Helicobacter hepaticus Cytolethal Distending Toxin Causes Cell Death in Intestinal Epithelial Cells via Mitochondrial Apoptotic Pathway

Abstract

Helicobacter hepaticus, the prototype for enterohepatic Helicobacter species, colonizes the lower intestinal and hepatobiliary tracts 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. CDT of several Gram-negative bacteria is associated with double-stranded DNA breaks resulting in cell cycle arrest and death of a wide range of eukaryotic cells in vitro. We previously observed H. hepaticus CDT (HhCDT) mediated apoptosis in INT407 cells. However, the exact mechanism for the induction of the apoptotic pathway by HhCDT is unknown. The objective of this study was to identify the apoptotic signaling pathway induced by HhCDT in INT407 cells. INT407 cells were incubated with or without recombinant HhCDT for 0-72 hours. H2AX phosphorylation and apoptotic parameters were analyzed. H2AX was phosphorylated 24 hours postexposure to HhCDT. Expression of pro-apoptotic Bax protein was upregulated after 24 hours, while Bcl(2) expression decreased. Cytochrome c was released from mitochondria after 12-24 hours of exposure. Concurrently, caspase 3/7 and 9 were activated. However, pretreatment of INT407 cells with caspase inhibitor (Z-VAD-FMK) inhibited the activation of caspase 3/7 and 9. Significant activity of caspase 8 was not observed in toxin treated cells. Activation of caspase 3/7 and caspase 9 confirms the involvement of the mitochondrial apoptotic pathway in HhCDT-treated cells. These findings show, for the first time, the ability of HhCDT to induce apoptosis via the mitochondrial pathway.