Pathogen-enriched transcriptional regulators modulate biofilm formation and virulence in Escherichia coli O157:H7.
Pathogenic Escherichia coli O157:H7 encodes numerous lineage-enriched transcriptional regulatory proteins that are absent or poorly conserved in non-pathogenic strains, yet their functions in transcriptional programming and biofilm-associated virulence remain largely uncharacterized. Here, we identified eight previously unreported biofilm-associated regulatory proteins involved in biofilm formation in enterohemorrhagic E. coli (EHEC) O157:H7 via comparative genomic analysis. Conserved domain analysis revealed that these regulators belong to diverse DNA-binding protein families, including LysR-, GntR-, LuxR-, AraC/XylS-, Cro/CI-, and Ogr/Delta-like families. Among these, two regulators (ECs_2620 and ECs_4457) were confirmed as direct DNA-binding transcriptional regulators through in vitro and in vivo DNA-binding assays. Functional analyses showed that overexpression of these regulators enhanced biofilm formation, whereas gene deletion impaired biofilm development and reduced bacterial virulence in both THP-1 cell and mouse infection models. Transcriptomic analysis revealed that these regulators primarily modulate genes involved in bacterial chemotaxis and flagellar assembly, representing a regulatory framework distinct from the classical c-di-GMP-centered regulatory paradigm described in non-pathogenic E. coli. Furthermore, we identified that the specific DNA-binding motifs of two regulators with direct in vitro DNA-binding activity. Evolutionary analysis showed that these regulators are highly conserved among pathogenic E. coli lineages, including EHEC, enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC), but are absent or truncated in non-pathogenic E. coli K-12 strains. Collectively, this study characterizes a set of pathogen-enriched transcriptional regulatory proteins and provides new insights into the macromolecular regulatory mechanisms underlying biofilm formation and virulence in pathogenic E. coli.