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Significance
The rapid evolution of antibiotic resistance in bacteria like Enterococcus faecium, Staphylococcus aureus, and Klebsiella pneumoniae, among others, has rendered many standard treatments ineffective. These pathogens have developed intrinsic and extrinsic mechanisms to resist traditional antibiotics such as gentamycin, ciprofloxacin, and ampicillin. The failure of these drugs leads to complications in treating infections, posing a significant threat to public health. Antimicrobial peptides (AMP) have recently been recognized as potential alternatives to traditional antibiotics due to their unique modes of action. These peptides are usually cationic and amphipathic, comprising 10-100 amino acid residues. They exert their antimicrobial effects by disrupting bacterial cell walls and membranes or inhibiting DNA replication and protein synthesis. AMPs like defensins, LL-37, gramicidins D, and Renalexin have shown efficacy against resistant bacterial strains.
A new study published in the Journal Applied Microbiology and Biotechnology by Julius Kwesi Narh, Professor Nestor Casillas-Vega and led by Professor Xristo Zarate from the Autonomous University of Nuevo Leon in Mexico, designed a novel hybrid AMP named LL-37_Renalexin, which demonstrated potent antibacterial properties against various clinically relevant pathogens. The researchers used advanced bioengineering techniques to combine two known AMPs, LL-37 and Renalexin, using a GS peptide linker. This innovative design aimed to harness the strengths of both peptides in one potent compound. They expressed the new hybrid peptide using carrier proteins SmbP and CusF3H+. These proteins were used to enhance the stability and expression levels of LL-37_Renalexin in bacterial systems. The team employed Escherichia coli strains BL21(DE3) and SHuffle T7(DE3) as hosts for peptide expression. These strains are known for their efficiency in protein production. They used immobilized metal affinity chromatography for the purification of LL-37_Renalexin, ensuring high purity and yield.
The researchers synthesized DNA encoding the hybrid peptide and cloned it into expression vectors for production in E. coli. They conducted small-scale expressions to verify the presence of the peptide in the soluble cell lysate, followed by large-scale expression to produce sufficient quantities for testing. The fusion proteins were purified using chromatography techniques, ensuring that the peptide was isolated in its pure form. To obtain the tag-free peptide, they utilized enterokinase to cleave off the carrier proteins from the hybrid peptide.
The team tested the antimicrobial efficacy of LL-37_Renalexin against various bacterial strains, including S. aureus, E. coli, MRSA, and K. pneumoniae. The authors demonstrated that LL-37_Renalexin has a significant reduction in bacterial colony-forming units, showcasing its effectiveness against multiple bacterial strains. The hybrid peptide required lower concentrations to inhibit bacterial growth compared to its individual components, suggesting greater potency. LL-37_Renalexin exhibited a broad spectrum of activity against both Gram-positive and Gram-negative bacteria. The hybrid peptide’s mechanism involves disruption of the bacterial membrane, pore formation, and possible inhibition of vital cellular processes. The study emphasizes the multifunctional nature of LL-37_Renalexin, highlighting its ability to target various bacterial strains effectively. One key advantage of the design is the GS linker and carrier proteins which contributed to the stable and soluble expression of the hybrid peptide. Another advantage is safety where LL-37_Renalexin did not exhibit toxicity to human cells, indicating its potential for safe therapeutic use.
According to the authors, the newly developed hybrid peptide offers several advantages. Firstly, it addresses the urgent need for new antimicrobial agents in the face of rising antibiotic resistance. Secondly, its method of production via recombinant DNA technology makes it a viable option for large-scale production. Lastly, the peptide’s efficacy at lower concentrations could reduce potential side effects, making it a safer therapeutic option. In conclusion, the findings of Professor Xristo Zarate and colleagues are significant in the field of antimicrobial research and offer a promising avenue for developing new treatments for antibiotic-resistant infections. This research could pave the way for more effective, safe, and targeted antimicrobial therapies. Professor Xristo Zarate said “We’ve created a new superbug-fighting weapon: LL-37_Renalexin, a hybrid antimicrobial peptide that’s like a one-two punch for drug-resistant bacteria. It’s got the power of two existing AMPs, LL-37 and Renalexin, combined into one potent package. And the best part? It works at lower doses than either of its parent peptides, meaning less chance of side effects. This is a game-changer for the fight against antibiotic resistance”.
Reference
Julius Kwesi Narh, Nestor G Casillas-Vega and Xristo Zarate. LL-37_Renalexin hybrid peptide exhibits antimicrobial activity at lower MICs than its counterpart single peptides. Applied Microbiology and Biotechnology 108, 126 (2024)