest Virus (RPV) and Newcastle Disease Virus (NDV), showed that the peptides possessed antimicrobial and antiviral activities. Minimum inhibitory concentration (MIC) values varied according to the peptide amounts and the exposure time. Furthermore, an increase in the levels of these cationic antimicrobial peptides was apparent in milk obtained from natural cases of mastitis, as compared to the levels in normal milk. MALDI-TOF-based amino acid sequencing confirmed the expression of two beta-defensins (LAP and BNBD-2) in mastitis milk. A comparison of peptide sequences revealed that buffalo LAP and BNBD-2 share 98.6% and 100% sequence identity, respectively, with those of cattle. In vitro, Bovine Viral Diarrhoea Virus (BVDV) infection was shown to induce the expression of the beta-defensin gene, as evidenced by the PCR amplification of cDNA with specific primers. The determination of the enhanced expression of beta-defensin peptides in mastitis milk and in PMN cells, can be considered as an advanced approach to the assessment of cellular and molecular responses to cell injury. It is hoped that in vitro studies on phagocytes such as PMN cells and other cell lines, will eventually replace the use of animals in elucidating the roles of these cytokines in response to microbe-derived cell damage. It will also be possible to use defensins as biomarkers to correlate failure in host cell defence systems with peptide heterogeneity.     

Four plant defensins from an indigenous South African Brassicaceae species display divergent activities against two test pathogens despite high sequence similarity in the encoding genes

Background

Plant defensins are an important component of the innate defence system of plants where they form protective antimicrobial barriers between tissue types of plant organs as well as around seeds. These peptides also have other activities that are important for agricultural applications as well as the medical sector. Amongst the numerous plant peptides isolated from a variety of plant species, a significant number of promising defensins have been isolated from Brassicaceae species. Here we report on the isolation and characterization of four defensins from Heliophila coronopifolia, a native South African Brassicaceae species.

Results

Four defensin genes (Hc-AFP1-4) were isolated with a homology based PCR strategy. Analysis of the deduced amino acid sequences showed that the peptides were 72% similar and grouped closest to defensins isolated from other Brassicaceae species. The Hc-AFP1 and 3 peptides shared high homology (94%) and formed a unique grouping in the Brassicaceae defensins, whereas Hc-AFP2 and 4 formed a second homology grouping with defensins from Arabidopsis and Raphanus. Homology modelling showed that the few amino acids that differed between the four peptides had an effect on the surface properties of the defensins, specifically in the alpha-helix and the loop connecting the second and third beta-strands. These areas are implicated in determining differential activities of defensins. Comparing the activities after recombinant production of the peptides, Hc-AFP2 and 4 had IC₅₀ values of 5-20 μg ml^-1 against two test pathogens, whereas Hc-AFP1 and 3 were less active. The activity against Botrytis cinerea was associated with membrane permeabilization, hyper-branching, biomass reduction and even lytic activity. In contrast, only Hc-AFP2 and 4 caused membrane permeabilization and severe hyper-branching against the wilting pathogen Fusarium solani, while Hc-AFP1 and 3 had a mild morphogenetic effect on the fungus, without any indication of membrane activity. The peptides have a tissue-specific expression pattern since differential gene expression was observed in the native host. Hc-AFP1 and 3 expressed in mature leaves, stems and flowers, whereas Hc-AFP2 and 4 exclusively expressed in seedpods and seeds.

Conclusions

Two novel Brassicaceae defensin sequences were isolated amongst a group of four defensin encoding genes from the indigenous South African plant H. coronopifolia. All four peptides were active against two test pathogens, but displayed differential activities and modes of action. The expression patterns of the peptide encoding genes suggest a role in protecting either vegetative or reproductive structures in the native host against pathogen attack, or roles in unknown developmental and physiological processes in these tissues, as was shown with other defensins.     

Functional interaction of human neutrophil peptide-1 with the cell wall precursor lipid II

Defensins constitute a major class of cationic antimicrobial peptides in mammals and vertebrates, acting as effectors of innate immunity against infectious microorganisms. It is generally accepted that defensins are bactericidal by disrupting the anionic microbial membrane. Here, we provide evidence that membrane activity of human α-defensins does not correlate with antibacterial killing. We further show that the α-defensin human neutrophil peptide-1 (HNP1) binds to the cell wall precursor lipid II and that reduction of lipid II levels in the bacterial membrane significantly reduces bacterial killing. The interaction between defensins and lipid II suggests the inhibition of cell wall synthesis as a novel antibacterial mechanism of this important class of host defense peptides.     

Modes of antifungal action and in planta functions of plant defensins and defensin-like peptides

Plant defensins are small basic peptides that are inhibitory against a range of plant and human pathogens. Their in vitro antimicrobial activity and structural similarity with human and insect defensins indicated an important role for plant defensins in the innate immune system of plants. Regarding their mode of antimicrobial action, most plant defensins interact with a specific microbial surface receptor, resulting in microbial cell death via e.g. induction of apoptosis. However, accumulating evidence suggests additional in vivo functions of these plant defensins, and by extension of the more recently discovered defensin-like peptides, in general plant development. In this review we will discuss both, the functional roles of defensins in the plant and their modes of antimicrobial action.     

Evolutionary selective trends of insect/mosquito antimicrobial defensin peptides containing cysteine-stabilized alpha/beta motifs

Insect defensins containing cysteine-stabilized alpha/beta motifs (Cs-alpha/beta defensin) are cationic, inducible antibacterial peptides involved in humoral defence against pathogens. To examine trends in molecular evolution of these antimicrobial peptides, sequences similar to the well-characterized Cs-alpha/beta defensin peptide of Anopheles gambiae, using six cysteine residues as landmarks, were retrieved from genomic and protein databases. These sequences were derived from different orders of insects. Genes of insect Cs-alpha/beta defensin appear to constitute a multigene family in which the copy number varies between insect species. Phylogenetic analysis of these sequences revealed two main lineages, one group comprising mainly lepidopteran insects and a second, comprising Hemiptera, Coleoptera, Diptera and Hymenoptera insects. Moreover, the topology of the phylogram indicated dipteran Cs-alpha/beta defensins are diverse, suggesting diversity in immune mechanisms in this order of insects. Overall evolutionary analysis indicated marked diversification and expansion of mature defensin isoforms within the species of mosquitoes relative to non-mosquito defensins, implying the presence of finely tuned immune responses to counter pathogens. The observed higher synonymous substitution rate relative to the nonsynonymous rate in almost all the regions of Cs-alpha/beta defensin of mosquitoes suggests that these peptides are predominately under purifying selection. The maximum-likelihood models of codon substitution indicated selective pressure at different amino acid sites in mosquito mature Cs-alpha/beta defensins is differ and are undergoing adaptive evolution in comparison to non-mosquito Cs-alpha/beta defensins, for which such selection was inconspicuous; this suggests the acquisition of selective advantage of the Cs-alpha/beta defensins in the former group. Finally, this study represents the most detailed report on the evolutionary strategies of Cs-alpha/beta defensins of mosquitoes in particular and insects in general, and indicates that insect Cs-alpha/beta defensins have evolved by duplication followed by divergence, to produce a diverse set of paralogues.     

Paneth cells of the human small intestine express an antimicrobial peptide gene.

Mucosal surfaces of several organ systems are important interfaces for host defense against microbes. Recent evidence suggests that antimicrobial peptides contribute to the defense of these surfaces. Defensins are one family of antimicrobial peptide, but their known distribution in humans has been limited to four members found in cells of myeloid origin. We sought to determine if the human defensin family was more complex. We found that the family of human defensins is diverse and is not restricted to expression in leukocytes. Southern blot and genomic clone analyses reveal that numerous defensin-related sequences are present in the human genome. A gene for a new human defensin family member was characterized. This gene, designated human defensin-5, is highly expressed in Paneth cells of the small intestine. This is the first example of an antimicrobial peptide gene expressed in an epithelial cell in humans. The data support the hypotheses that epithelial defensins equip the human small bowel with a previously unrecognized defensive capability which would augment other antimicrobial defenses.     

Defensins are natural peptide antibiotics of higher eukaryotes

The goal of this review is to characterize defensins representing an evolutionary the most ancient family of antimicrobial peptides. It gives general information on functional and structural features of defensins as the main components of the first-line defense of higher eukaryote organisms against infectious agents. The review not only considers current situation in the defensin research but also perspectives of creation of recombinant antimicrobial peptides of biomedical application.     

Attenuated virulence of Streptococcus agalactiae deficient in D-alanyl-lipoteichoic acid is due to an increased susceptibility to defensins and phagocytic cells

D-alanylation of lipoteichoic acid (LTA), allows Gram-positive bacteria to modulate their surface charge, regulate ligand binding and control the electromechanical properties of the cell wall. In this study, the role of D-alanyl LTA in the virulence of the extracellular pathogen Streptococcus agalactiae was investigated. We demonstrate that a DltA- isogenic mutant displays an increased susceptibility to host defence peptides such as human defensins and animal-derived cationic peptides. Accordingly, the mutant strain is more susceptible to killing by mice bone marrow-derived macrophages and human neutrophils than the wild-type strain. In addition, the virulence of the DltA- mutant is severely impaired in mouse and neonatal rat models. This mutant was eliminated more rapidly than the wild-type strain from the lung of three-week-old mice inoculated intranasally and, consequently, is unable to induce a pneumonia. Finally, after intravenous injection of three-week-old mice, the survival of the DltA- mutant is markedly reduced in the blood in comparison to that of the wild-type strain. We hypothesize that the decreased virulence of the DltA- mutant is a consequence of its increased susceptibility to cationic antimicrobial peptides and to killing by phagocytes. These results demonstrate that the D-alanylation of LTA contributes to the virulence of S. agalactiae.