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.     

Screening Antimicrobial Activities of Basic Protein Fractions from Dry and Germinated Wheat Seeds

Two small cationic peptide fractions (5 kDa) were isolated from dry and germinated seeds of wheat, named WAP and GWAP, respectively. The antifungal and antibacterial activities of the peptides were analyzed using disk diffusion and turbidity measurement assays. The peptides in vitro exhibited effective antifungal activity against four plant pathogenic fungi at minimum concentration of 15 g(protein) cm^–3. Their antimicrobial activity was negatively affected by the presence of 5 mM CaCl₂. The peptides were less effective against Gram-negative bacterium Erwinia carotovora subsp. carotovora, but they demonstrated inhibitory activity against Gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus. The antimicrobial activity of GWAP was more effective than WAP.     

Antimicrobial activity of omwaprin, a new member of the waprin family of snake venom proteins

We have isolated and characterized omwaprin, a 50-amino-acid cationic protein from the venom of inland taipan (Oxyuranus microlepidotus). It is a new member of the waprin family of snake venom proteins. A synthetic gene was designed and constructed for expressing the recombinant protein in Escherichia coli. Recombinant omwaprin was used for carrying out functional analyses. The protein is non-toxic to Swiss albino mice at doses of up to 10 mg/kg when administered intraperitoneally. However, it shows selective and dose-dependant antibacterial activity against Gram-positive bacteria. The minimum inhibitory doses were in the range 2-10 microg for selected species of bacteria in radial diffusion assays. The antibacterial activity is salt-tolerant up to 350 mM NaCl. However, omwaprin lost its antibacterial activity upon reduction and alkylation of its cysteine residues, or upon deletion of six N-terminal amino acid residues, four of which are positively charged. These observations indicate that the three-dimensional structure constrained by four disulfide bonds and the N-terminal residues are essential for its activity. The mechanism of action is via membrane disruption, as shown by scanning electron microscopy. Importantly, omwaprin lacks haemolytic activity on human erythrocytes. This demonstrates the specificity of omwaprin for bacterial membranes. Unlike other reported WAP (whey acidic protein) domain-containing antibacterial proteins, including elafin, EPPIN (epididymal proteinase inhibitor), SWAM1 and SWAM2 [single WAP (whey acidic protein) motif proteins 1 and 2] and SLPI (secretory leucocyte proteinase inhibitor), omwaprin shows species-specific activity on the Gram-positive bacteria tested.     

Molecular cloning and characterization of crustin from mud crab<Emphasis Type="Italic"> Scylla paramamosain</Emphasis>

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. In the present study, we report the identification and characterization of a crustin (CrusSp) from the hemocyte of mud crab, Scylla paramamosain using an expressed sequence tag (EST) and rapid amplification cDNA end (RACE) approaches. Analysis of the nucleotide sequence revealed seven different variances of the CrusSp cDNA in mud crab. The open reading frame encodes a protein of 111 amino acids with 21 residues signal sequence. The predicted molecular mass of the mature protein (90 amino acids) is 10.27 kDa with an estimated pI of 8.54. Analysis of the protein domain features indicated typical conserved cysteine residues containing a single whey acidic protein (WAP) domain at the C-terminus. A neighbour-joining tree showed that S. paramamosain crustin is closely related to other crustin homologues, and displays the highest similarity to crustin antimicrobial peptide in shore crab Carcinus maenas. Four exons and three introns were identified within the 999 bp genomic DNA sequence of CrusSp. Tissue distribution analysis showed that CrusSp was highly expressed in hemocytes, gills, intestines and muscle but it was not expressed in hepatopancreas and eyestalks. To gain insight into the in vitro antimicrobial activities of CrusSp, the mature peptide coding region was cloned into E. coli for heterologous expression. The recombinant CrusSp could inhibit the growth of gram-positive bacteria but had no inhibition activity against gram-negative bacteria. These results indicated the involvement of CrusSp in the innate immunity of S. paramamosain.     

The introduction of l-phenylalanine into antimicrobial peptide protonectin enhances the selective antibacterial activity of its derivative phe-Prt against Gram-positive bacteria

Protonectin was a typical amphiphilic antimicrobial peptide with potent antimicrobial activity against Gram-positive and Gram-negative bacteria. In the present study, when its eleventh amino acid in the sequence was substituted by phenylalanine, the analog named phe-Prt showed potent antimicrobial activity against Gram-positive bacteria, but no antimicrobial activity against Gram-negative bacteria, indicating a significant selectivity between Gram-positive bacteria and Gram-negative bacteria. However, when Gram-negative bacteria were incubated with EDTA, the bacteria were susceptible to phe-Prt. Next, the binding effect of phe-Prt with LPS was determined. Our result showed that LPS could hamper the bactericidal activity of phe-Prt against Gram-positive bacteria. The result of zeta potential assay further confirmed the binding effect of phe-Prt with LPS for it could neutralize the surface charge of E. coli and LPS. Then, the effect of phe-Prt on the integrity of outer membrane of Gram-negative bacteria was determined. Our results showed that phe-Prt had a much weaker disturbance to the outer membrane of Gram-negative bacteria than the parent peptide protonectin. In summary, the introduction of l-phenylalanine into the sequence of antimicrobial peptide protonectin made phe-Prt show significant selectivity against Gram-positive bacteria, which could partly be attributed to the delay effect of LPS for phe-Prt to access to cell membrane. Although further study is still needed to clarify the exact mechanism of selectivity, the present study provided a strategy to develop antimicrobial peptides with selectivity toward Gram-positive and Gram-negative bacteria.

     

RNA interference of an antimicrobial peptide, gloverin, of the beet armyworm, Spodoptera exigua, enhances susceptibility to Bacillus thuringiensis

Gloverin is known to be an inducible antimicrobial peptide. This study reports a gloverin gene (Seglv) identified from the beet armyworm, Spodoptera exigua. Seglv encodes 175 amino acids with a signal peptide. Its amino acid sequence is highly homologous (>95%) to other known gloverins. Seglv was expressed from egg to adult stages even without immune challenge. Especially, in larval stage, it was expressed in all tested tissues, such as hemocyte, fat body, gut, and epidermis. However, the constitutive expression level was significantly elevated in response to bacterial challenge. Expression of a Toll gene was required for expression of Seglv. A recombinant Seglv protein was synthesized using a bacterial expression system and purified with an affinity chromatography. The recombinant protein showed a specific antibacterial activity against a Gram-positive bacterium, but no activity against a Gram-negative Escherichia coli. Injection of specific double stranded RNA (dsRNA) against Seglv could suppress its expression. Knockdown of Seglv expression induced a significant developmental retardation and resulted in hypotrophy pupae. The larvae treated with dsRNA were much more susceptible to Bacillus thuringiensis than the control larvae. These results suggest that Seglv acts as an antimicrobial peptide especially against Gram-positive bacteria including B. thuringiensis.     

中国明对虾溶菌酶基因克隆、重组表达与性质分析

溶菌酶是机体先天免疫系统中一个重要的效应分子, 参与机体多种免疫反应, 在溶菌过程中形成一个水解体系, 破坏和消除侵入体内的病原, 从而实现机体的免疫防御。从中国明对虾中克隆得到了溶菌酶基因(称为FcLyz基因), 该基因全长709 bp, 其完整的阅读框为477 bp, 编码158个氨基酸, 前18个氨基酸(-1~-18)为信号肽, 成熟肽由140个氨基酸组成(1-140aa), 其分子量为16.2 kD。经SMART分析,该基因具有1个溶菌酶1(LYZ1)结构域(19-130aa)。半定量RT-PCR分析结果表明溶菌酶虽在多种组织中有较低水平的组成性表达, 但在细菌诱导的血细胞、心脏、肝胰腺和鳃等多种组织中表达上调。将中国明对虾溶菌酶基因的成熟肽亚克隆进原核表达载体pET-30a (+)中, 转化大肠杆菌BL21(DE3), 再进行诱导表达和亲和纯化, 得到了纯化的重组溶菌酶, 并进行了抑菌活性检测。结果表明, 重组对虾溶菌酶对革兰氏阳性菌的抑菌能力较强, 最小抑菌浓度达到3.43 mmol/L, 但对革兰氏阴性菌抑制作用较小。上述结果表明, 该溶菌酶作为一种重要的免疫效应分子, 参与了对虾的免疫防御反应。     

中国明对虾溶菌酶基因克隆、重组表达与性质分析

溶菌酶是机体先天免疫系统中一个重要的效应分子, 参与机体多种免疫反应, 在溶菌过程中形成一个水解体系, 破坏和消除侵入体内的病原, 从而实现机体的免疫防御。从中国明对虾中克隆得到了溶菌酶基因(称为FcLyz基因), 该基因全长709 bp, 其完整的阅读框为477 bp, 编码158个氨基酸, 前18个氨基酸(-1~-18)为信号肽, 成熟肽由140个氨基酸组成(1-140aa), 其分子量为16.2 kD。经SMART分析,该基因具有1个溶菌酶1(LYZ1)结构域(19-130aa)。半定量RT-PCR分析结果表明溶菌酶虽在多种组织中有较低水平的组成性表达, 但在细菌诱导的血细胞、心脏、肝胰腺和鳃等多种组织中表达上调。将中国明对虾溶菌酶基因的成熟肽亚克隆进原核表达载体pET-30a (+)中, 转化大肠杆菌BL21(DE3), 再进行诱导表达和亲和纯化, 得到了纯化的重组溶菌酶, 并进行了抑菌活性检测。结果表明, 重组对虾溶菌酶对革兰氏阳性菌的抑菌能力较强, 最小抑菌浓度达到3.43 mmol/L, 但对革兰氏阴性菌抑制作用较小。上述结果表明, 该溶菌酶作为一种重要的免疫效应分子, 参与了对虾的免疫防御反应。     

Crustin,a WAP domain containing antimicrobial peptide from freshwater prawn Macrobrachium rosenbergii: Immune characterization

Crustin (MrCrs) was sequenced from a freshwater prawn Macrobrachium rosenbergii. The MrCrs protein contains a signal peptide region at N-terminus between 1 and 22 and a long whey acidic protein domain (WAP domain) at C-terminus between 57 and 110 along with a WAP-type ‘four-disulfide core’ motif. Phylogenetic results show that MrCrs is clustered together with other crustacean crustin groups. MrCrs showed high sequence similarity (77%) with crustin from Pacific white shrimp Litopenaeus vannamei and Japanese spiny lobster Panulirus japonicas. I-TASSER uses the best structure templates to predict the possible structures of MrCrs along with PDB IDs such as 2RELA and 1FLEI. The gene expressions of MrCrs in both healthy M. rosenbergii and those infected with virus including infectious hypodermal and hematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV) and bacteria Aeromonas hydrophila (Gram-negative) and Enterococcus faecium (Gram-positive) were examined using quantitative real time PCR. To understand its biological activity, the recombinant MrCrs gene was constructed and expressed in Escherichia coli BL21 (DE3). The recombinant MrCrs protein agglutinated with the bacteria considered for analysis at a concentration of 25 μg/ml, except Lactococcus lactis. The bactericidal results showed that the recombinant MrCrs protein destroyed all the bacteria after incubation, even less than 6 h. These results suggest that MrCrs is a potential antimicrobial peptide, which is involved in the defense system of M. rosenbergii against viral and bacterial infections.     

Phylogeny of whey acidic protein (WAP) four-disulfide core proteins and their role in lower vertebrates and invertebrates

Proteins containing WAP (whey acidic protein) domains with a characteristic WFDC (WAP four-disulfide core) occur not only in mammals (including marsupials and monotremes) but also in birds, reptiles, amphibians and fish. In addition, they are present in numerous invertebrates, from cnidarians to urochordates. Many of those from non-mammalian groups are poorly understood with respect to function or phylogeny. Those well characterized so far are waprins from snakes, perlwapins from bivalves and crustins from decapod crustaceans. Waprins are venom proteins with a single WAP domain at the C-terminus. They display antimicrobial, rather than proteinase inhibitory, activities. Perlwapins, in contrast, possess three WAP domains at the C-terminus and are expressed in the shell nacre of abalones. They participate in shell formation by inhibiting the growth of calcium crystals in the shell. The crustin group is the largest of all WFDC-containing proteins in invertebrates with the vast majority being highly expressed in the haemocytes. Most have a single WAP domain at the C-terminus. The presence and type of the domains between the signal sequence and the C-terminus WAP domain separate the different crustin types. Most of the Type?I and II crustins are antimicrobial towards Gram-positive bacteria, whereas the Type?III crustins tend to display protease inhibition. Expression studies show that at least some crustins have other important biological effects, as levels change with physiological stress, wound repair, tissue regeneration or ecdysis. Thus WAP domains are widely distributed and highly conserved, serving in diverse physiological processes (proteinase inhibition, bacterial killing or inhibition of calcium transport).     

A New Amino Acid Antibiotic KT–151, Produced by Streptomyces luteogriseus,Strain No. KT–151

From the results of taxonomic studies, Streptomyces sp. strain No. KT–151 isolated from a soil sample collected in Kumamoto City, was identified as a strain belonging to Streptomyces luteogriseus Schmitz, Deak, Crook and Hooper 1964. A new antibiotic, produced by this strain, was isolated as a leaflet crystal by ion-exchange chromatography and found to be an amino acid with the molecular formula, C₅H₁₂N₂O₂, and named antibiotic KT–151 (refered to as KT–151 hereinafter). The antibiotic showed antimicrobial activity against various Gram-positive and Gram-negative bacteria in a chemically defined medium but it was antagonized by several amino acids such as valine, leucine, isoleucine and threonine.     

An outer membrane protein (OmpA) of Escherichia coli can be translocated across the cytoplasmic membrane of Bacillus subtllis

The translocation of secretory proteins derived from a Gram-positive (Staphylococcus hyicus prolipase) or a Gram-negative (Escherichia coli pre-OmpA protein) bacterium across the cytoplasmic membrane was studied in E. coli and Bacillus subtilis. in both microorganisms, the prolipase was found to be secreted across the plasma membrane when either the pre-prolipase signal peptide (38 amino acids in length) or the pre-OmpA signal peptide (21 amino acids in length) was used. Expression of the gene encoding the authentic pre-OmpA protein in B. subtilis resulted in the translocation of mature OmpA protein across the plasma membrane. Processing of the OmpA precursor in B. subtilis required the electrochemical potential and was sensitive to sodium azide, suggesting that the B. subtilis SecA homologue was involved in the translocation process. The mature OmpA protein, which was most likely present in an aggregated state, was fully accessible to proteases in protoplasted cells. Therefore, our results clearly demonstrate that an outer membrane protein can be secreted by B. subtilis, supporting the notion that the basic mechanism of protein translocation is highly conserved in Gram-positive and Gram-negative bacteria.     

A novel protein with a fibrinogen-like domain involved in the innate immune response of Marsupenaeus japonicus

Fibrinogen-related proteins play important roles in innate immunity. We isolated a fibrinogen-related protein gene (MjFREP1) in kuruma shrimp Marsupenaeus japonicus. MjFREP1 encoded a protein of 270 amino acids, including a 223 amino acid fibrinogen-like domain. Quantitative real-time polymerase chain reaction analysis shows that MjFREP1 is mainly expressed in the gills and the expression is significantly upregulated by Vibrio anguillarum, Staphylococcus aureus, or white spot syndrome virus (WSSV) challenge. Recombinant MjFREP1 fibrinogen-like domain agglutinates Gram-positive bacteria Bacillus subtilis, Bacillus thuringiensis, Bacillus megaterium, and S. aureus in the presence of calcium ions. The fibrinogen-like domain of MjFREP1 binds peptidoglycans, LPS, bacteria, and the VP28 of WSSV. These results suggest that the MjFREP1 may play an important role in the shrimp immune response against different pathogens.     

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.