The rise and spread of antibiotic-resistant bacteria, which can undergo a profound change in their biology by secreting extracellular polymeric substance on a surface (e.g., catheters, implants, etc.) to form biofilms that presumably allow microbial cells to survive in hostile environments, leading to unbeatable infections, is a serious threat to public health. Nowadays, peptide-based molecules are good models and provide excellent tools for a wide range of bioengineering and biomedical applications. Here, we describe the design of short heptad-based amphiphiles that is inherently antimicrobial and can eradicate bacterial biofilm on contact. The heptad regions of the designed amphiphiles contain hydrophobic residues at "a" and "d" positions and cationic residues in other positions of a heptad such that in the folded state, one face of the helix is hydrophobic and the opposing face is positively charged, presenting an amphiphilic structure. The introduction and location of tryptophan residue, as the linker, in the middle position of paired heptads was to enable the peptide's membrane-seeking ability and facilitate the amphiphile to form defined structures. We find that the designed amphiphilesshow strong helical rigidity under both aqueous and membrane-mimicking environments, by comparing the molecular mean residual ellipticity values of the peptides at 222 nm wavelength, e.g., [θ]_(222) for LW3 of-20,450 in PBS compared with -27,413 in SDS, while [θ]_(222) for Melittin of-6,198 in PBS compared with -30,121 in SDS. Results from antimicrobial assays demonstrate that the amphiphiles are strong active against a series of P. aeruginosa, including clinical and drug-resistant strains. Among these amphiphiles, the peptides with Leu residues at "a" and "d" positions and longer linkers have excellent bacteriostatic activity and salt stability. Optimized amphiphiles also exhibit excellent selectivity for the microbial membrane, reduce sessile biofilm biomass, and neutralize endotoxins. Fluoroscopic and microscopic assays further demonstrate that these amphiphilespermeabilize the bacterial membrane and disrupt membrane integrity. In conclusion, the new series of heptad-based amphiphiles described here can be used to develop therapeutic agents that have potent activity against P. aeruginosa, while being nontoxic to mammalian cells, and can be applied to anti-infective treatments to inhibit potential infections and biofilm formation.
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