Contribution of Helicobacter hepaticus Cytolethal Distending Toxin Subunits to Human Epithelial Cell Cycle Arrest and Apoptotic Death in vitro

Abstract

Cytolethal distending toxin (CDT) is the only known virulence factor found in H.hepaticus, the cause of chronic typhlocolitis and hepatitis leading to colonic and hepatocellular carcinomas in mice. Interaction of the tripartite polypeptide CdtA, CdtB, and CdtC subunits produced by H.hepaticus CDT (HhepCDT) causes cell cycle arrest and apoptotic death of cultured cells; however, the contribution of individual subunit to these processes has not been investigated. The temporal relationship between cell cycle and apoptotic death of human epithelial HeLa and INT407 cells intoxicated with HhepCDT holotoxin or reconstituted recombinant HhepCDT was compared by flow cytometry. The genotoxic activity of individual and combinations of recombinant HhepCDT protein subunits or increasing concentrations of individual recombinant HhepCDT protein subunits transfected into HeLa cells was assessed at 72hours post-treatment by flow cytometry. Similar time course of HhepCDT-induced G2 /M cell cycle arrest and apoptotic death was found with both cell lines which reached a maximum at 72hours. The presence of all three HhepCDT subunits was required for maximum cell cycle arrest and apoptosis of both cell lines. Transfection of HeLa cells with HhepCdtB, but not with HhepCdtA or HhepCdtC, resulted in a dose-dependent G2 /M arrest and apoptotic death. All three subunits of HhepCDT are required for maximum epithelial cell cycle arrest and progression to apoptotic death, and HhepCdtB subunit alone is necessary and sufficient for epithelial cell genotoxicity.