LAUSR

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.