Refinement statistics.

Translocation of Campylobacter concisus from the oral cavity to the intestinal tract is increasingly recognised as a contributor to inflammatory bowel disease (IBD). The C. concisus secreted protein Csep1 has emerged as a molecular marker of C. concisus strains associated with Crohn’s disease, a form of IBD. However, its structure and role in inflammation remain unknown. Here, we report the X-ray crystal structure of plasmid-encoded Csep1P that reveals a unique α-helical fold with structural similarity to Helicobacter pylori cysteine-rich proteins HcpB and HcpC. Because HcpA, another Hcp family member, is known to affect monocyte differentiation, this structural similarity led us to hypothesise that Csep1P may modulate monocyte differentiation and macrophage function. Transcriptomic analysis revealed that Csep1P induced a chemokine-dominant inflammatory state in macrophages, M1-chem. Protein-level validation in both THP-1-derived and primary human macrophages confirmed this selective chemokine response. While Csep1P alone did not upregulate proinflammatory cytokines, THP-1-derived macrophages pre-incubated with Csep1P produced a higher level of proinflammatory cytokines in response to commensal Escherichia coli, which was validated on primary human macrophages. Furthermore, silencing the delta like canonical notch ligand 4 (DLL4) gene decreased the proinflammatory response of Csep1P-mediated macrophages to E. coli. Collectively, our data demonstrate that the structurally unique Csep1P reprograms macrophage response, which provides a mechanistic link between C. concisus infection and Crohn’s disease pathogenesis, and identifies Csep1P as a potential target for therapeutic intervention.