Cellular responses to the DNA damaging Cytolethal Distending Toxin

Abstract: Cytolethal distending toxin (CDT) is a genotoxin, which belongs to a group of bacterial protein toxins called cyclomodulins. These are characterized by their interference with the eukaryotic cell cycle. CDT causes DNA damage, which induces cell cycle arrest and apoptosis. The active holotoxin consists of three subunits CdtA, CdtB and CdtC, where CdtB is the active subunit and has structural and functional similarities with DNase I. We demonstrated that CDT uses the same internalization pathway as several other bacterial toxins do, such as cholera toxin and Shiga toxin. The binding on the plasma membrane is dependent on cholesterol. The toxin is internalized via the Golgi complex, and retrogradely transported to the endoplasmic reticulum (ER) and found in the nucleoplasmic reticulum. The translocation from the ER to the nucleus does not require either the ER-associated (ERAD) pathway or the Derlin-1 protein. Additionally, we showed that CDT is not farnesylated, a modification known to occur in the cytosol. In contrast, to other AB toxins, CdtB was demonstrated to have heat-stable properties and is not degraded by the 20S proteasome. All these evidence suggest that the toxin is translocated directly from the ER to the nucleus. In adherent cells the cellular response to the CDT-induced DNA damage involved activation of the RhoA GTPase. We showed that the RhoA-specific Guanine nucleotide exchange factor (GEF) Net1 is dephosphorylated and translocated from the nucleus to the cytosol upon DNA damage. Knock down of Net1 by RNAi prevents RhoA activation, inhibits the formation of stress fibers, and enhances cell death. This indicates that Net1 activation is required for RhoAmediated response to genotoxic stress. The Net1 and the RhoA dependent signals converge the activation of mitogen-activated protein kinase p38 (p38 MAPK) and its downstream target MAPK-activated protein kinase 2 (MK2). To further investigate this novel cell survival pathway in response to CDT we screened a yeast deletion library for CdtB-sensitive strains. Approximately 4500 yeast deletion strains were transformed with a plasmid containing CdtB. The screen shows that 78 mutated strains were hypersensitive to CdtB. Twenty of the human ortholog genes were found to interact with the actin cytoskeleton regulation network. Our analysis focused on TSG101, FEN1 and Vinculin (VCL). We demonstrated that they are all required to induce actin stress fiber formation in response to DNA damage. FEN1 and VCL also regulate the RhoA GTPase and p38 MAPK activation, and delay cell death in response to CDT intoxication. In response to DNA damage, Ataxia-telangiectasia mutated (ATM) and ATM and Rad-3-related kinases (ATR) are activated and orchestrate DNA damage response. The transcription factor Myc has multi-functions such as inducing apoptosis in response to DNA damage. The Mycregulated effectors acting upstream of the mitochorial apoptotic pathway are still unknown. We demonstrated that Myc is required for activation of the ATM-dependent DNA damage checkpoint response in cells exposed to ionizing radiation or CDT. Activation of ATM effectors, such as histone H2AX and the nuclear foci formation of the Nijmegen Berakage Syndrome (Nbs)1 protein, were abolished in the absence of Myc. The cellular response to UV irradiation, known to activate an ATR-dependent checkpoint, was not delayed in the absent of the Myc expression. This data demonstrate that Myc is required for activation of the ATM-dependent pathway. Our studies highlight the importance of understanding the CDT biology and its mode of action. This knowledge could provide new tools to elucidate the putative involvement of bacteria in carcinogenesis.