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Molecular characterization of a novel big defensin from clam Venerupis philippinarum.

ABSTRACT: Antimicrobial peptides (AMPs) are important mediators of the primary defense mechanism against microbial invasion. In the present study, a big defensin was identified from Venerupis philippinarum haemocytes (denoted as VpBD) by RACE and EST approaches. The VpBD cDNA contained an open reading frame (ORF) of 285 bp encoding a polypeptide of 94 amino acids. The deduce amino acid sequence of VpBD shared the common features of big defensin including disulfide array organization and helix structure, indicating that VpBD should be a new member of the big defensin family. The mRNA transcript of VpBD was up-regulated significantly during the first 24 hr after Vibrio anguillarum challenge, which was 7.4-fold increase compared to that of the control group. Then the expression decreased gradually from 24 hr to 96 hr, and the lowest expression level was detected at 96 hr post-infection, which was still 3.9-fold higher than that of control. The mature peptide of VpBD was recombined in Escherichia coli and purified for minimum inhibitory concentration (MIC) determination. The rVpBD displayed broad-spectrum inhibitory activity towards all tested bacteria with the highest activity against Staphyloccocus aureus and Pseudomonas putida. These results indicated that VpBD was involved in the host immune response against bacterial infection and might contribute to the clearance of invading bacteria.

SUBMITTER: Zhao J

PROVIDER: S-EPMC2958110 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Molecular characterization of a novel big defensin from clam Venerupis philippinarum.

Zhao Jianmin J Li Chenghua C Chen Aiqin A Li Lingyun L Su Xiurong X Li Taiwu T

PloS one 20101020 10

Antimicrobial peptides (AMPs) are important mediators of the primary defense mechanism against microbial invasion. In the present study, a big defensin was identified from Venerupis philippinarum haemocytes (denoted as VpBD) by RACE and EST approaches. The VpBD cDNA contained an open reading frame (ORF) of 285 bp encoding a polypeptide of 94 amino acids. The deduce amino acid sequence of VpBD shared the common features of big defensin including disulfide array organization and helix structure, i ...[more]

PMID: 20975988

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Project description:Parasites of the genus Perkinsus spp. cause high mortalities and economic losses to the most noticeable bivalves produced in the worldwide aquaculture. In this study, we analyze how P. olseni influences the gene expression profiles of hemocytes from Manila clam (Venerupis philippinarum) using experimental infections along a temporal series and a Manila clam immune-enriched DNA microarray. Healthy and Perkinsus-infected clams (V. philippinarum) were obtained from Carril and Pontevedra shellfish farms, respectively (Galicia, NW Spain). The presence-absence of P. olseni was confirmed using the Ray`s fluid thioglycollate medium assay (RFTM) (Ray, 1966). Healthy clams were maintained in an open circuit filtered sea water tanks at 15°C with aeration. Natural infected animals were maintained in the same conditions using closed circuit sea water. All animals were fed daily with a mixture of microalgae containing Phaeodactylum tricornutum, Isochrysis galbana and Rhodomonas lens. Clams were acclimatized to the aquarium conditions for one week before the experiments were conducted. Perkinsus trophozoites were isolated from naturally infected animals following the protocol established by Ford et al., (2002). The concentration was adjusted to 5x104 trophozoites /ml in filtered sea water (FSW). Healthy clams (P. olseni free animals) (n=100) with a weight of 2.25 ± 0.64 g soft tissue, were notched in the shell and intramuscularly injected with 100 µl of the trophozoites suspension. Control animals (n=100) were injected with 100 µl of FSW. After infection, clams were maintained in 50 l tanks with aeration.Twenty animals from each experimental group and time point were sampled at 5, 10, 14, and 31 days post infection (pi).Hemolymph were extracted to perform microarrays experiments. In each condition hemolymph from three five individuals was pooled. Total RNA isolation was conducted following the manufacturer’s specifications. Isolated RNAs were treated with DNase I and purified again using the RNeasy Mini kit (Qiagen). A 8x15K Agilent 60-mer oligo-microarray (GPL16450) was used to compare gene expression profiles of clams after P. olseni infection with uninfected animals. The Agilent Feature Extraction Software (version 9.5.1) was used for the data extraction and background subtraction following standard procedures. The GeneSpring software (Agilent) was used to normalize and analyze the microarray fluorescence data.

Dataset Information

Molecular characterization of a novel big defensin from clam Venerupis philippinarum.

Publications

Molecular characterization of a novel big defensin from clam Venerupis philippinarum.

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