Adherence and gentamicin protection assays were employed to quantify the levels of adherence to and invasion into host cells. C. ureolyticus UNSWCD was able to adhere to the Caco-2 intestinal epithelial cell line with a value of 5.341% ± 0.74% but was not able to invade the Caco-2 cells.
The addition of two proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), to the cell line did not affect attachment or invasion, with attachment levels being 4.156% ± 0.61% (P = 0.270) for TNF-α and 6.472% ± 0.61% (P = 0.235) for IFN-γ. Scanning electron microscopy visually confirmed attachment and revealed that C. ureolyticus UNSWCD colonizes and adheres to intestinal cells, inducing cellular damage and microvillus degradation.
Purification and identification of the C. ureolyticus UNSWCD secretome detected a total of 111 proteins, from which 29 were bioinformatically predicted to be secretory proteins.
Functional classification revealed three putative virulence and colonization factors: the surface antigen CjaA, an outer membrane fibronectin binding protein, and an S-layer RTX toxin.
These results suggest that C. ureolyticus has the potential to be a pathogen of the gastrointestinal tract.