High-throughput transposon sequencing identifies HprR as a key regulator of the LEE operon and virulence in E. coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) comprises a prominent group of extracellular human pathogens that specifically colonize the colonic epithelium, leading to severe gastrointestinal diseases and representing a significant global public health threat. During infection, EHEC O157:H7-the most prevalent serotype - tightly adheres to intestinal epithelial cells and induces the formation of characteristic attaching and effacing (A/E) lesions. However, the molecular mechanisms governing this close interaction remain incompletely understood. In this study, we employed transposon-directed insertion-site sequencing (TraDIS) to conduct a genome-wide screen for genes essential for EHEC O157:H7 adherence. Among the identified candidates, disruption of hprR (encoding the response regulator of the HprS/HprR two-component system) resulted in markedly reduced epithelial colonization. Subsequent mechanistic analyses revealed that HprR positively regulates expression of genes within the locus of enterocyte effacement (LEE) pathogenicity island in a Ler-dependent manner, thereby promoting type III secretion system (T3SS) activity and epithelial colonization. Additionally, during luminal survival, HprR was found to directly bind to the promoter region of hiuH, a gene encoding a transthyretin-like protein, thereby enhancing bacterial resistance to oxidative stress and promoting persistence in the colonic lumen. Collectively, this study defines a comprehensive genomic framework for understanding EHEC O157:H7 colonization and identifies HprR as a dual-function regulatory protein that coordinates both epithelial adherence and luminal survival, thereby shaping host-pathogen interactions.