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Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization
Published on: August 11, 2018
A truncated peptide Spgillcin177-189 derived from mud crab Scylla paramamosain exerting multiple antibacterial
Xiaofei Wang1, Xiao Hong1, Fangyi Chen1,2,3
1State Key Laboratory of Marine Environmental Science, College of Ocean and Earth Sciences, Xiamen University, Xiamen, China.
Abstract:
Antimicrobial peptides (AMPs) may be the most promising substitute for antibiotics due to their effective bactericidal activity and multiple antimicrobial modes against pathogenic bacteria. In this study, a new functional gene named Spgillcin was identified in Scylla paramamosain, which encoded 216 amino acids of mature peptide. In vivo, Spgillcin was dominantly expressed in the gills of male and female crabs, offering the highest expression level among all tested organs or tissues. The expression pattern of Spgillcin was significantly altered when challenged by Staphylococcus aureus, indicating a positive immune response. In vitro, a functional truncated peptide Spgillcin177-189 derived from the amino acid sequence of Spgillcin was synthesized and showed a broad-spectrum and potent antibacterial activity against several bacterial strains, including the clinical isolates of multidrug-resistant (MDR) strains, with a range of minimum inhibitory concentrations from 1.5 to 48 μM. Spgillcin177-189 also showed rapid bactericidal kinetics for S. aureus and Pseudomonas aeruginosa but did not display any cytotoxicity to mammalian cells and maintained its antimicrobial activity in different conditions. Mechanistic studies indicated that Spgillcin177-189 was mainly involved in the disruption of cell membrane integrity where the membrane components lipoteichoic acid and lipopolysaccharide could significantly inhibit the antimicrobial activity in a dose-dependent manner. In addition, Spgillcin177-189 could change the membrane permeability and cause the accumulation of intracellular reactive oxygen species. No resistance was generated to Spgillcin177-189 when the clinical isolates of methicillin-resistant S. aureus and MDR P. aeruginosa were treated with Spgillcin177-189 and then subjected to a long term of continuous culturing for 50 days. In addition, Spgillcin177-189 exerted a strong anti-biofilm activity by inhibiting biofilm formation and was also effective at killing extracellular S. aureus in the cultural supernatant of RAW 264.7 cells. Taken together, Spgillcin177-189 has strong potential as a substitute for antibiotics in future aquaculture and medical applications.
Insights
A novel antimicrobial peptide, Spgillcin, derived from the mud crab Scylla paramamosain, demonstrates potent broad-spectrum antibacterial activity against multidrug-resistant strains. This peptide shows promise as a safe and effective alternative to conventional antibiotics in aquaculture and medicine.
Area of Science:
- Marine biology
- Biochemistry
- Immunology
Background:
- Antimicrobial peptides (AMPs) are crucial in innate immunity and represent promising alternatives to antibiotics.
- Pathogenic bacteria, including multidrug-resistant (MDR) strains, pose a significant threat to human health and aquaculture.
- The mud crab, Scylla paramamosain, is a valuable aquaculture species with potential for novel antimicrobial discoveries.
Purpose of the Study:
- To identify and characterize a novel antimicrobial peptide from Scylla paramamosain.
- To evaluate the antibacterial activity, mechanism of action, and safety of the peptide.
- To assess the potential of the peptide as a substitute for antibiotics.
Main Methods:
- Gene cloning and sequencing to identify the Spgillcin gene.
- Quantitative real-time PCR to analyze Spgillcin expression in vivo.
- Synthesis and in vitro antibacterial assays of the truncated peptide Spgillcin177-189.
- Cytotoxicity assays on mammalian cells.
- Mechanistic studies involving membrane integrity and reactive oxygen species.
- Antibiotic resistance development assays and anti-biofilm activity tests.
Main Results:
- The Spgillcin gene was identified, encoding a 216-amino acid peptide predominantly expressed in crab gills and upregulated upon Staphylococcus aureus challenge.
- The synthesized peptide Spgillcin177-189 exhibited potent, broad-spectrum activity against various bacterial strains, including MDR isolates, with MICs ranging from 1.5 to 48 μM.
- Spgillcin177-189 demonstrated rapid bactericidal kinetics, no cytotoxicity to mammalian cells, stability under various conditions, and efficacy against biofilms.
- No resistance developed against Spgillcin177-189 after prolonged exposure to resistant bacterial strains.
- Mechanisms involved cell membrane disruption, increased permeability, and reactive oxygen species accumulation.
Conclusions:
- Spgillcin is a novel antimicrobial peptide from Scylla paramamosain with significant potential.
- The truncated peptide Spgillcin177-189 displays potent antibacterial, anti-biofilm, and safety profiles.
- Spgillcin177-189 is a promising candidate for developing new antimicrobial agents for aquaculture and medical applications.
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