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Updated: Dec 22, 2025
Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds
Published on: September 8, 2021
A potent enzybiotic against methicillin-resistant Staphylococcus aureus
Jasjeet Kaur1, Prashant Singh2, Deepak Sharma2
1Department of Microbiology, Panjab University, Chandigarh, India.
Abstract:
Staphylococcus aureus is one of the most dreadful infectious agents, responsible for high mortality and morbidity in both humans and animals. The increased prevalence of multidrug-resistant (MDR) Staphylococcus aureus strains has limited the number of available treatment options, which calls for the development of alternative and effective modalities against MDR S. aureus. Endolysins are bacteriophage-derived antibacterials, which attack essential conserved elements of peptidoglycan that are vital for bacterial survival, making them promising alternatives or complements to existing antibiotics for tackling such infections. For developing endolysin lysin-methicillin-resistant-5 (LysMR-5) as an effective antimicrobial agent, we evaluated its physical and chemical characteristics, and its intrinsic antibacterial activity against staphylococcal strains, including methicillin-resistant Staphylococcus aureus (MRSA). In this study, we cloned, expressed, and purified LysMR-5 from S. aureus phage MR-5. In silico analysis revealed that LysMR-5 harbors two catalytic and one cell wall-binding domain. Biochemical characterization and LC-MS analysis showed that both catalytic domains were active and had no dependence on divalent ions for their action, Zn2+ exerted a negative effect. The optimal lytic activity of the endolysin was at 37 °C/pH 7.0 and in the presence of ≥ 300 mM concentration of NaCl. Circular dichroism (CD) demonstrated a loss in secondary structure with an increase in temperature confirming the thermosensitive nature of endolysin. Antibacterial assays revealed that LysMR-5 was active against diverse clinical isolates of staphylococci. It showed high lytic efficacy against S. aureus ATCC 43300, as an endolysin concentration as low as 15 µg/ml was sufficient to achieve maximum lytic activity within 30 min and it was further confirmed by scanning electron microscopy. Our results indicate that rapid and strong bactericidal activity of LysMR-5 makes it a valuable candidate for eradicating multidrug-resistant S. aureus.
Insights
This study introduces lysin-methicillin-resistant-5 (LysMR-5), a novel bacteriophage-derived endolysin effective against multidrug-resistant Staphylococcus aureus. LysMR-5 demonstrates potent bactericidal activity, offering a promising alternative to conventional antibiotics for treating resistant infections.
Area of Science:
- Microbiology
- Biochemistry
- Drug Discovery
Background:
- Staphylococcus aureus is a significant cause of human and animal infections.
- Multidrug-resistant (MDR) strains, including MRSA, pose a growing therapeutic challenge.
- Bacteriophage-derived endolysins are emerging as potent antibacterials targeting conserved bacterial structures.
Purpose of the Study:
- To characterize the physical, chemical, and antibacterial properties of LysMR-5.
- To evaluate LysMR-5 as a potential therapeutic agent against MDR Staphylococcus aureus.
- To assess the lytic efficacy of LysMR-5 against clinical isolates.
Main Methods:
- Cloning, expression, and purification of LysMR-5.
- In silico analysis of protein domains.
- Biochemical characterization, including optimal activity conditions and ion effects.
- Circular dichroism for thermal stability assessment.
- Antibacterial assays against various staphylococcal strains.
Main Results:
- LysMR-5 possesses two active catalytic domains and a cell wall-binding domain.
- Optimal activity observed at 37°C, pH 7.0, and high salt concentrations (≥300 mM NaCl).
- LysMR-5 exhibits potent bactericidal activity against diverse staphylococcal isolates, including MRSA.
- High lytic efficacy demonstrated at low concentrations (15 µg/ml) within 30 minutes.
Conclusions:
- LysMR-5 is a thermostable endolysin with significant antibacterial activity.
- Its rapid and potent bactericidal action makes it a promising candidate for combating MDR S. aureus infections.
- LysMR-5 represents a viable alternative or adjunct to current antibiotic therapies.
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