Design of a cyclic peptide targeting intracellular Staphylococcus aureus

Targeted Design of Novel Antimicrobial Peptides against Staphylococcal Membranes

Due to the rising worldwide concern of antibiotic resistance, the creation of new antimicrobial agents has become an essential necessity. Novel antimicrobial peptides (AMPs), celebrated for their extensive efficacy and minimal likelihood of resistance development, are considered among the most promising treatment options. This paper introduced the rational design and synthesis of a new antimicrobial peptide, RK7 (RKKYWLL), which targeted the biophysical differences between human and staphylococcal cell membranes to achieve selective disturbance and damage of the membrane. Models of human and staphylococcal cell membranes were first developed, subsequently followed by molecular docking screens to discover peptides with a strong affinity for staphylococcal membranes while demonstrating inertness toward human membranes. Molecular dynamics (MD) simulations showed that RK7 could specifically interact with the bacterial membrane. AI-assisted design suggested that RK7 is nontoxic and has good stability. Following antibacterial and cytotoxicity testing, RK7 was shown to be nontoxic to normal human cells while exhibiting significant staphylococcal inhibition. RK7 demonstrated a 98.14% inhibition rate against Staphylococcus at a dose of 62.5 μg/mL. This research provides a novel strategy for designing antimicrobial peptides based on compositional differences in cell membranes, offering an innovative approach for the targeted design of future antimicrobial agents.

Design, synthesis, and evaluation of membrane-targeted resveratrol-antimicrobial peptide mimic conjugates for MRSA-infected wound healing.

Wound infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are notoriously difficult to treat due to biofilm formation and multidrug resistance, necessitating the development of novel antimicrobial agents. To address this challenge, we designed and synthesized a series of resveratrol-antimicrobial peptide mimic conjugates using a molecular splicing strategy. Among them, lead compound III-5 exhibited a minimum inhibitory concentration (MIC) of 6.25 μg/mL against MRSA, which is an approximately 40-fold improvement in antimicrobial activity over the precursor resveratrol. Moreover, III-5 demonstrated low hemolytic activity, a low propensity to induce drug resistance, and favorable anti-inflammatory properties. The membrane-targeted III-5 effectively disrupted bacterial cell membrane integrity and significantly inhibited both biofilm formation and the eradication of preformed mature biofilms. Transcriptomic analysis indicated a membrane-targeted mechanism, interfering with lipoteichoic acid biosynthesis, cell wall organization, and two-component systems. Together, these membrane-targeted actions synergistically impair cell wall integrity and suppress biofilm formation. Furthermore, in a murine model of MRSA-infected wounds, treatment with III-5-loaded PVA-SA hydrogel achieved a 96% wound healing rate by day 14, significantly accelerating wound closure and reducing the bacterial burden. Collectively, these findings position resveratrol-antibacterial peptide mimic conjugates as a promising antimicrobial candidate for combating MRSA-associated wound infections and provide valuable insights for the development of novel antimicrobial agents.

Extracellular injection system combined with peptides for intracellular Staphylococcus aureus treatment

Assessing Vancomycin‐Formyl Peptide Conjugate Binding to the Surface of Resistant Staphylococcus aureus via Flow Cytometry

Staphylococcus aureus strains have emerged with resistance mechanisms that reduce the efficacy of last resort antibiotics and evade the immune system. One strategy to combat antimicrobial resistance is to modulate host immunity to eliminate infections more effectively. This has led to the development of immunotherapeutics consisting of vancomycin conjugated to formyl peptides (fPeps), with vancomycin targeting the cell wall and the fPeps engaging host innate immunity. Here, we used flow cytometry to quantify the binding of vancomycin=fPep conjugates to S. aureus clinical isolates. This revealed reduced binding of vancomycin=fPeps compared to vancomycin alone and quantified the interaction between the conjugates and the bacterial cell surface, which is important to quantify to then control the chemotactic gradient established by the fPep cargo. The direct antimicrobial activity of these conjugates was also reduced when compared to vancomycin, reflecting the reduced binding of these conjugates to S. aureus. This flow cytometry method allows quantification of vancomycin=fPep binding to bacteria and will assist in future studies to understand how attached fPeps and other immune signalling cargoes can stimulate innate immune cell activation leading to bacterial phagocytosis.

Dual-Mechanism Antimicrobial Peptides from a Nature-Inspired Scaffold

Antimicrobial peptides are promising alternatives to conventional antibiotics, yet systematic strategies to enhance their potency and elucidate their mechanisms of action remain limited. Here, we generated and evaluated a focused library of 20 peptides derived from the lead peptide L3. Across clinically relevant pathogens, including Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Candida albicans, several variants showed enhanced antibacterial activity, reducing MIC values to as low as 32 μg/mL (G2-4). Additional candidates (G1-8, G2-1, G2-2, G2-10) achieved MICs of 64 μg/mL against E. coli. Studies in environmental Escherichia isolates revealed species-specific susceptibility patterns. Mechanistic investigations demonstrated minimal membrane-lytic activity at concentrations exceeding their MICs, indicating that membrane disruption is not their primary mode of action. In contrast, in vitro transcription/translation assays demonstrated potent inhibition of protein expression. These results demonstrate how targeted sequence refinement can substantially enhance antimicrobial potency while modulating interactions with bacterial membranes and the transcription/translation machinery.

Screening of Cell-Penetrating Peptides To Enhance the Therapeutic Efficacy of α-Helical Antimicrobial Peptides against Intracellular Bacterial Infections.

Intracellular bacterial infections pose a major health challenge. Antimicrobial peptides (AMPs) are promising anti-infective agents, but their efficacy against intracellular pathogens is limited by poor cellular uptake. Cell-penetrating peptides (CPPs) have been employed to improve AMP delivery and intracellular bactericidal activity. However, due to the diversity in CPP types and cellular uptake mechanisms, the optimal CPPs for AMP delivery remain unknown. Herein, six representative CPPs were conjugated to the model α-helical AMP HC1, and the resulting conjugates were systematically evaluated for cellular uptake efficacy and in vitro and in vivo intracellular antibacterial activity. Bac7-HC1 emerged as the most effective conjugate against intracellular bacterial infections. The results highlight that cationic or amphipathic CPPs, particularly those with intrinsic antimicrobial activity and high positive charge, can enhance the anti-infective performance of HC1-based conjugates in this context. This work identifies Bac7-HC1 as a lead candidate for treating intracellular infections and provides preliminary insights for the design of CPP-based AMP therapeutics.