E. coli strains harboring Stx2 prophages.

The type VI secretion system (T6SS) is a specific protein secretion apparatus that contributes to bacterial virulence. Enterohemorrhagic Escherichia coli O157:H7 (EHEC) harbors multiple prophages and can cause severe human diseases worldwide. Here, we compared the EHEC T6SS main gene cluster with its ancestral strain E. coli O55:H7 (aEPEC) and predicted 26 mutation loci in protein-coding regions. Sequence analysis of these mutation loci indicated a degenerative trend in T6SS function in EHEC. Notably, a 28-bp tandem repeat insertion in the T6SS core gene tssM significantly compromised T6SS secretion activity. Inactivation of the T6SS significantly enhanced EHEC cytotoxicity and accelerated epithelial cell death. Mechanistically, inactivation of T6SS promotes EHEC Stx2-converting prophage (Φstx2) expression, and deletion of Φstx2 weakens the T6SS-deficient strain’s cytotoxicity. Analysis of EHEC evolutionary path revealed that tssM mutation may occur after Φstx2 integration, and this mutation is widely distributed in E. coli bearing Φstx2 (E. coliΦstx2), suggesting T6SS degeneration may be closely associated with Φstx2 integration in E. coliΦstx2. Crucially, degenerative T6SS could render Φstx2 more sensitive to activation, and in turn activate EHEC major virulence factors such as Shiga toxin and type III secretion system. Taken together, our findings suggest that the ancestral aEPEC strain acquired Φstx2 and underwent T6SS degeneration, ultimately evolving into a highly cytotoxic EHEC lineage.