A key step in enteropathogenic Escherichia coli (EPEC) infection of intestinal cells involves a Tir-induced actin reorganization. Nck mediates this event by binding with WIP through its second SH3 domain (Nck-SH3.2). Recently we have developed a preventative antibacterial mechanism that safeguards intestinal cells by shutting down this intracellular signal through a site-selective covalent peptide-protein reaction, a new antibacterial strategy that acts on the host cells instead of bacterium cells. Here we present the experimental details of the design and synthesis of cysteine-reactive peptides to selectively block Nck-SH3.2 but not the other two SH3 domains. Procedures of EPEC infection, covalent reaction inside Caco-2 cells, and bacterial counting to check the antibacterial effect are also described.
Keywords: Bacterial infection; Covalent peptide inhibitor; Cysteine; EPEC; Nck; Protein–protein interaction; Site-specific protein reaction.
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.