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Abstract
Helicobacter hepaticus (H. hepaticus) the prototype for enterohepatic Helicobacter species (EHS), colonizes the lower intestinal and hepatobiliary tract of mice and causes typhlocolitis, hepatitis, and hepatocellular carcinoma in susceptible mouse strains. Cytolethal distending toxin (CDT) is the only known virulence factor found in H. hepaticus; however, the role of CDT in disease is incompletely understood. CDT is a tripartite polypeptide complex of subunits A, B, and C and is expressed in many pathogenic Gram-negative bacteria. CDT is associated with irreversible double-stranded DNA breaks, cell cycle arrest and death of a wide range of cultured eukaryotic cells. We studied the kinetics of cell cycle arrest and apoptosis induced by H. hepaticus cytolethal distending toxin (HhCDT) in HeLa and INT407 cells, and here we reported the initiation of cell cycle arrest followed by apoptosis in HhCDT treated epithelial cells.
To determine the molecular basis and mechanism of H. hepaticus CDT-mediated cell death in INT407 cells, we studied the regulation of different signaling molecules in INT407 cells following exposure to the toxin. First we reported the elevation of phosphorylated H2AX level of INT407 cells in response to the toxin confirming in-vivo DNA double-stranded breaks. We observed elevated phospho-p53 and pro-apoptotic Bax protein levels after the treatment. Furthermore, cytochrome c was released from the mitochondria to the cytoplasm and caspase 3/7 and 9 were activated. These observations strongly suggest the involvement of mitochondrial mediated apoptosis in HhCDT-treated INT407 cells. Finally we observed an in-vitro PIP3 phosphatase activity of the recombinant HhCdtB subunit and down regulation of phosphorylated Akt levels in HhCDT-treated INT407 cells. These observations suggest the possible regulation of cellular activities such as cell cycle modulation and apoptosis via the Akt-mediated signaling pathways in HhCDT-treated cells. In conclusion our work improves the understanding of H. hepaticus CDT cellular toxicity and the cytotoxic signaling mechanism of CDT.
