Molecular characterization of four Helicobacter cetorum strains from dolphins compared to human Helicobacter pylori

Helicobacter species colonize the stomachs of many aquatic and terrestrial mammals, including Helicobacter pylori in humans and Helicobacter cetorum in dolphins. There are several H. cetorum genome sequences in databases, but a detailed molecular characterization of these bacteria is missing. Here, we compared four H. cetorum isolates from dolphins with H. pylori strains using electron microscopy as well as structural and functional analyses. All strains expressed similarly high urease activity and were hemolytic to erythrocytes. Western blots revealed conserved expression of flagellin-A, neutrophil-activating protein NapA, serine protease HtrA, γ-glutamyl-transpeptidase GGT and toxin VacA. In contrast, the virulence-associated cag pathogenicity island of H. pylori is missing in H. cetorum. 3D-modeling revealed similar structures of hexameric VacA from both species with minor differences. H. cetorum VacA expression was associated with vacuole formation in epithelial cells similar to that of s1/m2, but not as strong as H. pylori s1/m1 vacA strains, and complementation of H. pylori with H. cetorum vacA restored the s1/m2-like VacA phenotype. While H. pylori infection robustly activated toll-like receptors TLR1, TLR2, TLR4, TLR5, TLR9, and TLR10, H. cetorum only stimulated TLR1/2, TLR4, and TLR10, but much less pronounced than H. pylori. Accordingly, infection of epithelial cells with H. pylori induced strong DNA damage, NF-κB activation, and IL-8 secretion, but these responses were barely detectable in H. cetorum-infected cells. Activation of only few TLRs and significantly weaker pro-inflammatory responses than H. pylori suggest that H. cetorum is a commensal or only moderately virulent pathobiont in the stomach of dolphins, comparable to the less pathogenic cagPAI-negative H. pylori strains in humans. Since H. cetorum is evolutionarily older than H. pylori, we propose that H. cetorum represents a direct ancestor of H. pylori that arose after a host jump over 623,000 years ago, which is the coalescence time of the two species.