# An invasive phenotype induced by relaxation of DNA supercoiling in Campylobacter jejuni triggers disruption of tight junctions of intestinal spheroids

## TL;DR

Relaxing DNA supercoiling in Campylobacter jejuni makes it more invasive, disrupting intestinal cell junctions and suggesting a new route of infection.

## Contribution

The study identifies a novel invasive phenotype in C. jejuni linked to DNA supercoiling relaxation and paracellular invasion.

## Key findings

- Relaxed DNA supercoiling increases C. jejuni invasion and tight junction disruption in epithelial cells.
- Microtubule inhibition rescues β-catenin disruption, indicating microtubules are key targets for C. jejuni virulence.
- SI C. jejuni penetrates 3D spheroids but does not significantly disrupt their overall structure.

## Abstract

The relaxation of DNA supercoiling in Campylobacter jejuni leads to increased protein secretion and a more invasive phenotype, but little is known about the specific mechanisms involved. The aim of this study was to elucidate how these induced bacteria interact with epithelial cells to mediate invasion using different cell models. In HT29 epithelial cell monolayers, pre-treatment of C. jejuni with novobiocin to relax DNA supercoiling significantly increased bacterial association and invasion, forming clusters at cell junctions. This invasive phenotype, which we term C. jejuni supercoiling induced (SI), led to marked disruption of tight junctions (TJs) and adherens junctions, as evidenced by the loss of occludin and β-catenin signal during infection. In a 3D spheroid model, C. jejuni (SI) displayed increased association with and penetration into the centre of spheroids, although significant disruption of their integrity was not observed. Further investigation revealed that cytoskeletal dynamics play a pivotal role in this process; inhibition of microtubule polymerization, but not actin polymerization, rescued the β-catenin disruption induced by C. jejuni (SI), highlighting microtubules as key targets for C. jejuni virulence. This study reveals that SI invasion by C. jejuni is associated with the disruption of TJs, suggesting a paracellular route of invasion.