Molecular Characterization of a Newly Identified Subfamily Member of Penaeidin from two Penaeid Shrimps, <Emphasis Type="Italic">Fenneropenaeus indicus</Emphasis> and <Emphasis Type="Italic">Metapenaeus monoceros</Emphasis>

Penaeidins are a major group of antimicrobial peptides found in penaeid shrimps. This study reports a new isoform of penaeidin from the hemocytes of Indian white shrimp, Fenneropenaeus indicus (Fi-PEN, JX657680), and the pink shrimp, Metapenaeus monoceros (Mm-PEN, KF275674). Mm-PEN is also the first antimicrobial peptide to be identified from M. monoceros. The complete coding sequences of the newly identified Fi-PEN and Mm-PEN consisted of an ORF of 338 bp encoding 71 amino acids with a predicted molecular weight of 5.66 kDa and a pI of 9.38. The penaeidins had its characteristic signal peptide region (19 amino acids), which was followed by a mature peptide with a proline-rich domain (24 amino acids) at the N-terminal region and a cysteine-rich domain (28 amino acids) at the C-terminal region, designating it to penaeidin-3 subgroup. Structural analysis revealed an alpha-helix in its secondary structure and an extended structure at the proline-rich domain. The newly identified penaeidin isoform showed maximum similarity of 63 % to a penaeidin-3 isoform of P. monodon, which further proves it to be a new isoform. Phylogenetic analysis showed that it possessed similar evolutionary status like other penaeidins, which has subsequently diverged at different phases of evolution. The wide distribution of penaeidins in penaeid shrimps indicates the importance of these AMPs in the innate immunity.     

Processing in transgenic Arabidopsis thaliana plants of polyproteins with linker peptide variants derived from the Impatiens balsamina antimicrobial polyprotein precursor

We have previously developed a method for expression in Arabidopsis thaliana L. of transgenes encoding cleavable chimaeric polyprotein precursors. The polyprotein precursors consisted of a leader peptide and two different antimicrobial proteins (AMPs), DmAMP1 originating from Dahlia merckii seeds and a variant form of RsAFP2 originating from Raphanus sativus seeds, which were linked by an intervening linker peptide sequence originating from a natural polyprotein occurring in seeds of Impatiens balsamina. By altering the amino acid sequence of the linker peptide separating the two AMPs, we now show that it is possible to improve the accuracy of polyprotein precursor cleavage, leading to the release of both the AMPs with either no or a few additional amino acids derived from the linker peptide. Furthermore, subcellular localization indicated that both the AMPs are predominantly present in the extracellular fluid of the transgenic plants.     

Two novel families of antimicrobial peptides from skin secretions of the Chinese torrent frog, Amolops jingdongensis

The characterization of new natural antimicrobial peptides (AMPs) can help to solve the serious problem of bacterial resistance to currently used antibiotics. In the current study, we analyzed two families of AMPs from the Chinese torrent frog Amolops jingdongensis with a range of bioactivities. The first family of peptides, named jindongenin-1a, is 24 amino acids in length; a BLAST search of jindongenin-1a revealed no sequence similarity with other AMPs. The second family consists of two peptides containing 29 amino acid residues each. These peptides have high sequence similarity with the AMPs of palustrin-2 and are therefore designated palustrin-2AJ1 and palustrin-2AJ2. The cDNA sequences encoding these AMPs have been cloned and the deduced protein sequence of each AMP has been determined by protein sequencing. Sequence and structural analysis showed that each precursor is composed of a putative signal peptide, an N-terminal spacer, a processing site and a disulfide-bridged heptapeptide segment at the C-terminus. We synthesized jindongenin-1a and palustrin-AJ1 to test their antimicrobial, hemolytic, antioxidative and cytotoxic activities. These two peptides showed broad-spectrum antimicrobial activity to standard and clinically isolated strains of bacteria. In addition, they exhibited weak hemolytic activity to human and rabbit erythrocytes under our experimental conditions. Moreover, these peptides also displayed cytotoxic activity against the K562 and HT29 mammalian cell lines and low anti-oxidant activity. These findings provide helpful insight that will be useful in the design of anti-infective peptide agents.     

Cloning of a cluster of chitinase genes from Aeromonas sp. No. 10S-24

A gene encoding chitinases from Aeromonas sp. No. 10S-24 was cloned into Escherichia coli DH5α using pUC19, and its nucleotides were sequenced. The chitinase gene was clustered in ORFs (open reading frame) 1 to 4, in a 8-kb fragment of DNA. ORF-1 consisted of 1608 bp encoding 535 amino acid residues, and ORF-2 consisted of 1425 bp encoding 474 amino acid residues. ORF-3 was 1617 bp long and encodes a protein consisting of 538 amino acids. ORF-4 encodes 287 amino acids of the N-terminal region. The amino acid sequences of ORF-1 and ORF-3 share sequence homology with chitinase D from Bacillus circulans, and chitinase A and B from Streptomyces lividans. The amino acid sequence of ORF-2 shared sequence homology with chitinase II from Aeromonas sp. No. 10S-24, and chitinase from Saccharopolyspora erythraea. A region of the sequence starting from Ala-28 of the amino acid sequence of ORF-3 coincided with the N-terminal amino acid sequence of chitinase III from Aeromonas sp. No. 10S-24.     

Molecular cloning and sequence analysis of a mannitol dehydrogenase gene and isolation of mdh promoter from Candida magnoliae

The mdh gene encodes mannitol dehydrogenase (MDH), which catalyzes the conversion of fructose into mannitol. The putative mdh gene of Candida magnoliae was isolated by PCR using the primers deduced from the N-terminal amino acid sequences of an intact MDH and its tryptic peptides, cloned in E. coli, and sequenced. The mdh gene consisted of 852 bp encoding for 283 amino acids. Analysis of the amino acid sequence revealed that MDH consisted of typical NADPH-dependent short chain dehydrogenases/reductases (SDRs). To develop a strong promoter to induce expression of the foreign genes in C. magnolia, the putative promoter was isolated. The reporter protein, GFP, was well-expressed under the control of the putative mdh promoter of 153 bp in C. magnoliae.     

Identification and molecular characterization of a C-type lectin-like protein from Chinese shrimp (Fenneropenaeus chinensis)

A C-type lectin-like protein was cloned and characterized from the Chinese shrimp Fenneropenaeus chinensis, and named as FcCTL. The results indicated that the full length cDNA of 859 bp had an open reading frame encoded a polypeptide of 220 amino acids with one carbohydrate-recognition domain, six conserved Cys and one key motif EPGD. The theoretical molecular weight and pI of mature protein was 25.3 kDa and 5.4. Sequence comparison of the deduced amino acid sequence of FcCTL showed varied identity of 26–34, 34, 31 and 30 % with those of F. chinensis, Portunus trituberculatus, Tetraodon nigroviridis, Penaeus monodon, respectively. qRT-PCR analysis indicated that FcCTL was expressed highest in hepatopancreas of normal shrimp, and it’s expression was up-regulated in hepatopancreas and gills post white spot syndrome virus challenge. The purified recombinant FcCTL showed higher antimicrobial activity against Gram-positive bacteria than against Gram-negative bacteria and fungi. And the hemagglutinating activity of rFcCTL could be completely inhibited by GlcNAc (5 μg/ml), LPS (2.5 μg/ml), d-galactose (100 mM) and maltose (100 mM). These data suggested that FcCTL might play an important role in shrimp immune and would be helpful to better understand the innate immunity mechanism of shrimp.     

Preliminary study on a potential antibacterial peptide derived from histone H2A in hemocytes of scallop Chlamys farreri

Histone H2A is reported to participate in host defense response through producing novel antimicrobial peptides (AMPs) from its N-terminus in vertebrates and invertebrates, while the AMPs derived from H2A have not to our knowledge been reported in mollusca. In the present study, gene cloning, mRNA expression of H2A from scallop Chlamys farreri, and the recombinant expression of its N-terminus were conducted to investigate whether a similar mechanism exists in mollusca. The full-length DNA of H2A was identified by the techniques of homology cloning and genomic DNA walking. The full-length DNA of the scallop H2A was 696bp long, including a 5'-terminal untranslated region (UTR) of 90bp, a 3'-terminal UTR of 228bp with a stem-loop structure and a canonical polyadenylation signal sequence AATAAA, and an open reading frame of 375bp encoding a polypeptide of 125 amino acids. The mRNA expression of H2A in the hemocytes of scallop challenged by microbe was measured by semi-quantitative RT-PCR. The expression of H2A was not upregulated after stimulation, suggesting that H2A did not participate in immunity response directly. The DNA fragment of 117bp encoding 39 amino acids corresponding to the N-terminus of scallop H2A, which was homologous to buforin I in vertebrates, was cloned into Pichia pastoris GS115. The transformants (His(+) Mut(+)) containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418. The peptide of 39 amino acids was expressed by induction of 0.5% methanol. The recombinant product exerted antibacterial activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria. The antibacterial activity toward G(+) bacteria was 2.5 times more than that against G(-) bacteria. The results elucidated that N-terminus of H2A was a potential AMP and provided a promising candidate for a new antibiotic screening. However, whether H2A is really involved in scallop immune response mechanisms needs to be further investigated.     

Molecular cloning and characterization of three crustins from the Chinese white shrimp,Fenneropenaeus chinensis

Antibacterial peptides crustins are the effector molecules of innate immunity in decapods. In this study, three crustin cDNA sequences (Fc-crus 1, Fc-crus 2, and Fc-crus 3) were cloned from the Chinese white shrimp Fenneropenaeus chinensis. The full-length cDNAs of Fc-crus 2 and 3 are 473 bp and 574 bp, respectively. The deduced peptides of Fc-crus 2 and 3 contain a signal peptide and a crustin domain at the C-terminal formed by twelve conserved cysteine residues. The partial sequence of Fc-cru 1 is 575 bp long and the deduced amino acids also contain a crustin domain. The expression profiles of these three crustins were studied with RT-PCR. Fc-crus 1 and Fc-crus 2 constitutively expressed in hemocytes with high levels, and the expression level is increased in the heart, stomach, intestine and ovaries when shrimp was challenged with Vibrio anguillarum, The expression of Fc-crus 1 and Fc-crus 2 was detected in each developmental stage. Fc-crus 3 was constitutively expressed in the ovaries and induced as an expression in the stomach. Unlike Fc-crus 1 and Fc-crus 2, the mRNA of Fc-crus 3 was not detected in the developmental stages extending from nauplii and mysis to post-larvae. The recombinant proteins containing mature Fc-crus 2 and Fc-crus 3 were recombinantly expressed in Escherichia coli and respectively purified. The antibacterial assays revealed that the recombinant mFc-crus could inhibit the growth of Gram-positive bacteria in vitro.     

EcDBS1R6: A novel cationic antimicrobial peptide derived from a signal peptide sequence

BackgroundBacterial infections represent a major worldwide health problem the antimicrobial peptides (AMPs) have been considered as potential alternative agents for treating these infections. Here we demonstrated the antimicrobial activity of EcDBS1R6, a peptide derived from a signal peptide sequence of Escherichia coli that we previously turned into an AMP by making changes through the Joker algorithm.MethodsAntimicrobial activity was measured by broth microdilution method. Membrane integrity was measured using fluorescent probes and through scanning electron microscopy imaging. A sliding window of truncated peptides was used to determine the EcDBS1R6 active core. Molecular dynamics in TFE/water environment was used to assess the EcDBS1R6 structure.ResultsSignal peptides are known to naturally interact with membranes; however, the modifications introduced by Joker transformed this peptide into a membrane-active agent capable of killing bacteria. The C-terminus was unable to fold into an α-helix whereas its fragments showed poor or no antimicrobial activity, suggesting that the EcDBS1R6 antibacterial core was located at the helical N-terminus, corresponding to the signal peptide portion of the parent peptide.ConclusionThe strategy of transforming signal peptides into AMPs appears to be promising and could be used to produce novel antimicrobial agents.General significanceThe process of transforming an inactive signal peptide into an antimicrobial peptide could open a new venue for creating new AMPs derived from signal peptides.     

Cloning and characterization of calreticulin and its association with salinity stress in P. trituberculatus

Calreticulin (CRT) is a highly conserved and multifunctional endoplasmic reticulum (ER) chaperone protein and plays important roles in salinity stress response. Portunus trituberculatus is a commercially important fishery species, and water salinity conditions influence its commercial farming significantly. In order to research the function of calreticulin under salinity stress, the full-length cDNA sequence of calreticulin from P. trituberculatus (PtCRT) was firstly cloned and characterized. The complete cDNA sequence of PtCRT is 1676 bp with 1218 bp open reading frame (ORF), encoding a polypeptide of 405 amino acids. Multiple sequence alignments showed that the deduced acid amino sequences of PtCRT shared the highest homology to CRT of Fenneropenaeus chinensis (89 %). Fluorescent quantitative real-time PCR analysis indicated that PtCRT was expressed in all detected tissues and showed the highest expression level in hepatopancreas. In addition, salinity challenge significantly influenced the expression level of PtCRT in gill. Six single nucleotide polymorphisms (SNPs) were detected in cDNA sequence of PtCRT, and one SNP was associated with the salt tolerant trait. All results indicated that PtCRT plays an important role in mediating the salinity adaption of P. trituberculatus.     

Enhancement of the antimicrobial activity and selectivity of GNU7 against Gram-negative bacteria by fusion with LPS-targeting peptide

Antimicrobial peptides (AMPs) provide a potential source of new antimicrobial therapeutics for the treatment of multidrug-resistant pathogens. To develop Gram-negative selective AMPs that can inhibit the effects of lipopolysaccharide (LPS)-induced sepsis, we added various rationally designed LPS-targeting peptides [amino acids 28–34 of lactoferrin (Lf28–34), amino acids 84–99 of bactericidal/permeability increasing protein (BPI84–99), and de novo peptide (Syn)] to the potent AMP, GNU7 (RLLRPLLQLLKQKLR). Compared to our original starting peptide GNU7, hybrid peptides had an 8- to 32-fold improvement in antimicrobial activity against Gram-negative bacteria, such as Escherichia coli and Salmonella typhimurium. Among them, Syn-GNU7 showed the strongest LPS-binding and -neutralizing activities, thus allowing it to selectively eliminate Gram-negative bacteria from within mixed cultures. Our results suggest that LPS-targeting peptides would be useful to increase the antimicrobial activity and selectivity of other AMPs against Gram-negative bacteria.     

Functional Characterization of a c-type Lysozyme from Indian Shrimp Fenneropenaeus indicus

Lysozyme gene from Fenneropenaeus indicus was cloned, expressed in Escherichia coli and characterized. The cDNA consists of 477 base pairs and encodes amino acid sequence of 159 residues. F. indicus lysozyme had high identity (98 %) with Fenneropenaeus merguiensis and Fenneropenaeus chinensis and exhibits low to moderate identities with lysozymes of other invertebrates and vertebrates. This lysozyme is presumed to be chicken types as it possesses two catalytic and eight cysteine residues that are conserved across c-type lysozymes and a c-terminal extension, which is a characteristic of lysozymes from marine invertebrates. Further, the antimicrobial properties of the recombinant lysozyme from F. indicus were determined in comparison with recombinant hen egg white lysozyme. This exhibited high activity against a Gram-negative pathogenic bacterium Salmonella typhimurium and two fungal strains Pichia pastoris and Saccharomyces cerevisiae in turbidimetric assay. Distribution of lysozyme gene and protein in tissues of shrimps infected with white spot syndrome virus revealed that the high levels of lysozyme are correlated with low and high viral load in abdominal muscle and tail, respectively. In conclusion, lysozyme from F. indicus has a broad spectrum of antimicrobial properties, which once again emphasizes its role in shrimp innate immune response.     

Stepwise identification of potent antimicrobial peptides from human genome

The increasing incidence of hospital acquired infections caused by antibiotic resistant pathogens has led to an increase in morbidity and mortality, finding alternative antibiotics unaffected by resistance mechanisms is fundamentally important for treating this problem. Naturally occurring proteins usually carry short peptide fragments that exhibit noticeable biological activity against a wide variety of microorganisms such as bacteria, fungi and protozoa. Traditional discovery of such antimicrobially active fragments (i.e. antimicrobial peptides, AMPs) from protein repertoire is either random or led by chance. Here, we report the use of a rational protocol that combines in silico prediction and in vitro assay to identify potential AMPs with high activity and low toxicity from the entire human genome. In the procedure, a three-step inference strategy is first proposed to perform genome-wide analysis to infer AMPs in a high-throughput manner. By employing this strategy we are able to screen more than one million peptide candidates generated from various human proteins, from which we identify four highly promising samples, and subsequently their antibacterial activity on five strains as well as cytotoxicity on human myoblasts are tested experimentally. As a consequence, two high-activity, low-toxicity peptides are discovered, which could be used as the structural basis to further develop new antibiotics. In addition, from 1491 known AMPs we also derive a quantitative measure called antibacterial propensity index (API) for 20 naturally occurring amino acids, which shows a significant allometric correlation with the theoretical minimal inhibitory concentration of putative peptides against Gram-positive and Gram-negative bacteria. This study may provide a proof-of-concept paradigm for the genome-wide discovery of novel antimicrobial peptides by using a combination of in silico and in vitro analyses.     

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).     

Cloning and characterization of hemerythrin gene from Sipuncula Phascolosoma esculenta

Hemerythrin is a non-heme respiratory protein involved in oxygen storage and transport in invertebrates. In the present study, the hemerythrin cDNA was cloned from Phascolosoma esculenta (denoted as PeHr) by PCR and rapid amplification of cDNA ends approaches. The full-length PeHr consisted of 770 bp containing of a 5′-terminal untranslated region (UTR) of 83 bp, a 3′-terminal UTR of 327 bp, and a coding domain sequence of 360 bp encoding a polypeptide of 120 amino acids with estimated molecular mass of 13.6 kDa and theoretical isoelectric point of 5.78. The expression profiles of PeHr were evaluated by real-time RT-PCR under blood loss stress. The expression level of PeHr was significantly up-regulated from 45 to 48 h, then slightly recovered to its original level. The coding sequence of the PeHr was cloned and expressed in Escherichia coli BL21 (DE3) for antibodies preparation. Western blotting analysis conformed that the generated antibodies could specifically identify not only recombinant product, but also native protein from the total protein extraction. Our results indicated that PeHr might be involved into haemocytes regeneration, and its function roles should be further investigated by the generated antibodies.