Antimicrobial Activity of Novel α/β Hybrid Peptides Against Methicillin-Resistant Staphylococcus aureus (MRSA)

Research Background

With the widespread use and misuse of antibiotics, multi-drug resistance (MDR) has become a major global public health threat. Particularly, ESKAPE pathogens (including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are increasingly resistant to existing antibiotics, leading to the failure of traditional antibiotics in clinical treatments. Therefore, the development of novel antimicrobial agents has become an urgent priority.

Antimicrobial peptides (AMPs), as an essential component of the innate immune system, exhibit broad-spectrum antimicrobial activity and are less likely to induce resistance. However, natural AMPs are prone to degradation by proteases in vivo, limiting their clinical application. To address this issue, researchers have begun exploring the incorporation of non-natural amino acids (such as β-amino acids) into peptide chains to enhance their proteolytic stability and antimicrobial activity. This study was conducted in this context, aiming to synthesize α/β hybrid peptides containing tbu-β3,3ac6c and evaluate their antimicrobial activity against drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA).

Research Source

This study was conducted by Aminur Rahman Sarkar, Jyoti Kumari, Arti Rathore, Rubina Chowdhary, Rakshit Manhas, Shifa Firdous, Avisek Mahapa, and Rajkishor Rai. The research team is affiliated with the Indian Institute of Integrative Medicine (IIIM) under the Council of Scientific and Industrial Research (CSIR) and the Academy of Scientific & Innovative Research (AcSIR). The findings were published online on October 29, 2024, in The Journal of Antibiotics.

Research Process and Results

1. Peptide Synthesis and Characterization

The research team synthesized nine α/β hybrid peptides (P1-P9) containing tbu-β3,3ac6c. The C-terminus of these peptides was modified with phenylethylamine, while the N-terminus was linked to a C12 alkyl chain via an amide bond (P4-P6) or a urea bond (P7-P9). The peptides were synthesized using solution-phase synthesis, and their purity was assessed using high-performance liquid chromatography (HPLC), with all peptides exceeding 95% purity. Additionally, the peptides were characterized using high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR).

2. Antimicrobial Activity Evaluation

The antimicrobial activity of P1-P9 against various pathogens was evaluated using resazurin-based minimum inhibitory concentration (MIC) assays. The results showed that P4, P6, and P7 exhibited significant antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus, and MRSA, with MIC values ranging from 6.25 to 12.5 μM. Further studies demonstrated that P4, P6, and P7 also showed good efficacy against multi-drug resistant (MDR) clinical isolates of S. aureus, with MIC values ranging from 6.25 to 50 μM.

3. Minimum Bactericidal Concentration (MBC) and Time-Kill Kinetics

The research team further evaluated the bactericidal effects of P4, P6, and P7 against MRSA. MBC assays revealed that the MBC values for P4 and P6 were 12.5 μM, while for P7, it was 25 μM. Time-kill kinetics experiments showed that P4 reduced MRSA growth by 99.99% within 3 hours, while P7 completely eradicated MRSA within 1 hour. These results indicate that P4, P6, and P7 possess rapid bactericidal capabilities, outperforming the standard drug vancomycin.

4. Biofilm Inhibition and Disruption

P4, P6, and P7 also demonstrated significant biofilm inhibition capabilities. At 4×MIC concentrations, P6 and P7 inhibited 70% and 77% of MRSA biofilm formation, respectively. However, these peptides showed limited ability to disrupt pre-formed biofilms, suggesting their primary role in preventing biofilm formation.

5. Hemolytic Activity Test

To assess the safety of P4, P6, and P7, the research team conducted hemolysis assays. The results showed that at MIC concentrations, P4, P6, and P7 induced approximately 19% hemolysis, while at 50 μM, P6 exhibited the highest hemolysis rate of 36%. Although these peptides showed some hemolytic activity at higher concentrations, the balance between their antimicrobial and hemolytic activities remains promising for potential applications.

6. Scanning Electron Microscopy (SEM) Analysis

SEM observations revealed that P4- and P6-treated MRSA cells exhibited significant surface roughness and lysis, indicating that these peptides exert their bactericidal effects by disrupting bacterial cell membranes. In contrast, P7 showed weaker membrane disruption at MIC concentrations but demonstrated similar bactericidal effects at 2×MIC concentrations.

7. Synergistic Effects with Vancomycin

The synergistic effects of P4, P6, and P7 with vancomycin were evaluated using a checkerboard assay. The results showed that P4, P6, and P7 exhibited synergistic effects with vancomycin in multiple combinations, with no antagonistic interactions observed. This suggests that these peptides can be used in combination with vancomycin to enhance their efficacy against MRSA.

Research Conclusions and Significance

This study successfully synthesized nine α/β hybrid peptides containing tbu-β3,3ac6c and evaluated their antimicrobial activity against MRSA and other drug-resistant bacteria. Among them, P4, P6, and P7 demonstrated significant antimicrobial efficacy, rapidly killing MRSA and inhibiting its biofilm formation. Additionally, the synergistic effects of these peptides with vancomycin provide potential for their use in clinical combination therapies.

The highlight of this study lies in the incorporation of tbu-β3,3ac6c and C12 alkyl chains, which significantly enhanced the antimicrobial activity and proteolytic stability of the peptides. Furthermore, the research team used SEM to reveal the bactericidal mechanism of these peptides, showing that they exert their effects by disrupting bacterial cell membranes. These findings provide important theoretical and experimental foundations for the development of novel antimicrobial agents.

Research Value

The scientific value of this study lies in revealing the potential of α/β hybrid peptides in combating multi-drug resistant bacteria, particularly their rapid bactericidal and biofilm-inhibiting capabilities against MRSA. Additionally, the synthesis methods and experimental models developed by the research team provide valuable references for future studies. From an application perspective, P4, P6, and P7, as potential antimicrobial drug candidates, hold promise for future clinical treatments, especially in addressing multi-drug resistant bacterial infections.

This study offers new insights and methods for tackling the global issue of antibiotic resistance, with significant scientific and practical implications.