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Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids
Published on: May 4, 2018
Purification and Characterization of Novel Anti-MRSA Peptides Produced by Brevibacillus sp. SPR-20
Nuttapon Songnaka1, Monthon Lertcanawanichakul2, Albert M Hutapea3
1School of Pharmacy, Walailak University, Nakhon Si Thammarat 80161, Thailand.
Abstract:
Methicillin-resistant Staphylococcus aureus (MRSA) is listed as a high-priority pathogen because its infection is associated with a high mortality rate. It is urgent to search for new agents to treat such an infection. Our previous study isolated a soil bacterium (Brevibacillus sp. SPR-20), showing the highest antimicrobial activity against S. aureus TISTR 517 and MRSA strains. The present study aimed to purify and characterize anti-MRSA substances produced by SPR-20. The result showed that five active substances (P1-P5) were found, and they were identified by LC-MS/MS that provided the peptide sequences of 14-15 residues. Circular dichroism showed that all peptides contained β-strand and disordered conformations as the major secondary structures. Only P1-P4 adopted more α-helix conformations when incubated with 50 mM SDS. These anti-MRSA peptides could inhibit S. aureus and MRSA in concentrations of 2-32 μg/mL. P1 (NH2-VVVNVLVKVLPPPVV-COOH) had the highest activity and was identified as a novel antimicrobial peptide (AMP). The stability study revealed that P1 was stable in response to temperature, proteolytic enzymes, surfactant, and pH. The electron micrograph showed that P1 induced bacterial membrane damage when treated at 1× MIC in the first hour of incubation. The killing kinetics of P1 was dependent on concentration and time. Mechanisms of P1 on tested pathogens involved membrane permeability, leakage of genetic material, and cell lysis. The P1 peptide at a concentration up to 32 μg/mL showed hemolysis of less than 10%, supporting its safety for human erythrocytes. This study provides promising anti-MRSA peptides that might be developed for effective antibiotics in the post-antibiotic era.
Insights
Researchers discovered novel antimicrobial peptides (AMPs) from soil bacteria effective against methicillin-resistant Staphylococcus aureus (MRSA). These peptides, particularly P1, show potent activity, stability, and safety, offering potential new antibiotics for MRSA infections.
Area of Science:
- Microbiology
- Biochemistry
- Pharmacology
Background:
- Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant global health threat due to high mortality rates.
- Urgent need for novel therapeutic agents to combat MRSA infections.
- Previous isolation of a soil bacterium, Brevibacillus sp. SPR-20, with high antimicrobial activity against S. aureus and MRSA.
Purpose of the Study:
- To purify and characterize anti-MRSA substances from Brevibacillus sp. SPR-20.
- To identify and evaluate the properties of novel antimicrobial peptides (AMPs) against MRSA.
Main Methods:
- Isolation and purification of active compounds.
- LC-MS/MS for peptide sequencing.
- Circular dichroism for structural analysis.
- Antimicrobial activity assays (MIC determination).
- Stability testing (temperature, enzymes, pH, surfactant).
- Electron microscopy for mechanism of action studies.
- Hemolysis assays for safety evaluation.
Main Results:
- Five active peptide substances (P1-P5) were purified, with sequences of 14-15 residues.
- Peptides P1-P4 showed increased alpha-helix content in the presence of SDS.
- Effective inhibition of S. aureus and MRSA strains at concentrations of 2-32 μg/mL.
- Peptide P1 identified as a novel AMP with potent activity and broad stability.
- P1 demonstrated membrane damage, genetic material leakage, and cell lysis in MRSA.
- P1 exhibited low hemolysis (<10% at 32 μg/mL), indicating safety for erythrocytes.
Conclusions:
- Brevibacillus sp. SPR-20 produces novel antimicrobial peptides with significant anti-MRSA activity.
- Peptide P1 is a promising candidate for developing new antibiotics against MRSA.
- The study highlights the potential of AMPs in addressing the challenge of antibiotic resistance.
