Harnessing chirality for enhanced stability: An all-D protease resistant antimicrobial peptide active against drug-resistant skin bacteria.

The increasing prevalence of antimicrobial resistance has driven the search for new therapeutic alternatives to conventional antibiotics. Here, we improved the proteolytic stability of the archaeal antimicrobial peptide VLL28 by bioinformatically guiding sequence optimization, integrating truncation, charge-modifying mutations, and chirality inversion. Four variants, i.e. VLL28 and its shortened derivative KRK23, each in L- and D-form, were assessed for antibacterial activity, membrane interactions and serum stability. All peptides retained potent, low-micromolar activity, and were found to be able to permeabilize the bacterial outer-membranes as well as depolarize the cytoplasmic membranes. In addition, only D-enantiomers resisted proteolysis. Among them, the truncated all-D derivative, (D)KRK23, exhibited the most balanced overall profile, combining serum stability, broad-spectrum antimicrobial potency and negligible hemolytic activity. (D)KRK23 also inhibited biofilm formation at sub minimum inhibitory concentrations (MIC) and reduced bacterial survival in keratinocyte infection models involving both antibiotic-sensitive and -resistant bacteria. In addition, it modulated host innate immune response, affecting the expression of the proinflammatory cytokine CXCL8 and of endogenous antimicrobial peptides (AMPs) produced by keratinocytes. Collectively, these results identify (D)KRK23 as a serum-stable multifunctional AMP active against antibiotic-resistant skin pathogens.