Molecular Characterisation and Phylogenetic Analysis of a Novel Isoform of Hepatic Antimicrobial Peptide, Hepcidin (Zc-hepc1), from the Coral Fish Moorish idol, Zanclus cornutus (Linnaeus, 1758)

Hepcidin is a family of short cysteine-rich antimicrobial peptides (AMPs) participating in various physiological functions with inevitable role in host immune responses. Present study deals with identification and characterisation of a novel hepcidin isoform from coral fish Zanclus cornutus. The 81 amino acid (aa) preprohepcidin obtained from Z. cornutus consists of a hydrophobic aa rich 22 mer signal peptide, a highly variable proregion of 35 aa and a bioactive mature peptide with 8 conserved cysteine residues which contribute to the disulphide back bone. The mature hepcidin, Zc-hepc1 has a theoretical isoelectric point of 7.46, a predicted molecular weight of 2.43 kDa and a net positive charge of +1. Phylogenetic analysis grouped Z. cornutus hepcidin with HAMP2 group hepcidins confirming the divergent evolution of hepcidin-like peptide in fishes. Zc-hepc1 can attain a β-hairpin-like structure with two antiparallel β-sheets. This is the first report of an AMP from the coral fish Z. cornutus.     

Amphibian cathelicidin fills the evolutionary gap of cathelicidin in vertebrate

Cathelicidins comprise a family of antimicrobial peptides (AMPs) sharing a highly conserved cathelin domain, and play a central role in the innate defense against infection in most of vertebrates. But so far it has not yet been found in amphibians although a large number of other groups of AMPs have been identified. In the current work, the first amphibian cathelicidin (cathelicidin-AL) has been characterized from the frog skin of Amolops loloensis. Cathelicidin-AL (RRSRRGRGGGRRGGSGGRGGRGGGGRSGAGSSIAGVGSRGGGGGRHYA) is a cationic peptide containing 48 amino acid residues (aa) with 12 basic aa and no acidic aa. The chemical synthesized peptide efficiently killed bacteria and some fungal species including clinically isolated drug-resistance microorganisms. The cDNA encoding cathelicidin-AL precursor was cloned from the skin cDNA library of A. loloensis. As other cathelicidins, the precursor of cathelicidin-AL also contains highly conserved anionic cathelin domain of cysteine proteinase inhibitor followed by the AMP fragment at C-terminus. Phylogenetic analysis revealed that as connecting link, the amphibian cathelicidin predates reptilia but postdates fish cathelicidin. The peptide purification combined with gene cloning results confirms the presence of cathelicidin in amphibians and filled the evolutionary gap of cathelicidin in vertebrate, considering amphibians' special niche as the animals bridging the evolutionary land-water gap.     

Cloning and characterization of a novel calcium channel toxin-like gene BmCa1 from Chinese scorpion Mesobuthus martensii Karsch

Many studies have been carried on peptides and genes encoding scorpion toxins from the venom of Mesobuthus martensii Karsch (synonym: Buthus martensii Karsch, BmK), such as Na+, K+ and Cl- channel modulators. In this study, a novel calcium channel toxin-like gene BmCa1 was isolated and characterized from the venom of Mesobuthus martensii Karsch. First, a partial cDNA sequence of the Ca2+ channel toxin-like gene was identified by random sequencing method from a venomous gland cDNA library of Mesobuthus martensii Karsch. The full-length sequence of BmCa1 was then obtained by 5'RACE technique. The peptide deduced from BmCa1 precursor nucleotide sequence contains a 27-residue signal peptide and a 37-residue mature peptide. Although BmCa1 and other scorpion toxins are different at the gene and protein primary structure levels, BmCa1 has the same precursor nucleotide organization and cysteine arrangement as that of the first subfamily members of calcium channel scorpion toxins. Genomic DNA sequence of BmCa1 was also cloned by PCR. Sequence analysis showed that BmCa1 gene consists of three exons separated by two introns of 72 bp and 1076 bp in length, respectively. BmCa1 is the first calcium channel toxin-like gene cloned from the venom of Mesobuthus martensii Karsch and potentially represents a novel class of calcium channel toxins in scorpion venoms.     

Identification and characterization of a novel murine beta-defensin-related gene

Beta-defensins comprise a family of cationic peptides, which are predominately expressed at epithelial surfaces and have a broad-range antimicrobial activity. We have assembled two BAC-based contigs from the chromosomal region 8A4 that contain the murine defensins, and we have mapped six reported beta-defensin genes. In addition, we have isolated and functionally characterized a novel beta-defensin gene that deviates from the canonical six cysteine motif present in the mature functional peptide of all other beta-defensins. This defensin-related gene (Defr1) is most highly expressed in testis and heart. The genomic organization is highly similar to Defb3, 4, 5, and 6, and the exon 1 sequence is very highly conserved. A synthetic Defr1 peptide displayed antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Burkholderia cepacia. The antimicrobial activity of Defr1 against S. aureus, E.coli, and B. cepacia was found to be reduced in raised concentration of NaCl, but its action against P. aeruginosa was independent of NaCl concentration. This is the first report of a functional beta defensin that lacks one of the conserved cysteine residues in its predicted mature peptide. This study has major implications for the structure and functions of these important host defense molecules.     

Characterization and Structural Analysis of Hepcidin Like Antimicrobial Peptide From Schizothorax richardsonii (Gray)

Innate immune system is a primary line of defense in fish that protects it from the invading pathogens. Antimicrobial peptides (AMPs) are widely distributed in nature and are essential components of innate immunity. These molecules enable the host’s innate immune system to fight against a variety of infectious agents. One such AMP, hepcidin, is a cysteine rich amphipathic peptide. We have amplified, cloned and characterized hepcidin like AMP from Schizothorax richardsonii that inhabits one of the most difficult aquatic ecosystems in the Indian Himalayas. The cDNA encoding hepcidin like peptide was amplified as a 371 bp fragment with an open reading frame (ORF) of 279 nucleotides flanked by 5′ and 3′ UTRs of 70 and 22 bases respectively. This ORF encodes a peptide of 93 amino acids with a signal peptide of 24 amino acids and a mature peptide of 25 amino acids. The mature hepcidin like peptide of S. richardsonii has eight cystine residues that participate in the formation of four disulfide bonds, a unique feature of hepcidin like AMPs. A 3D model of hepcidin like mature peptide was generated using Modeller 9.10 which was validated using PROCHECK and ERRAT. Phylogenetic analysis of hepcidin like AMP from S. richardsonii revealed that it was closely related to hepcidin from olive barb (Puntius sarana).     

Advances in antimicrobial peptide immunobiology

Antimicrobial peptides are ancient components of the innate immune system and have been isolated from organisms spanning the phylogenetic spectrum. Over an evolutionary time span, these peptides have retained potency, in the face of highly mutable target microorganisms. This fact suggests important coevolutionary influences in the host-pathogen relationship. Despite their diverse origins, the majority of antimicrobial peptides have common biophysical parameters that are likely essential for activity, including small size, cationicity, and amphipathicity. Although more than 900 different antimicrobial peptides have been characterized, most can be grouped as belonging to one of three structural classes: (1) linear, often of alpha-helical propensity; (2) cysteine stabilized, most commonly conforming to beta-sheet structure; and (3) those with one or more predominant amino acid residues, but variable in structure. Interestingly, these biophysical and structural features are retained in ribosomally as well as nonribosomally synthesized peptides. Therefore, it appears that a relatively limited set of physicochemical features is required for antimicrobial peptide efficacy against a broad spectrum of microbial pathogens.During the past several years, a number of themes have emerged within the field of antimicrobial peptide immunobiology. One developing area expands upon known microbicidal mechanisms of antimicrobial peptides to include targets beyond the plasma membrane. Examples include antimicrobial peptide activity involving structures such as extracellular polysaccharide and cell wall components, as well as the identification of an increasing number of intracellular targets. Additional areas of interest include an expanding recognition of antimicrobial peptide multifunctionality, and the identification of large antimicrobial proteins, and antimicrobial peptide or protein fragments derived thereof. The following discussion highlights such recent developments in antimicrobial peptide immunobiology, with an emphasis on the biophysical aspects of host-defense polypeptide action and mechanisms of microbial resistance.     

Selection for Antimicrobial Peptide Diversity in Frogs Leads to Gene Duplication and Low Allelic Variation

Antimicrobial peptides are highly diverse pathogen-killing molecules. In many taxa, their evolution is characterized by positive selection and frequent gene duplication. It has been proposed that genes encoding antimicrobial peptides might be subject to balancing selection and/or an enhanced mutation rate, but these hypotheses have not been well evaluated because allelic variation has rarely been studied at antimicrobial peptide loci. We present an evolutionary analysis of novel antimicrobial peptide genes from leopard frogs, Rana. Our results demonstrate that a single genome contains multiple homologous copies, among which there is an excess of nonsynonymous nucleotide site divergence relative to that expected from synonymous site divergence. Thus, we confirm the trends of recurrent duplication and positive selection. Allelic variation is quite low relative to interspecies divergence, indicating a recent positive selective sweep with no evidence of balancing selection. Repeated gene duplication, rather than a balanced maintenance of divergent allelic variants at individual loci, appears to be how frogs have responded to selection for a diverse suite of antimicrobial peptides. Our data also support a pattern of enhanced synonymous site substitution in the mature peptide region of the gene, but we cannot conclude that this is due to an elevated mutation rate.     

Molecular characterization of two novel antibacterial peptides inducible upon bacterial challenge in an annelid, the leech Theromyzon tessulatum

Two novel antimicrobial peptides named theromacin and theromyzin were isolated and characterized from the coelomic liquid of the leech Theromyzon tessulatum. Theromacin is a 75-amino acid cationic peptide containing 10 cysteine residues arranged in a disulfide array showing no similarities with other known antimicrobial peptides. Theromyzin is an 86-amino acid linear peptide and constitutes the first anionic antimicrobial peptide observed in invertebrates. Both peptides exhibit activity directed against Gram-positive bacteria. Theromacin and theromyzin cDNAs code precursor molecules containing a putative signal sequence directly followed by the mature peptide. The enhancement of theromacin and theromyzin mRNA levels has been observed after blood meal ingestion and upon bacterial challenge. In situ hybridization revealed that both genes are expressed in large fat cells in contact with coelomic cavities. Gene products were immunodetected in large fat cells, in intestinal epithelia, and at the epidermis level. In addition, a rapid release of the peptides into the coelomic liquid was observed after bacterial challenge. The presence of antimicrobial peptide genes in leeches and their expression in a specific tissue functionally resembling the insect fat body provide evidence for the first time of an antibacterial response in a lophotrochozoan comparable to that of holometabola insects.     

Overexpression of Jatropha curcas Defensin (JcDef) Enhances Sheath Blight Disease Resistance in Tobacco

Plant defensins are small, basic, cysteine‐rich peptides, belonging to the antimicrobial peptide superfamily, commonly found in the plant kingdom. In this study, we cloned and characterized a plant defensin gene from Jatropha curcas (JcDef). JcDef carried conserved receptor binding sites and a cysteine motif, and it was phylogenetically grouped together with defensin Ec‐AMP‐D2‐like in Elaeis guineensis. JcDef is localized to cytoplasm and highly expressed in young tissues with fast metabolism such as cotyledons and stem apexes. Transgenic expression of JcDef in tobacco showed enhanced resistance against sheath blight disease caused by R. solani, indicating the antibacterial function.     

Mytilus galloprovincialis myticin C: a chemotactic molecule with antiviral activity and immunoregulatory properties

Previous research has shown that an antimicrobial peptide (AMP) of the myticin class C (Myt C) is the most abundantly expressed gene in cDNA and suppressive subtractive hybridization (SSH) libraries after immune stimulation of mussel Mytilus galloprovincialis. However, to date, the expression pattern, the antimicrobial activities and the immunomodulatory properties of the Myt C peptide have not been determined. In contrast, it is known that Myt C mRNA presents an unusual and high level of polymorphism of unidentified biological significance. Therefore, to provide a better understanding of the features of this interesting molecule, we have investigated its function using four different cloned and expressed variants of Myt C cDNA and polyclonal anti-Myt C sera. The in vivo results suggest that this AMP, mainly present in hemocytes, could be acting as an immune system modulator molecule because its overexpression was able to alter the expression of mussel immune-related genes (as the antimicrobial peptides Myticin B and Mytilin B, the C1q domain-containing protein MgC1q, and lysozyme). Moreover, the in vitro results indicate that Myt C peptides have antimicrobial and chemotactic properties. Their recombinant expression in a fish cell line conferred protection against two different fish viruses (enveloped and non-enveloped). Cell extracts from Myt C expressing fish cells were also able to attract hemocytes. All together, these results suggest that Myt C should be considered not only as an AMP but also as the first chemokine/cytokine-like molecule identified in bivalves and one of the few examples in all of the invertebrates.     

Genomic and Structural Characterization of Kunitz-Type Peptide LmKTT-1a Highlights Diversity and Evolution of Scorpion Potassium Channel Toxins

Background

Recently, a new subfamily of long-chain toxins with a Kunitz-type fold was found in scorpion venom glands. Functionally, these toxins inhibit protease activity and block potassium channels. However, the genomic organization and three-dimensional (3-D) structure of this kind of scorpion toxin has not been reported.

Principal Findings

Here, we characterized the genomic organization and 3-D nuclear magnetic resonance structure of the scorpion Kunitz-type toxin, LmKTT-1a, which has a unique cysteine pattern. The LmKTT-1a gene contained three exons, which were interrupted by two introns located in the mature peptide region. Despite little similarity to other Kunitz-type toxins and a unique pattern of disulfide bridges, LmKTT-1a possessed a conserved Kunitz-type structural fold with one α-helix and two β-sheets. Comparison of the genomic organization, 3-D structure, and functional data of known toxins from the α-KTx, β-KTx, γ-KTx, and κ-KTx subfamily suggested that scorpion Kunitz-type potassium channel toxins might have evolved from a new ancestor that is completely different from the common ancestor of scorpion toxins with a CSα/β fold. Thus, these analyses provide evidence of a new scorpion potassium channel toxin subfamily, which we have named δ-KTx.

Conclusions/Significance

Our results highlight the genomic, structural, and evolutionary diversity of scorpion potassium channel toxins. These findings may accelerate the design and development of diagnostic and therapeutic peptide agents for human potassium channelopathies.     

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.     

Structural and functional characterization of hBD-1(Ser35), a peptide deduced from a DEFB1 polymorphism

beta-Defensins are mammalian antimicrobial peptides that share a unique disulfide-bonding motif of six conserved cysteines. An intragenic polymorphism of the DEFB1 gene that changes a highly conserved Cys to Ser in the peptide coding region has recently been described. The deduced peptide cannot form three disulfide bonds, as one of the cysteines is unpaired. We have determined the cysteine connectivities of a corresponding synthetic hBD-1(Ser35) peptide, investigated the structure by circular dichroism spectroscopy, and assayed the in vitro antimicrobial activity. Despite a different arrangement of the disulfides, hBD-1(Ser35) proved as active as hBD-1 against the microorganisms tested. This activity likely depends on the ability of hBD-1(Ser35) to adopt an amphipathic conformation in hydrophobic environment, similar to the wild type peptide, as suggested by CD spectroscopy.     

Sonorensin: an Antimicrobial Peptide,Belonging to the Heterocycloanthracin Subfamily of Bacteriocins,from a New Marine Isolate,Bacillus sonorensis MT93

Marine environments are the greatest fronts of biodiversity, representing a resource of unexploited or unknown microorganisms and new substances having potential applications. Among microbial products, antimicrobial peptides (AMPs) have received great attention recently due to their applications as food preservatives and therapeutic agents. A new marine soil isolate producing an AMP was identified as Bacillus sonorensis based on 16S rRNA gene sequence analysis. It produced an AMP that showed a broad spectrum of activity against both Gram-positive and Gram-negative bacteria. The peptide, named sonorensin, was purified to homogeneity using a combination of chromatographic techniques. The intact molecular mass of the purified peptide, 6,274 Da, as revealed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF), was in agreement with Tricine-SDS-PAGE analysis. A PCR array of primers was used to identify AMP structural genes, which allowed the successful amplification of the related genes from strain MT93. The putative open reading frame of sonorensin was amplified, cloned into the pET-32a(+) vector, expressed as a thioredoxin (Trx) fusion protein in Escherichia coli, and then purified. Sequence alignment analysis revealed that the bacteriocin being reported could belong to new subfamily of bacteriocins, heterocycloanthracin. The peptide indicated its potential as a biocontrol agent or food antimicrobial agent, due to its antimicrobial activity against bacteria such as Listeria monocytogenes and Staphylococcus aureus. This is the first report of the production, purification, and characterization of wild-type and recombinant bacteriocin by B. sonorensis and the first bacteriocin of the heterocycloanthracin subfamily to be characterized.     

Mammalian antimicrobial peptide protegrin‐4 self assembles and forms amyloid‐like aggregates: Assessment of its functional relevance

Protegrin‐4 (PG‐4) is a member of the porcine leukocyte protegrins family of cysteine‐rich antimicrobial peptides (AMPs) isolated from Sus scrofa. It consists of 18 amino acid residues and works as a part of innate immune system. In this study, we examined the intrinsic aggregation propensity of this AMP using multiple computational algorithms, namely, TANGO, AGGRESCAN, FOLDAMYLOID, AMYLPRED, and ZYGGREGATOR, and found that the peptide is predicted to have a high propensity for the β sheet formation that disposes this peptide to be amyloidogenic. Under in vitro conditions, PG‐4 formed visible aggregates and displayed the hallmark properties of typical amyloids such as enhanced binding of Congo red, increased fluorescence with Thioflavin‐T, and fibrillar morphology under transmission electron microscopy. Then we examined its antimicrobial activity against Bacillus subtilis and found that the aggregated peptide retained its antimicrobial activity. Additionally, the aggregates remain non‐toxic to the HEK293 and Caco2 cells. Our study suggests that the inherent aggregation properties of AMP can rationally be explored as a potential source of peptide‐based antimicrobials with enhanced stability.     

Solution structure of LCI, a novel antimicrobial peptide from Bacillus subtilis

LCI, a 47-residue cationic antimicrobial peptide (AMP) found in Bacillus subtilis, is one of the main effective components that have strong antimicrobial activity against Xanthomonas campestris pv Oryzea and Pseudomonas solanacearum PE1, etc. To provide insight into the activity of the peptide, we used nuclear magnetic resonance spectroscopy to determine the structure of recombinant LCI. The solution structure of LCI has a novel topology, containing a four-strand antiparallel β-sheet as the dominant secondary structure. It is the first structure of the LCI protein family. Different from any known β-structure AMPs, LCI contains no disulfide bridge or circular structure, suggesting that LCI is also a novel β-structure AMP.     

Novel alternatives to antibiotics: bacteriophages, bacterial cell wall hydrolases, and antimicrobial peptides

Extensive research has been conducted on the development of three groups of naturally occurring antimicrobials as novel alternatives to antibiotics: bacteriophages (phages), bacterial cell wall hydrolases (BCWH), and antimicrobial peptides (AMP). Phage therapies are highly efficient, highly specific, and relatively cost-effective. However, precautions have to be taken in the selection of phage candidates for therapeutic applications as some phages may encode toxins and others may, when integrated into host bacterial genome and converted to prophages in a lysogenic cycle, lead to bacterial immunity and altered virulence. BCWH are divided into three groups: lysozymes, autolysins, and virolysins. Among them, virolysins are the most promising candidates as they are highly specific and have the capability to rapidly lyse antibiotic-resistant bacteria on a generally species-specific basis. Finally, AMP are a family of natural proteins produced by eukaryotic and prokaryotic organisms or encoded by phages. AMP are of vast diversity in term of size, structure, mode of action, and specificity and have a high potential for clinical therapeutic applications.     

Detection of the antimicrobial peptide gene in different Amaranthus species

Using primers to amplify the gene AMP2 in Amaranthus caudatus, we found the gene to be present in seven other species of the Amaranthus genus (A. albus, A. cruentus, A. blitum, A. hybridus, A. hypochondriacus, A. retroflexus and A. tricolor), in which it had not been described previously. The PCR products were sequenced and it was established that all the sequences were identical, except for two polymorphisms. These single nucleotide polymorphisms occurred at nucleotide positions 45 and 246. This exchange of one nucleotide for another was manifested in an amino acid change in both cases. Due to the fact that both polymorphisms lay outside the region encoding the chitin-binding peptide domain, which is crucial for antimicrobial peptide function, they will not likely affect the proper functioning of the peptide. With the exception of the above-mentioned polymorphisms, all sequences were identical to the sequence of the AMP2 gene that codes for the A. caudatus Ac-AMP2 (antimicrobial peptide isolated from Amaranthus caudatus seeds). The detection of sequences with high degree of sequence similarity to A. caudatus AMP2 gene leads us to the assumption that an antimicrobial peptide could also be produced by other amaranth species.