# O‑Island 28 encodes a type I secretion and RTX adhesion system regulated by RstA and required for early EHEC O157:H7 adherence

## TL;DR

This study identifies O-Island 28 as a key genomic region in EHEC O157:H7 that helps the bacteria stick to and colonize the gut, and its activity is controlled by calcium and a protein called RstA.

## Contribution

The study reveals OI-28 as a calcium-responsive, RstA-activated type I secretion system essential for EHEC adherence and colonization.

## Key findings

- Deletion of OI-28 reduces epithelial adherence and intestinal colonization in mice.
- OI-28 is activated by RstA, which binds directly to its regulatory region.
- Calcium enhances OI-28 expression and T1SS-dependent adherence.

## Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a leading foodborne pathogen worldwide that causes severe diarrheal disease, hemorrhagic colitis, and hemolytic uremic syndrome, representing a significant threat to public health. O-islands are discrete genomic regions absent from nonpathogenic E. coli K-12 but present in EHEC O157:H7, many of which correspond to or overlap with pathogenicity islands (PAIs) that contribute to virulence. Among these O-islands, O‑Island 28 (OI‑28) in EHEC O157:H7 is a conserved genomic island predicted to encode a complete type I secretion system (T1SS) and two RTX family proteins, but its role in pathogenesis has remained unclear. Here, we show that deletion of OI‑28 markedly reduces epithelial adherence and intestinal colonization in mice without affecting in vitro growth. Mechanistically, OI‑28 is activated by the response regulator RstA, and RstA binds directly to the OI‑28 regulatory region. Consistent with the calcium-dependent folding of RTX adhesins, extracellular Ca2+ enhances OI‑28 expression and T1SS-dependent adherence in an RstA-dependent manner, and dietary calcium depletion reduces early colonization in vivo. Comparative genomics further demonstrated that OI‑28 is required for the colonization of multiple pathogenic E. coli strains. Collectively, these findings demonstrate that OI‑28 is an RstA‑activated, calcium‑responsive T1SS secretion system that is conserved across pathogenic E. coli strains and is essential for efficient epithelial adherence and early intestinal colonization.