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Published on: May 13, 2020
Molecular cloning, genomic organization and recombinant expression of a crustin-like antimicrobial peptide from black
Piti Amparyup1, Hidehiro Kondo, Ikuo Hirono
1National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Paholyothin Rd., Klong1, Klong Luang, Pathumthani 12120, Thailand.
Abstract:
A novel crustin-like antimicrobial peptide (Crus-likePm) was identified from haemocytes of Penaeus monodon. The deduced amino acid sequence of a Crus-likePm consists of 124 amino acid residues of the mature peptide and a signal peptide of 17 amino acid residues. The mature peptide contains a glycine-rich domain at the N-terminus and 12 conserved cysteine residues containing a single WAP domain at the C-terminus. Phylogenetic tree and sequence comparison clearly confirmed a distinct between a Crus-likePm and other shrimp crustins. Genomic organization and upstream region of a Crus-likePm gene was investigated. The gene consisted of two exons and one intron. The 5'-flanking regions of a Crus-likePm gene contain multiple putative transcription factor binding sites. mRNA transcript of a Crus-likePm was found to be abundantly expressed in haemocyte and highly up-regulated after Vibrio harveyi injection. The mature Crus-likePm was cloned into the pET28b with an N-terminal hexa-histidine tag fused in-frame, and expressed in E. coli. The purified recombinant Crus-likePm showed strong antimicrobial activity against both Gram-positive and Gram-negative bacteria including V. harveyi, a major pathogenic bacteria in shrimp aquaculture.
Insights
A novel antimicrobial peptide, Crus-likePm, was discovered in shrimp. This peptide effectively combats Gram-positive and Gram-negative bacteria, including Vibrio harveyi, offering potential for aquaculture disease control.
Area of Science:
- Marine biology
- Immunology
- Biochemistry
Background:
- Shrimp aquaculture faces significant losses due to bacterial pathogens like Vibrio harveyi.
- Antimicrobial peptides (AMPs) are crucial components of innate immunity in invertebrates.
- Understanding shrimp AMPs can lead to novel disease management strategies.
Purpose of the Study:
- To identify and characterize a novel crustin-like antimicrobial peptide (Crus-likePm) from Penaeus monodon.
- To investigate the gene structure, expression, and antimicrobial activity of Crus-likePm.
- To assess the potential of Crus-likePm in combating shrimp bacterial infections.
Main Methods:
- Identification and sequence analysis of Crus-likePm from shrimp haemocytes.
- Phylogenetic analysis to compare Crus-likePm with other crustins.
- Gene structure analysis, including exon-intron organization and promoter region investigation.
- mRNA expression analysis via quantitative PCR after bacterial challenge.
- Recombinant expression of Crus-likePm in E. coli and purification.
- Antimicrobial activity assays against various bacterial strains.
Main Results:
- A novel Crus-likePm was identified, featuring a glycine-rich N-terminus and a WAP domain with 12 conserved cysteines.
- Phylogenetic analysis confirmed Crus-likePm as distinct from other known shrimp crustins.
- The Crus-likePm gene comprises two exons and one intron, with putative transcription factor binding sites in its 5'-flanking region.
- Crus-likePm mRNA was highly expressed in haemocytes and significantly upregulated following Vibrio harveyi infection.
- Recombinant Crus-likePm exhibited potent antimicrobial activity against both Gram-positive and Gram-negative bacteria, including Vibrio harveyi.
Conclusions:
- Crus-likePm is a novel antimicrobial peptide with a unique structure and significant antimicrobial properties.
- Its expression is inducible by bacterial infection, highlighting its role in shrimp innate immunity.
- Recombinant Crus-likePm demonstrates broad-spectrum antibacterial activity, showing promise as a therapeutic agent for shrimp aquaculture.
