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The escalating threat of multidrug-resistant pathogens necessitates innovative approaches to combat infectious diseases. In this study, we examined peptides R23F S *, V31K S *, and R44K S *, which were engineered to include an amyloidogenic fragment sourced from the S1 protein of S. aureus , along with one or two cell-penetrating peptide (CPP) components. We assessed the antimicrobial efficacy of these peptides in a liquid medium against various strains of both Gram-positive bacteria, including S. aureus (209P and 129B strains), MRSA (SA 180 and ATCC 43300 strains), and B. cereus (strain IP 5832), and Gram-negative bacteria such as P. aeruginosa (ATCC 28753 and 2943 strains) and E. coli (MG1655 and K12 strains). Peptides R23F S *, V31K S *, and R44K S * exhibited antimicrobial activity comparable to gentamicin and meropenem against all tested bacteria at concentrations ranging from 24 to 48 μM. The peptides showed a stronger antimicrobial effect against B. cereus . Notably, peptide R44K S * displayed high efficacy compared to peptides R23F S * and V31K S *, particularly evident at lower concentrations, resulting in significant inhibition of bacterial growth. Furthermore, modified peptides V31K S * and R44K S * demonstrated enhanced inhibitory effects on bacterial growth across different strains compared to their unmodified counterparts V31K S and R44K S . These results highlight the potential of integrating cell-penetrating peptides, amyloidogenic fragments, and amino acid residue modifications to advance the innovation in the field of antimicrobial peptides, thereby increasing their effectiveness against a broad spectrum of pathogens.

Combining antimicrobial peptides (AMPs) with cell-penetrating peptides (CPPs) has shown promise in boosting antimicrobial potency, especially against Gram-negative bacteria. We examined the CPP-AMP interaction with distinct bacterial types based on cell wall differences. Our investigation focused on AMPs incorporating penetratin CPP and dihybrid peptides containing both cell-penetrating TAT protein fragments from the human immunodeficiency virus and Antennapedia peptide (Antp). Assessment of the peptides TAT-AMP, AMP-Antp, and TAT-AMP-Antp revealed their potential against Gram-positive strains ( Staphylococcus aureus , Methicillin-resistant Staphylococcus aureus (MRSA), and Bacillus cereus ). Peptides TAT-AMP and AMP-Antp using an amyloidogenic AMP from S1 ribosomal protein Thermus thermophilus , at concentrations ranging from 3 to 12 μM, exhibited enhanced antimicrobial activity against B. cereus . TAT-AMP and TAT-AMP-Antp, using an amyloidogenic AMP from the S1 ribosomal protein Pseudomonas aeruginosa , at a concentration of 12 µM, demonstrated potent antimicrobial activity against S. aureus and MRSA. Notably, the TAT-AMP, at a concentration of 12 µM, effectively inhibited Escherichia coli ( E. coli ) growth and displayed antimicrobial effects similar to gentamicin after 15 h of incubation. Peptide characteristics determined antimicrobial activity against diverse strains. The study highlights the intricate relationship between peptide properties and antimicrobial potential. Mechanisms of AMP action are closely tied to bacterial cell wall attributes. Peptides with the TAT fragment exhibited enhanced antimicrobial activity against S. aureus , MRSA, and P. aeruginosa. Peptides containing only the Antp fragment displayed lower activity. None of the investigated peptides demonstrated cytotoxic or cytostatic effects on either BT-474 cells or human skin fibroblasts. In conclusion, CPP-AMPs offer promise against various bacterial strains, offering insights for targeted antimicrobial development., Competing Interests: The authors declare no conflict of interest.