Global expression profile of enterohemorrhagic Escherichia coli O157:H7 in phagosome of murine macrophages.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes foodborne outbreaks leading to hemolytic uremic syndrome (HUS) via Shiga toxin (Stx). During HUS pathogenesis, EHEC O157:H7 is phagocytized by intestinal macrophages, although only a small proportion of internalized bacteria survive within the phagosome. To investigate this persistence, we performed global RNA-seq analysis of intracellular EHEC O157:H7 within murine macrophage phagosomes, complemented by in vitro assays simulating key phagosomal stressors. Our results reveal that EHEC employs a sophisticated survival strategy characterized by metabolic reprogramming and stress resistance. Inside macrophages, EHEC activates the SOS pathway, synthesizes membrane lipids, and engages NO detoxification mechanisms while repressing energetically costly virulence systems -motility, Type 3 Secretion System (T3SS), and Type 6 Secretion System (T6SS)-to prioritize survival. Notably, Shiga toxin gene regulation proved highly context-dependent: phagocytosis selectively upregulated the stx2a A subunit via the SOS response, whereas stx1a remained uninduced, likely restrained by NO-mediated repression. In contrast, under in vitro multi-stress conditions (pH 4.5) engaged the AR2-AR5 acid resistance systems and triggered robust expression of both stx1a subunits (A and B). These findings highlight the dynamic, stress-specific regulatory networks EHEC relies on to persist within the host.