Disabling iron uptake and pilus assembly in uropathogenic Escherichia coli using CRISPR-Cas9: a step toward antivirulence therapy

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), driven by virulence factors such as iron acquisition systems and adhesive pili. In this study, we employed CRISPR-Cas9-mediated genome editing to functionally inactivate two critical virulence genes— iucD , involved in aerobactin-mediated iron uptake, and papC , encoding the outer membrane usher protein essential for P pilus assembly. Using a clinical UPEC isolate, we introduced premature stop codons via homologous repair templates guided by gene-specific single-guide RNAs. Colony PCR and Sanger sequencing confirmed precise site-specific editing, leading to truncated protein variants. In silico analyses using InterPro and Swiss-Model revealed a complete loss of essential domains in both proteins. Molecular docking studies demonstrated a marked reduction in binding affinities of truncated IucD for NAD(P)H and impaired protein-protein interaction between truncated PapC and PapG. This study highlights the utility of CRISPR-Cas9 as a powerful tool for dissecting bacterial pathogenesis and supports the potential of targeting virulence determinants like iucD and papC as part of an antivirulence strategy for managing UPEC infections.