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Characterization and antimicrobial evaluation of SpPR-AMP1, a proline-rich antimicrobial peptide from the mud crab
Chanprapa Imjongjirak1, Pawanrat Amphaiphan1, Walaiporn Charoensapsri2
1Department of Food Technology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand.
Abstract:
Antimicrobial peptide (AMP) is an important molecule in the innate immune system. Here, we report the cloning and functional studies of proline-rich AMPs (PR-AMPs) from the three species of mud crab: Scylla paramamosain, S. serrata, and the swimming crab Portunus pelagicus. The deduced peptides revealed that they contain the putative signal peptides and encode for mature peptides, which contain sequence architecture similar to a 6.5-kDa proline-rich AMP of the shore crab, Carcinus maenas which showed similarity with the bactenecin7. Tissue distribution analysis indicated that the SpPR-AMP1 was expressed in a wide range of adult tissues, with the highest expression levels in the crab hemocyte. Challenge experiments showed that the levels of SpPR-AMP1 mRNA expression were up-regulated in the hemocyte after peptidoglycan stimulation. To evaluate the biological properties of mature SpPR-AMP1, peptides were chemically synthesized and recombinantly expressed. SpPR-AMP1 showed strong antibacterial activity against both Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Vibrio harveyi. The results indicate that the SpPR-AMP1 plays a role in crab immunity.
Insights
Researchers identified proline-rich antimicrobial peptides (PR-AMPs) in mud crabs. These PR-AMPs, particularly SpPR-AMP1, demonstrate significant antibacterial activity and are crucial components of the crab innate immune system.
Area of Science:
- Marine Biology
- Immunology
- Biochemistry
Background:
- Antimicrobial peptides (AMPs) are vital for innate immunity.
- Proline-rich AMPs (PR-AMPs) are a specific class of AMPs with unique structures.
- Crustaceans possess innate immune systems that rely on AMPs for defense.
Purpose of the Study:
- To clone and characterize proline-rich antimicrobial peptides (PR-AMPs) from three crab species.
- To investigate the expression patterns and immune-related functions of a specific PR-AMP (SpPR-AMP1).
- To evaluate the antibacterial activity of the identified PR-AMPs.
Main Methods:
- Cloning of PR-AMP genes from Scylla paramamosain, S. serrata, and Portunus pelagicus.
- Tissue distribution analysis of SpPR-AMP1 mRNA expression.
- Peptidoglycan stimulation experiments to assess immune response.
- Chemical synthesis and recombinant expression of mature SpPR-AMP1.
- In vitro antibacterial assays against Gram-positive and Gram-negative bacteria.
Main Results:
- Deduced PR-AMP sequences showed similarity to known AMPs like bactenecin7.
- SpPR-AMP1 exhibited highest expression in hemocytes and was upregulated after bacterial stimulation.
- Synthesized SpPR-AMP1 displayed potent antibacterial activity against Micrococcus luteus and Vibrio harveyi.
- The identified PR-AMPs possess functional roles in crab immunity.
Conclusions:
- Proline-rich AMPs are present and functional in mud crabs and swimming crabs.
- SpPR-AMP1 is a key component of the crab innate immune system, effective against diverse bacterial pathogens.
- This study expands our understanding of AMP diversity and function in marine invertebrates.
