EruA, a Regulator of Adherent-Invasive E. coli, Enhances Bacterial Pathogenicity by Promoting Adhesion to Epithelial...

Adherent-invasive E. coli (AIEC) is closely related to inflammatory bowel disease (IBD). However, its pathogenic mechanism has not yet been fully elucidated. Using a BLASTP search, we discovered that the amino acid sequence of a putative protein (UFP37798.1) in the AIEC LF82 strain is highly homologous to some regulators in the SlyA family. We named it EruA. We displayed the secondary structures of EruA using bioinformatics, overexpressed the His₆-tagged EruA protein using SDS-PAGE, and dissected the genetic organization of the eruA chromosomal region using 5'RACE. We constructed an eruA deletion mutant (ΔeruA) and a complementary strain (CΔeruA) of the LF82 strain. The transcriptomes of wild-type (WT) and ΔeruA bacteria were compared using RNA sequencing and qRT-PCR, thereby identifying 32 differentially expressed genes (DEGs). Based on YASARA software and EMSA analysis, EruA directly binds to the consensus sequences (P_fimA and P_tnaB) in the promoter region of the fimA and tnaB genes from these DEGs. By using a super-resolution confocal microscope (SCM), counting CFUs of colonies on plates, indole quantification, and crystal violet staining of biofilms adhered to tubes or 96-well plates, we found that EruA activates the fimA to promote bacterial adhesion to intestinal epithelial cells and activates the tnaB to enhance bacterial indole production and biofilm formation. Moreover, EruA helps AIEC resist environmental stress and enhances bacterial survival within macrophages as well as loading in mouse tissues. Notably, EruA promotes AIEC colonization in the colons of mice and exacerbates intestinal inflammation caused by bacterial infection in mice with DSS-induced inflammatory colitis, manifested by weight loss, colon length shortening, and pathological changes in colon tissues. Therefore, EruA plays a key role in the pathogenicity of AIEC.