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 ...