EruA, a Regulator of Adherent-Invasive E. coli, Enhances Bacterial Pathogenicity by Promoting Adhesion to Epithelial Cells and Survival Within Macrophages

Adherent-invasiveE. 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 theeruAchromosomal region using 5′RACE. We constructed aneruAdeletion 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 (PfimAand PtnaB) in the promoter region of thefimAandtnaBgenes 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 thefimAto promote bacterial adhesion to intestinal epithelial cells and activates thetnaBto 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.