Isopeptide-Blocker Impairs the Mechanics of Recently Translated Pilin Proteins

ABSTRACT Gram-positive bacteria, such as Streptococcus pyogenes, use their adhesive pili to attach to host cells during early stages of a bacterial infection. These extracellular hair-like appendages experience mechanical stresses of hundreds of picoNewtons; however, the presence of an internal isopeptide bond prevents the protein from unfolding. Here, we designed a peptide to intervene in the formation of the isopeptide bond on the pilin Spy0128 from Streptococcus pyogenes, preventing folding and rendering the Spy0128 susceptible to protease digestion. By using a combination of protein engineering and single-molecule force spectroscopy, we demonstrate that the isopeptide-blocker peptide interferes with the formation of the isopeptide bond. While the intact Spy0128 is inextensible under mechanical forces, the intervened Spy0128 is completely extensible and lacks of mechanical stability. We propose that this isopeptide-blocker affords a novel strategy for mechanically-targeted antibiotics which, by blocking the folding structure of bacterial pili, could prevent the colonization of infectious microorganisms.