Molecular switches meet multivalency: Antimicrobial activity of multimeric peptide assemblies from the pneumococcal LytA autolysin.

Peptidic molecular switches are amino acid sequences that alter their conformation in response to external stimuli. One such sequence (P4A, 14 amino acids) is derived from a choline-binding repeat of the LytA autolysin from the respiratory pathogen Streptococcus pneumoniae (pneumococcus) and is directly involved in the recognition of this aminoalcohol by the enzyme. P4A shifts from a native β-hairpin to an amphiphilic α-helix in the presence of detergent micelles or phospholipid vesicles via an unprecedented molecular switching mechanism. Here we explore the application of this structural transition to develop a novel antimicrobial strategy based on the hypothesis of peptides that adopt a defined structure in aqueous solution but switch to an alternative amphiphilic conformation near bacterial membranes, leading to membrane disruption. With this aim we designed and tested linear and dendritic assemblies of P4A as antimicrobial agents against S. pneumoniae. Linear P4A constructs increased pneumococcal membrane permeability to the SYTOX Green fluorescent probe up to 70% at 10 μM, causing premature release of endogenous LytA amidase and inducing autolysis. Moreover, multivalent P4A dendrimers showed markedly stronger bactericidal activity (up to 56,000-fold compared to linear peptides) due to enhanced multivalent interactions. Additionally, a P4A dendrimer was effective in a zebrafish model of antibiotic-resistant pneumococcal infection at a non-toxic, 2.7 nM concentration dose. These results underscore the potential of integrating structural switching and multivalency in next-generation antimicrobial design.