Insect immunity. Isolation from a coleopteran insect of a novel inducible antibacterial peptide and of new members of the insect defensin family.

Injection of heat-killed bacteria into larvae of the large tenebrionid beetle Zophobas atratus (Insecta, Endopterygota, Coleoptera) results in the appearance in the hemolymph of a potent antibacterial activity as evidenced by a plate growth inhibition assay. We have isolated three peptides (A-C) from this immune hemolymph which probably account for most of this activity. Their primary structures were established by a combination of peptide sequencing and molecular mass determination by mass spectrometry. Peptide A, which is bactericidal against Gram-negative cells, is a 74-residue glycine-rich molecule with no sequence homology to known peptides. We propose the name coleoptericin for this novel inducible antibacterial peptide. Peptides B and C are isoforms of a 43-residue peptide which contains 6 cysteines and shows significant sequence homology to insect defensins, initially reported from dipteran insects. This peptide is active against Gram-positive bacteria. The results are discussed in connection with recent studies on inducible antibacterial peptides present in the three other major orders of the endopterygote clade of insects: the Lepidoptera, Diptera, and Hymenoptera.     

Insect immunity. Constitutive expression of a cysteine-rich antifungal and a linear antibacterial peptide in a termite insect

Two novel antimicrobial peptides, which we propose to name termicin and spinigerin, have been isolated from the fungus-growing termite Pseudacanthotermes spiniger (heterometabole insect, Isoptera). Termicin is a 36-amino acid residue antifungal peptide, with six cysteines arranged in a disulfide array similar to that of insect defensins. In contrast to most insect defensins, termicin is C-terminally amidated. Spinigerin consists of 25 amino acids and is devoid of cysteines. It is active against bacteria and fungi. Termicin and spinigerin show no obvious sequence similarities with other peptides. Termicin is constitutively present in hemocyte granules and in salivary glands. The presence of termicin and spinigerin in unchallenged termites contrasts with observations in evolutionary recent insects or insects undergoing complete metamorphosis, in which antimicrobial peptides are induced in the fat body and released into the hemolymph after septic injury.     

Structure-activity relationships of the intramolecular disulfide bonds in coprisin,a defensin from the dung beetle

Defensins, which are small cationic molecules produced by organisms as part of their innate immune response, share a common structural scaffold that is stabilized by three disulfide bridges. Coprisin is a 43-amino acid defensin-like peptide from Copris tripartitus. Here, we report the intramolecular disulfide connectivity of cysteine-rich coprisin, and show that it is the same as in other insect defensins. The disulfide bond pairings of coprisin were determined by combining the enzymatic cleavage and mass analysis. We found that the loss of any single disulfide bond in coprisin eliminated all antibacterial, but not antifungal, activity. Circular dichroism (CD) analysis showed that two disulfide bonds, Cys20-Cys39 and Cys24-Cys41, stabilize coprisin’s α-helical region. Moreover, a BLAST search against UniProtKB database revealed that coprisin’s α-helical region is highly homologous to those of other insect defensins. [BMB Reports 2014; 47(11): 625-630]     

Insect antimicrobial peptides and their applications

Insects are one of the major sources of antimicrobial peptides/proteins (AMPs). Since observation of antimicrobial activity in the hemolymph of pupae from the giant silk moths Samia Cynthia and Hyalophora cecropia in 1974 and purification of first insect AMP (cecropin) from H. cecropia pupae in 1980, over 150 insect AMPs have been purified or identified. Most insect AMPs are small and cationic, and they show activities against bacteria and/or fungi, as well as some parasites and viruses. Insect AMPs can be classified into four families based on their structures or unique sequences: the α-helical peptides (cecropin and moricin), cysteine-rich peptides (insect defensin and drosomycin), proline-rich peptides (apidaecin, drosocin, and lebocin), and glycine-rich peptides/proteins (attacin and gloverin). Among insect AMPs, defensins, cecropins, proline-rich peptides, and attacins are common, while gloverins and moricins have been identified only in Lepidoptera. Most active AMPs are small peptides of 20–50 residues, which are generated from larger inactive precursor proteins or pro-proteins, but gloverins (~14 kDa) and attacins (~20 kDa) are large antimicrobial proteins. In this mini-review, we will discuss current knowledge and recent progress in several classes of insect AMPs, including insect defensins, cecropins, attacins, lebocins and other proline-rich peptides, gloverins, and moricins, with a focus on structural-functional relationships and their potential applications.     

Isolation, characterization and chemical synthesis of a new insect defensin from Chironomus plumosus (Diptera)

Injection of low doses of bacteria into the aquatic larvae of the dipteran insect Chironomus plumosus induces the appearance in their hemolymph of a potent antibacterial activity. We have isolated two 36-residue peptides from this hemolymph which are active against Gram-positive bacteria. The peptides are novel members of the insect defensin family and their sequences present marked differences with those of insect defensins isolated from other dipteran species. We have developed a method for efficient renaturation of this cysteine-rich molecule and obtained a highly pure synthetic Chironomus defensin.     

Two novel insect defensins from larvae of the cupreous chafer, Anomala cuprea: purification, amino acid sequences and antibacterial activity.

A humoral immune response in larvae of the coleopteran insect, Anomala cuprea has been examined for exploring the molecular basis of host-pathogen interactions. The antibacterial activity against the Gram-positive strain, Micrococcus luteus was detected at a low level in absence of injection. The activity increased strikingly in the hemolymph of the larvae challenged with Escherichia coli, showing the fluctuating profile through a time course, which consists of the static induction phase, the production phase rising to a maximum level, and the reduction phase extending over a long duration. Two peptides were purified and characterized by reverse-phase HPLC, Edman degradation and mass spectrometry. They were isoforms, composed of similar sequences with two amino acid substitutions in 43 residues, and novel members of the insect defensins, cysteine-rich antibacterial peptides. Anomala defensins A and B showed potent activity against Gram-positive bacteria, with slight differences in activity against a few strains of tested bacteria. Anomala defensin B was active at high concentration of 40 microM against the Gram-negative strain, Xenorhabdus japonicus, a pathogen toward the host, A. cuprea larvae.     

Solution structure of termicin,an antimicrobial peptide from the termite Pseudacanthotermes spiniger

The solution structure of termicin from hemocytes of the termite Pseudacanthotermes spiniger was determined by proton two-dimensional nuclear magnetic resonance spectroscopy and molecular modeling techniques. Termicin is a cysteine-rich antifungal peptide also exhibiting a weak antibacterial activity. The global fold of termicin consists of an alpha-helical segment (Phe4-Gln14) and a two-stranded (Phe19-Asp25 and Gln28-Phe33) antiparallel beta-sheet forming a "cysteine stabilized alphabeta motif" (CSalphabeta) also found in antibacterial and antifungal defensins from insects and from plants. Interestingly, termicin shares more structural similarities with the antibacterial insect defensins and with MGD-1, a mussel defensin, than with the insect antifungal defensins such as drosomycin and heliomicin. These structural comparisons suggest that global fold alone does not explain the difference between antifungals and antibacterials. The antifungal properties of termicin may be related to its marked hydrophobicity and its amphipatic structure as compared to the antibacterial defensins. [SWISS-PROT accession number: Termicin (P82321); PDB accession number: 1MM0.]     

Cutting edge: the toll pathway is required for resistance to gram-positive bacterial infections in Drosophila

In Drosophila, the response against various microorganisms involves different recognition and signaling pathways, as well as distinct antimicrobial effectors. On the one hand, the immune deficiency pathway regulates the expression of antimicrobial peptides that are active against Gram-negative bacteria. On the other hand, the Toll pathway is involved in the defense against filamentous fungi and controls the expression of antifungal peptide genes. The gene coding for the only known peptide with high activity against Gram-positive bacteria, Defensin, is regulated by both pathways. So far, survival experiments to Gram-positive bacteria have been performed with Micrococcus luteus and have failed to reveal the involvement of one or the other pathway in host defense against such infections. In this study, we report that the Toll pathway, but not that of immune deficiency, is required for resistance to other Gram-positive bacteria and that this response does not involve Defensin.     

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.     

Lead optimization of antifungal peptides with 3D NMR structures analysis

Antimicrobial peptides are key components of the innate immune response in most multicellular organisms. These molecules are considered as one of the most innovative class of anti-infective agents that have been discovered over the last two decades, and therefore, as a source of inspiration for novel drug design. Insect cystine-rich antimicrobial peptides with the CS alpha beta scaffold (an alpha-helix linked to a beta-sheet by two disulfide bridges) represent particularly attractive templates for the development of systemic agents owing to their remarkable resistance to protease degradation. We have selected heliomicin, a broad spectrum antifungal CS alpha beta peptide from Lepidoptera as the starting point of a lead optimization program based on phylogenic exploration and fine tuned mutagenesis. We report here the characterization, biological activity, and 3D structure of heliomicin improved analogs, namely the peptides ARD1, ETD-135, and ETD-151. The ARD1 peptide was initially purified from the immune hemolymph of the caterpillars of Archeoprepona demophoon. Although it differs from heliomicin by only two residues, it was found to be more active against the human pathogens Aspergillus fumigatus and Candida albicans. The peptides ETD-135 and ETD-151 were engineered by site-directed mutagenesis of ARD1 in either cationic or hydrophobic regions. ETD-135 and ETD-151 demonstrated an improved antifungal activity over the native peptides, heliomicin and ARD1. A comparative analysis of the 3D structure of the four molecules highlighted the direct impact of the modification of the amphipathic properties on the molecule potency. In addition, it allowed to characterize an optimal organization of cationic and hydrophobic regions to achieve best antifungal activity.     

Solution structures of the antifungal heliomicin and a selected variant with both antibacterial and antifungal activities

In response to an experimental infection, the lepidopteran Heliothis virescens produces an antifungal protein named heliomicin. Heliomicin displays sequence similarities with antifungal plant defensins and antibacterial or antifungal insect defensins. To gain information about the structural elements required for either antifungal or antibacterial activity, heliomicin and selected point-mutated variants were expressed in yeast as fusion proteins. The effects of mutations, defined by comparing the primary structure of heliomicin with the sequences of members of the insect defensin family, were analyzed using antibacterial and antifungal assays. One of the variants shows significant activity against Gram-positive bacteria while remaining efficient against fungi. The three-dimensional structures of this variant and of the wild-type protein were determined by two-dimensional (1)H NMR to establish a correlation between structure and antibacterial or antifungal activity. Wild-type and mutated heliomicins adopt a similar scaffold, including the so-called cysteine-stabilized alphabeta motif. A comparison of their structures with other defensin-type molecules indicates that common hydrophobic characteristics can be assigned to all the antifungal proteins. A comparative analysis of various structural features of heliomicin mutant and of antibacterial defensins enables common properties to be assessed, which will help to design new mutants with increased antibacterial activity.     

Engineering of a linear inactive analog of human β‐defensin 4 to generate peptides with potent antimicrobial activity

Human β‐defensins (HBDs) are cationic antimicrobial peptides constrained by three disulfide bridges. They have diverse range of functions in the innate immune response. It is of interest to investigate whether linear analogs of defensins can be generated, which possess antimicrobial activity. In this study, we have designed linear peptides with potent antimicrobial activity from an inactive peptide spanning the N‐terminus of HBD4. Our results show that l ‐arginine to d ‐arginine substitution imparts considerable antimicrobial activity against both bacteria and Candida albicans. Increase in hydrophobicity by fatty acylation of the peptides with myristic acid further enhances their potency. In the presence of high concentrations of salt, antimicrobial activity of the myristoylated peptide with l ‐arginine is attenuated relatively to a lesser extent as compared with the linear active peptide with d ‐arginine. Substitution of cysteine with the hydrophobic helix‐promoting amino acid α‐aminoisobutyric acid favors candidacidal activity but not antibacterial activity. The mechanism of killing by d ‐arginine substituted unacylated analog involves transient interaction with the bacterial membrane followed by translocation into the cytoplasm without membrane permeabilization. Accumulation of peptides in the cytoplasm can affect various cellular processes that lead to cell death. However, the peptide causes membrane permeabilization in case of C. albicans. Myristoylation results in greater interaction of the peptide chain with the microbial cell surface and causes membrane permeabilization. Results described in the study demonstrate that it is possible to generate highly active linear analogs of defensins by selective introduction of d ‐amino acids and fatty acids, which could be attractive candidates for development as therapeutic agents. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Enteric defensins: antibiotic peptide components of intestinal host defense

Five intestinal defensins, termed cryptdins 1-5, have been purified from mouse small bowel, sequenced, and localized to the epithelium by immunohistochemistry. Although identified as members of the defensin peptide family by peptide sequencing, enteric defensins are novel in that four cryptdins have amino termini which are three to six residues longer than those of leukocyte-derived defensins. A fifth cryptdin is the first defensin to diverge from the previously invariant spacing of cysteines in the peptide structure. The most abundant enteric defensin, cryptdin-1, had antimicrobial activity against an attenuated phoP mutant of Salmonella typhimurium but was not active against the virulent wild-type parent. Immunohistochemical localization demonstrated that cryptdin-1, and probably cryptdins 2 and 3, occur exclusively in Paneth cells, where the peptides appear to be associated with cytoplasmic granules. Biochemical and immunologic analysis of the luminal contents of the small intestine suggest that cryptdin peptides are secreted into the lumen, similar to Paneth cell secretion of lysozyme. The presence of several enteric defensins in the intestinal epithelium, evidence of their presence in the lumen, and the antibacterial activity of cryptdin-1 suggest that these peptides contribute to the antimicrobial barrier function of the small bowel mucosa.     

A drosomycin-GFP reporter transgene reveals a local immune response in Drosophila that is not dependent on the Toll pathway.

A hallmark of the systemic antimicrobial response of Drosophila is the synthesis by the fat body of several antimicrobial peptides which are released into the hemolymph in response to a septic injury. One of these peptides, drosomycin, is active primarily against fungi. Using a drosomycin-green fluorescent protein (GFP) reporter gene, we now show that in addition to the fat body, a variety of epithelial tissues that are in direct contact with the external environment, including those of the respiratory, digestive and reproductive tracts, can express the antifungal peptide, suggesting a local response to infections affecting these barrier tissues. As is the case for vertebrate epithelia, insect epithelia appear to be more than passive physical barriers and are likely to constitute an active component of innate immunity. We also show that, in contrast to the systemic antifungal response, this local immune response is independent of the Toll pathway.     

Primary structure and in vitro antibacterial properties of the Drosophila melanogaster attacin C Pro-domain

In Drosophila melanogaster, seven distinct families of antimicrobial peptides with different structures and specificities are synthesized by the fat body and released into the hemolymph during the immune response. Using microscale high performance liquid chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and Edman degradation, we have isolated and characterized from immune-challenged Drosophila two novel induced molecules, under the control of the Imd pathway, that correspond to post-translationally modified antimicrobial peptides or peptide fragments. The first molecule is a doubly glycosylated form of drosocin, an O-glycosylated peptide that kills Gram-negative organisms. The second molecule represents a truncated form of the pro-domain of the Drosophila attacin C carrying two post-translational modifications and has significant structural similarities to proline-rich antibacterial peptides including drosocin. We have synthesized this peptide and found that it is active against Gram-negative bacteria. Furthermore, this activity is potentiated when the peptide is used in combination with the Drosophila antimicrobial peptide cecropin A. The synergistic action observed between these two molecules suggests that the truncated post-translationally modified pro-domain of attacin C by itself may play an important role in the antimicrobial defense of Drosophila.     

防御素的生物学特性及其抗病基因工程

防御素是一种富含半胱氨酸的小分子多肽,对细菌等微生物具有广谱抗性,且作用机制特殊。迄今为止,国内外在防御素方面进行了大量的研究,已经从各类生物体中分离出不同种类的防御素,并在基因工程和医药领域呈现广泛的应用前景。文章对防御素的分类、生物学特性,包括哺乳动物α-、β-、θ-防御素、昆虫以及植物防御素的分子结构及抗菌活性进行了综述,阐述了防御素的膜作用及与细胞内复合物结合的作用机制。总结和归纳了防御素基因的分离、表达研究进展及动、植物防御素基因在抗病基因工程领域的应用,并对防御素在未来的生物制药和植物抗病基因工程方面的应用前景进行了展望。     

Prokaryotic expression of a constitutively expressed <Emphasis Type="Italic">Tephrosia villosa</Emphasis> defensin and its potent antifungal activity

Plant defensins are small, highly stable, cysteine-rich antimicrobial peptides produced by the plants for inhibiting a broad-spectrum of microbial pathogens. Some of the well-characterized plant defensins exhibit potent antifungal activity on certain pathogenic fungal species only. We characterized a defensin, TvD1 from a weedy leguminous herb, Tephrosia villosa. The open reading frame of the cDNA was 228 bp, which codes for a peptide with 75 amino acids. Expression analyses indicated that this defensin is expressed constitutively in T. villosa with leaf, stem, root, and seed showing almost similar levels of high expression. The recombinant peptide (rTvD1), expressed in the Escherichia coli expression system, exhibited potent in vitro antifungal activity against several filamentous soil-borne fungal pathogens. The purified peptide also showed significant inhibition of root elongation in Arabidopsis seedlings, subsequently affecting the extension of growing root hairs indicating that it has the potential to disturb the plant growth and development.     

Structural characterization of plant defensin protein superfamily

Plant defensins represent a major innate immune protein superfamily with strong inhibitory effects on infectious diseases of humans, antifungal/antibacterial activities, proteinase and insect amylase inhibitory activities. They are generally defined by their conserved cysteine scaffold with α-helix and triple strand anti parallel β-sheet connected to the scaffold. With the genome of more plant species being fully sequenced, significant information about newly sequenced defensin proteins has been revealed. In this paper, we identify members of defensin protein families across plant species and use protein-modeling-based structural reconstitution to reveal specific three dimensional hidden features of plant defensins mediating defense responses and other interesting biological activities in plants. Our data revealed that plant defensins are structurally similar to their insect counterparts despite the low amino acid sequence similarity between these two organisms. The molecular and structural relationship among plant defensins and defensins from other species is discussed.