# Type VI secretion system degeneration accelerates intestinal epithelial cell death in Escherichia coli O157:H7

## TL;DR

The study shows that a degenerated protein secretion system in E. coli O157:H7 makes it more harmful by activating toxins that damage human cells.

## Contribution

The study reveals a novel evolutionary link between T6SS degeneration and prophage activation in enhancing EHEC virulence.

## Key findings

- Degeneration of the T6SS in EHEC increases cytotoxicity and epithelial cell death.
- T6SS degeneration correlates with Φstx2 prophage activation and Shiga toxin production.
- T6SS mutations are widespread in Φstx2-associated E. coli strains.

## Abstract

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.

Many Gram-negative bacterial pathogens can employ a type VI secretion system (T6SS) to exert their virulence. However, Escherichia coli O157:H7 (EHEC) T6SS has degenerated and exhibits higher cytotoxicity toward epithelial cells during infection. Our study demonstrates that the T6SS of EHEC has become significantly impaired compared to that of its ancestral strain, E. coli O55:H7. The degenerative T6SS made Shiga toxin 2-converting prophage (Φstx2) more sensitive to activation under host-derived reactive oxygen species (ROS) pressure, promoted Stx2 expression, and subsequently triggered other virulence factors such as the type III secretion system. The degenerative T6SS was widely distributed in Φstx2-associated E. coli, suggesting that T6SS degeneration might be closely related to Φstx2 integration during evolution. Overall, our findings reveal a novel mechanism regarding the role of the T6SS in virulence during the interactions between EHEC, prophages, and host cells.