| Enteric Campylobacter (ECS) and Helicobacter (EHS) species produce a novel family of bacterial toxins called cytolethal distending toxin (CDT). CDT consists of a heterotrimeric complex of CdtA, CdtB, and CdtC sub-units encoded by the corresponding cdtA , cdtB, and cdtC genes. Although CdtB, the structural toxin, has in vitro nuclease activity similar to mammalian deoxyribonuclease I (DNase I) and causes cell cycle arrest and apoptotic death of eukaryotic cells, the molecular mechanisms of toxicity are incompletely understood. We characterized the cdtB genes and cytotoxic activities of ECS and EHS isolated from humans, macaque monkeys, and pigs. The molecular mechanisms of cellular toxicity of CdtB from the prototypes ECS, C. jejuni and EHS, H. hepaticus were investigated in detail using recombinant fusion proteins and transfection of cultured cell lines.; Epithelial HeLa and INT407 cells incubated with CdtABC holotoxin of C. jejuni, C. hyointestinalis, and H. hepaticus , but not with holotoxin from C. coli, display characteristic cytoplasmic and nuclear enlargements together with G2/M cell cycle arrest and induction of apoptosis. Optimal conditions for in vitro dsDNA digestion were established by developing a novel quantitative fluorometric assay using native and recombinant H. hepaticus CdtB digestion of plasmid and eukaryotic DNA targets. Although the Ca2+- and Mg2+-dependence and neutral properties of CdtB were similar to DNase I, hydrolysis of DNA by CdtB was approximately 100-fold less active and considerably more resistant to inhibition by ZnCl2 and actin, suggesting functional differences between the enzymes. HeLa cells either constitutively expressing each sub-unit or transiently transfected with full-length, N-, or C-terminal truncated H. hepaticus CdtB-green fluorescent protein fusions revealed, for the first time, that nuclear localization of EHS CdtB alone is sufficient for cellular intoxication. Simultaneous quantitative analysis of cell cycle and apoptotic markers of INT407, HeLa, and monocyte/macrophage J774A.1 cell lines intoxicated with holotoxin or recombinant CdtB of H. hepaticus and C. jejuni revealed that apoptosis preceded cell cycle arrest, suggesting distinct effector pathways for each activity. The data provide new insights into the molecular mechanisms of a family of nuclease toxins produced by several important bacterial pathogens of humans and animals. |
