Molecular characterization of four <i>Helicobacter cetorum</i> strains from dolphins compared to human <i>Helicobacter pylori</i>

<i>Helicobacter</i> species colonize the stomachs of many aquatic and terrestrial mammals, including <i>Helicobacter pylori</i> in humans and <i>Helicobacter cetorum</i> in dolphins. There are several <i>H. cetorum</i> genome sequences in databases, but a detailed molecular characterization of these bacteria is missing. Here, we compared four <i>H. cetorum</i> isolates from dolphins with <i>H. pylori</i> 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 <i>cag</i> pathogenicity island of <i>H. pylori</i> is missing in <i>H. cetorum</i>. 3D-modeling revealed similar structures of hexameric VacA from both species with minor differences. <i>H. cetorum</i> VacA expression was associated with vacuole formation in epithelial cells similar to that of <i>s1/m2</i>, but not as strong as <i>H. pylori s1/m1 vacA</i> strains, and complementation of <i>H. pylori</i> with <i>H. cetorum vacA</i> restored the <i>s1/m2</i>-like VacA phenotype. While <i>H. pylori</i> infection robustly activated toll-like receptors TLR1, TLR2, TLR4, TLR5, TLR9, and TLR10, <i>H. cetorum</i> only stimulated TLR1/2, TLR4, and TLR10, but much less pronounced than <i>H. pylori</i>. Accordingly, infection of epithelial cells with <i>H. pylori</i> induced strong DNA damage, NF-κB activation, and IL-8 secretion, but these responses were barely detectable in <i>H. cetorum</i>-infected cells. Activation of only few TLRs and significantly weaker pro-inflammatory responses than <i>H. pylori</i> suggest that <i>H. cetorum</i> is a commensal or only moderately virulent pathobiont in the stomach of dolphins, comparable to the less pathogenic <i>cag</i>PAI-negative <i>H. pylori</i> strains in humans. Since <i>H. cetorum</i> is evolutionarily older than <i>H. pylori</i>, we propose that <i>H. cetorum</i> represents a direct ancestor of <i>H. pylori</i> that arose after a host jump over 623,000 years ago, which is the coalescence time of the two species.