Altered flagellin-TLR5 signaling pathway in interaction between host and laboratory Escherichia coli strains engineered for enhanced intestinal colonization in mouse

The intestinal microbiota is essential for maintaining intestinal homeostasis by regulating mucosal immune responses. Identifying bacterial traits that support stable host interactions is therefore crucial for developing next-generation mucosal probiotics. RNase T2 is a conserved ribonuclease with species-specific functions. In Escherichia coli, RNase T2 (RNase I) suppresses biofilm formation, a key process for intestinal colonization. We previously showed that an RNase I-deficient E. coli (Δrna) strain stably colonizes the mouse intestine. Here, we analyzed the immunogenic properties of the Δrna strain. Heat-killed Δrna induced an early IL-1β and TNF-α-skewed cytokine response in RAW264.7 macrophages, with reduced IL-6 expression compared to wild type. In mice, Δrna colonization caused no intestinal tissue injury but enhanced IL-6 and IL-22 expression in the cecum and colon. Selected TLR-specific reporter assays identified TLR5-dependent signaling driven by increased FliC expression. These findings show that RNase I deficiency modulates immune activation while maintaining non-pathogenic mucosal interactions.