Cysteine-rich low molecular weight antimicrobial peptides from <Emphasis Type="Italic">Brevibacillus</Emphasis> and related genera for biotechnological applications

The production of natural antimicrobial peptides (AMPs) is an innate immunity trait of all life forms including eukaryotes and prokaryotes. While these AMPs are usually called as defensins in eukaryotes, they are known as bacteriocins in prokaryotes. Bacteriocins are more diverse AMPs considering their varied composition and posttranslational modifications. Accordingly, this review is focused on cysteine-rich AMPs resembling eukaryotic defensins such as laterosporulin from Brevibacillus spp. and associated peptides secreted by the members of related genera. In fact, structural studies of laterosporulin showed the pattern typically observed in human defensins and therefore, should be considered as bacterial defensin. Although the biosynthesis mechanism of bacterial defensins displayed high similarities, variations in amino acid composition and structure provided the molecular basis for a better understanding of their properties. They are reported to inhibit Gram-positive, Gram-negative, non-multiplying and human pathogenic bacteria. The extreme stability is due to the presence of intra-molecular disulfide bonds in prokaryotic defensins and reveals their potential clinical and food preservation applications. Notably, they are also reported to have potential anticancer properties. Therefore, this review is focused on multitude of diverse applications of bacterial defensins, exploring the possible correlations between their structural, functional and possible biotechnological applications.     

Structure-activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against Fusarium graminearum

Plant defensins are small cysteine-rich antimicrobial proteins. Their three-dimensional structures are similar in that they consist of an α-helix and three anti-parallel β-strands stabilized by four disulfide bonds. Plant defensins MsDef1 and MtDef4 are potent inhibitors of the growth of several filamentous fungi including Fusarium graminearum. However, they differ markedly in their antifungal properties as well as modes of antifungal action. MsDef1 induces prolific hyperbranching of fungal hyphae, whereas MtDef4 does not. Both defensins contain a highly conserved γ-core motif (GXCX(3-9)C), a hallmark signature present in the disulfide-stabilized antimicrobial peptides, composed of β2 and β3 strands and the interposed loop. The γ-core motifs of these two defensins differ significantly in their primary amino acid sequences and in their net charge. In this study, we have found that the major determinants of the antifungal activity and morphogenicity of these defensins reside in their γ-core motifs. The MsDef1-γ4 variant in which the γ-core motif of MsDef1 was replaced by that of MtDef4 was almost as potent as MtDef4 and also failed to induce hyperbranching of fungal hyphae. Importantly, the γ-core motif of MtDef4 alone was capable of inhibiting fungal growth, but that of MsDef1 was not. The analysis of synthetic γ-core variants of MtDef4 indicated that the cationic and hydrophobic amino acids were important for antifungal activity. Both MsDef1 and MtDef4 induced plasma membrane permeabilization; however, kinetic studies revealed that MtDef4 was more efficient in permeabilizing fungal plasma membrane than MsDef1. Furthermore, the in vitro antifungal activity of MsDef1, MsDef1-γ4, MtDef4 and peptides derived from the γ-core motif of each defensin was not solely dependent on their ability to permeabilize the fungal plasma membrane. The data reported here indicate that the γ-core motif defines the unique antifungal properties of each defensin and may facilitate de novo design of more potent antifungal peptides.     

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.     

家蝇防御素基因的cDNA克隆及序列分析

Defensin is a kind of cationic.inducible antimicrobial peptide found in a large range of living organisms that contributes to host defense by disrupting the cytoplasmic membrane of microorganisms.with their broad antimicrobial spectrum and strong pharmaceutical effects.antimicrobial peptides,including defensins,represent a source of novel antibiotic agents.A novel full-length 430 base pairs cDNA of an insect defensin was cloned using polymerase chain reaction (PCR) from the cDnA library of houseflies(Musca domestica) that had been challenged by E.coli and staphylococcus taincd an NH2-terminal signal sequence(1-22)followed by a propeptide and the mature peptide(53-92),The sequence identity with other insect defensin is between 51% and 73%.The mature peptide,with a predicted molecular weight of 4.0kDa,and pI of 8.69,has 1 negative charged amino acid and 4 positice ones,the putative housefly defensin is characterized by 6 invariant cysteine residues forming 3 disulfide bonds,Cys1-Cys4,Cys2-Cys5 and Cys3-Cys6,These results suggest that the novel full-length cDNA of the defensin gene.Denominated Mdde,has been successfully cloned from houseflies.     

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.     

Molecular diversity and evolution of myticin-C antimicrobial peptide variants in the Mediterranean mussel, Mytilus galloprovincialis

Mussels have diverse groups of cysteine rich, cationic antimicrobial peptides (AMPs) (defensins, mytilins, myticins, and mytimycin) that constitute an important component of their innate immune defence. Despite the identification and characterization of these AMPs in mussels, the underlying genetic mechanisms that maintain high diversity among multiple variants of the myticin-C isoform are poorly understood. Using phylogeny-based models of sequence evolution and several site-by-site frequency spectrum statistical tests for neutrality, herein we report that positive selection has been the major driving force in maintaining high diversity among the allelic-variants of the myticin-C AMP of Mytilus galloprovincialis. The statistical tests rejected the hypothesis that all polymorphism within myticin-C loci is neutral. Although a majority of the codons constrained to purifying selection (rate of amino acid replacement to the silent substitution, omega < 1), approximately 8% of the codons with omega approximately equal to 5.5 are under positive selection (omega > 1), thus indicating adaptive evolution of certain amino acids. Direct interaction of these peptides with the surrounding pathogens and/or altered/new pathogens in the changing environment is the likely cause of molecular adaptation of certain amino acid sites in myticin-C variants.     

Structure of Petunia hybrida defensin 1, a novel plant defensin with five disulfide bonds

The structure of a novel plant defensin isolated from the flowers of Petunia hybrida has been determined by (1)H NMR spectroscopy. P. hybrida defensin 1 (PhD1) is a basic, cysteine-rich, antifungal protein of 47 residues and is the first example of a new subclass of plant defensins with five disulfide bonds whose structure has been determined. PhD1 has the fold of the cysteine-stabilized alphabeta motif, consisting of an alpha-helix and a triple-stranded antiparallel beta-sheet, except that it contains a fifth disulfide bond from the first loop to the alpha-helix. The additional disulfide bond is accommodated in PhD1 without any alteration of its tertiary structure with respect to other plant defensins. Comparison of its structure with those of classic, four-disulfide defensins has allowed us to identify a previously unrecognized hydrogen bond network that is integral to structure stabilization in the family.     

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.     

三角帆蚌Hc-GSK3β基因鉴定及内壳色性状相关SNP筛选

为了探究三角帆蚌(Hyriopsis cumingii)糖原合成激酶-3β(GSK3β)基因对壳色的影响,研究采用RACE技术获得Hc-GSK3β基因cDNA全长1867 bp,其中包含1261 bp的ORF区编码420个氨基酸, ORF中含有一个S＿TKc结构域,该结构域序列高度保守。组织差异表达分析发现Hc-GSK3β基因在紫色蚌鳃、斧足、内脏团和边缘膜组织中表达量高于白色蚌的表达量(P<0.05),且在斧足和边缘膜表达差异水平达到极显著(P<0.01),而在紫色蚌闭壳肌组织中表达量显著低于白色蚌(P<0.05)。原位杂交(ISH)实验结果显示在三角帆蚌外套膜的外褶、中褶、內褶、背膜区和腹膜区均有阳性信号产生,且在外褶的信号表达较强烈。该基因经重测序比较,共鉴定出6个SNP位点,其中在C+185A位点的CA基因型在紫色蚌的分布频率显著高于白色三角帆蚌(P<0.05);在紫色蚌中, T+341G位点TT基因型三角帆蚌内壳颜色参数b值显著低于TG基因型(P<0.05)。研究表明, Hc-GSK3β基因参与了三角帆蚌壳色形成,筛选的SNP标记可用于三角帆蚌壳...     

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.     

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 β 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.     

Three-dimensional structure of RTD-1, a cyclic antimicrobial defensin from Rhesus macaque leukocytes

Most mammalian defensins are cationic peptides of 29-42 amino acids long, stabilized by three disulfide bonds. However, recently Tang et al. (1999, Science 286, 498-502) reported the isolation of a new defensin type found in the leukocytes of rhesus macaques. In contrast to all the other defensins found so far, rhesus theta defensin-1 (RTD-1) is composed of just 18 amino acids with the backbone cyclized through peptide bonds. Antibacterial activities of both the native cyclic peptide and a linear form were examined, showing that the cyclic form was 3-fold more active than the open chain analogue [Tang et al. (1999) Science 286, 498-502]. To elucidate the three-dimensional structure of RTD-1 and its open chain analogue, both peptides were synthesized using solid-phase peptide synthesis and tert-butyloxycarbonyl chemistry. The structures of both peptides in aqueous solution were determined from two-dimensional (1)H NMR data recorded at 500 and 750 MHz. Structural constraints consisting of interproton distances and dihedral angles were used as input for simulated-annealing calculations and water refinement with the program CNS. RTD-1 and its open chain analogue oRTD-1 adopt very similar structures in water. Both comprise an extended beta-hairpin structure with turns at one or both ends. The turns are well defined within themselves and seem to be flexible with respect to the extended regions of the molecules. Although the two strands of the beta-sheet are connected by three disulfide bonds, this region displays a degree of flexibility. The structural similarity of RTD-1 and its open chain analogue oRTD-1, as well as their comparable degree of flexibility, support the theory that the additional charges at the termini of the open chain analogue rather than overall differences in structure or flexibility are the cause for oRTD-1's lower antimicrobial activity. In contrast to numerous other antimicrobial peptides, RTD-1 does not display any amphiphilic character, even though surface models of RTD-1 exhibit a certain clustering of positive charges. Some amide protons of RTD-1 that should be solvent-exposed in monomeric beta-sheet structures show low-temperature coefficients, suggesting the possible presence of weak intermolecular hydrogen bonds.     

Structure-based protein engineering for alpha-amylase inhibitory activity of plant defensin

The structure of a novel plant defensin isolated from the seeds of the mung bean, Vigna radiate, has been determined by (1)H nuclear magnetic resonance spectroscopy. The three-dimensional structure of VrD2, the V. radiate plant defensin 2 protein, comprises an alpha-helix and one triple-stranded anti-parallel beta-sheet stabilized by four disulfide bonds. This protein exhibits neither insecticidal activity nor alpha-amylase inhibitory activity in spite of showing a similar global fold to that of VrD1, an insecticidal plant defensin that has been suggested to function by inhibiting insect alpha-amylase. Our previous study proposed that loop L3 of plant defensins is important for this inhibition. Structural analyses and surface charge comparisons of VrD1 and VrD2 revealed that the charged residues of L3 correlate with the observed difference in inhibitory activities of these proteins. A VrD2 chimera that was produced by transferring the proposed functional loop of VrD1 onto the structurally equivalent loop of VrD2 supported this hypothesis. The VrD2 chimera, which differs by only five residues compared with VrD2, showed obvious activity against Tenebrio molitor alpha-amylase. These results clarify the mode of alpha-amylase inhibition of plant defensins and also represent a possible approach for engineering novel alpha-amylase inhibitors. Plant defensins are important constituents of the innate immune system of plants, and thus the application of protein engineering to this protein family may provide an efficient method for protecting against crop losses.     

Ha-DEF1, a sunflower defensin,induces cell death in <Emphasis Type="Italic">Orobanche</Emphasis> parasitic plants

Plant defensins are small basic peptides of 5–10 kDa and most of them exhibit antifungal activity. In a sunflower resistant to broomrape, among the three defensin encoding cDNA identified, SF18, SD2 and HaDef1, only HaDef1 presented a preferential root expression pattern and was induced upon infection by the root parasitic plant Orobanche cumana. The amino acid sequence deduced from HaDef1 coding sequence was composed of an endoplasmic reticulum signal sequence of 28 amino acids, a standard defensin domain of 50 amino-acid residues and an unusual C-terminal domain of 30 amino acids with a net positive charge. A 5.8 kDa recombinant mature Ha-DEF1 corresponding to the defensin domain was produced in Escherichia coli and was purified by means of a two-step chromatography procedure, Immobilized Metal Affinity Chromatography (IMAC) and Ion Exchange Chromatography. Investigation of in vitro antifungal activity of Ha-DEF1 showed a strong inhibition on Saccharomyces cerevisiae growth linked to a membrane permeabilization, and a morphogenetic activity on Alternaria brassicicola germ tube development, as already reported for some other plant defensins. Bioassays also revealed that Ha-DEF1 rapidly induced browning symptoms at the radicle apex of Orobanche seedlings but not of another parasitic plant, Striga hermonthica, nor of Arabidopsis thaliana. FDA vital staining showed that these browning areas corresponded to dead cells. These results demonstrate for the first time a lethal effect of defensins on plant cells. The potent mode of action of defensin in Orobanche cell death and the possible involvement in sunflower resistance are discussed.     

Purification of a novel arthropod defensin from the American oyster, Crassostrea virginica

An antimicrobial peptide was purified from acidified gill extract of a bivalve mollusk, the American oyster (Crassostrea virginica), by preparative acid-urea--polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography. The 4265.0 Da peptide had 38 amino acids, including 6 cysteines. It showed strongest activity against Gram-positive bacteria (Lactococcus lactis subsp. lactis and Staphylococcus aureus; minimum effective concentrations [MECs] 2.4 and 3.0 microg/ml, respectively) but also had significant activity against Gram-negative bacteria (Escherichia coli D31 and Vibrio parahemolyticus; MECs 7.6 and 15.0 microg/ml, respectively). Comparison of the amino acid sequence with those of other known antimicrobial peptides revealed that the novel peptide had high sequence homology to arthropod defensins, including those from other bivalves, the mussels Mytilus edulis and Mytilus galloprovincialis. This is the first antimicrobial peptide to be isolated from any oyster species and we have named it American oyster defensin (AOD).     

Determination of the disulfide array in the human defensin HNP-2. A covalently cyclized peptide

HNP-2 is a 29-residue peptide present in human neutrophils and is a member of the defensin family of antimicrobial peptides. All defensins contain an invariant disulfide infrastructure comprised of 6 half-cystine residues. The disulfide structure of HNP-2 was determined using a novel method to identify the cross-links involving the amino- and carboxyl-terminal cysteine residues. A derivative of HNP-2 was synthesized by covalent modification of the terminal cysteine residues. This derivative was purified, characterized, and subjected to exhaustive proteolytic digestion. Characterization of purified proteolytic fragments by amino acid analysis and/or sequence analysis identified an oligopeptide containing all 6 cystine residues. This oligopeptide was subjected to a single cycle of Edman degradation to cleave the peptide bond linking 2 adjacent cysteines. Purification and characterization of the Edman reaction products allowed for assignment of the disulfide array in HNP-2, revealing a cystine motif unique to the defensin peptide family. Further, the covalent structure of HNP-2 was found to be cyclic as one disulfide links the amino- and carboxyl-terminal cysteine residues. HNP-2 is the only polypeptide known to possess such a configuration.