The cytolethal distending toxin effects on Mammalian cells: a DNA damage perspective

Abstract

The cytolethal distending toxin (CDT) is produced by many pathogenic Gram-negative bacteria and is considered as a virulence factor. In human cells, CDT exposure leads to a unique cytotoxicity associated with a characteristic cell distension and induces a cell cycle arrest dependent on the DNA damage response (DDR) triggered by DNA double-strand breaks (DSBs). CDT has thus been classified as a cyclomodulin and a genotoxin. Whereas unrepaired damage can lead to cell death, effective, but improper repair may be detrimental. Indeed, improper repair of DNA damage may allow cells to resume the cell cycle and induce genetic instability, a hallmark in cancer. In vivo, CDT has been shown to induce the development of dysplastic nodules and to lead to genetic instability, defining CDT as a potential carcinogen. It is therefore important to characterize the outcome of the CDT-induced DNA damage and the consequences for intoxicated cells and organisms. Here, we review the latest results regarding the host cell response to CDT intoxication and focus on DNA damage characteristics, cell cycle modulation and cell outcomes.

Figure 1

Cytolethal distending toxin (CDT) internalization and trafficking. Depending on the bacteria, CDT may be secreted freely, into outer membrane vesicles (OMVs) or, in the particular case of Salmonella typhi, into intracellular vesicles. In the case of a CDT extracellular secretion, CdtA and CdtC are involved in the toxin binding to the eukaryotic membrane. Once bound, CdtA remains associated with the membrane, while CdtC and CdtB are internalized, with CdtB being relocated to the nucleus by a retrograde transport pathway, via early and late endosomes. This has been demonstrated using inhibitors, such as methyl-β-cyclodextrin, methylamine, bafilomycin A1, BFA, etc. For OMV-secreted CDT, the toxin is internalized into the host cell through the OMV fusion with the eukaryotic membrane. CdtB is relocated to the nucleus by an undescribed pathway (dotted arrow), and the CdtA and CdtC outcome in the cytoplasm is still unknown. The typhoid toxin production requires S. typhi internalization into the host cell; thereafter, the toxin must be secreted to be active. The typhoid toxin interacts with the eukaryotic membrane and is endocytosed, and CdtB is relocated to the nucleus.

Figure 2

Activation and crosstalk between the ATM and ATR pathways. (a) Pathway activation at the double-stranded break (DSB). DSB formation induces the MRN-dependent ATM recruitment. ATM then phosphorylates numerous substrates, including itself and H2AX near the DSB site. DSB repair occurs through non-homologous end joining (NHEJ) or homologous recombination (HR) mechanisms (see Section 3.2 for details). During HR, resection of the DSB extremities produces ssDNA stretches that are coated by RPA, leading to ATRIP-dependent ATR recruitment and activation. (b) Pathway activation during replicative stress. Replication fork (RF) stalling induces ssDNA formation that is rapidly coated by RPA. This structure is recognized by ATRIP, which drives the recruitment and activation of ATR. Besides phosphorylating its other substrates, ATR locally phosphorylates H2AX. RF can restart in case of transient stress or collapse after prolonged stresses. RF collapse induces DSB formation and ATM pathway activation, leading to a second and more important wave of H2AX phosphorylation.

Figure 3

The activation of the DNA damage response upon CDT exposure. This picture depicts the DDR molecular events induced after CDT intoxication. The dotted lines represent well-studied events occurring during the DDR, but not yet demonstrated in the context of CDT exposure. CdtB-induced DNA lesions are detected by sensors, such as the MRN complex and RPA, resulting in the recruitment and the activation of the PI3K related kinases, ATM and ATR. ATM and ATR then phosphorylate hundreds of substrates, including H2AX, CHK1, CHK2 and p53 (black arrows). This signaling cascade results in the regulation of cell cycle modulators (blue lines), through the inhibition of CDC25C phosphatase by CHK2 and, possibly, CHK1. Phosphorylated CDC25C is unable to activate the cyclin B/CDK1 complex (red crosses), essential for mitotic entry. Moreover, the p53-dependent accumulation of p21 blocks cells in G1 by inhibiting the CDK2/cyclin E complex. At the same time, the DSB repair mechanisms (NHEJ and HR) are activated by ATM and ATR (red arrows). However, if the level of DNA lesions is too severe, the process of cell death is initiated (green arrows). Apoptotic cell death can be induced by an intrinsic pathway involving p53 activation, leading to an increase in the BAX level, the sequestration and inactivation of Bcl-2, the mitochondrial release of cytochrome C and caspase 9 activation. Apoptosis can also be induced through the activation of the extrinsic pathway, involving caspase 8 activation. In both cases, this leads to caspase 3 activation and apoptotic cell death.

Figure 4

A model of CDT-mediated DSB formation and repair. (A) Direct DSB formation with high doses of CDT. CdtB induces a large number of nicks on the host cell DNA. When two closed nicks face each other on opposite strands (dashed rectangle), they directly create a DSB that is sensed by MRN and signaled by ATM, resulting in H2AX phosphorylation and 53BP1 recruitment. This lesion will be repaired by NHEJ or HR. (B) Low doses of CDT induce S-phase-associated DSB. At low doses, the number of nicks is not large enough to produce direct DSBs. However, if these nicks are left unrepaired or if CdtB induces nicks during the S-phase (hatched CdtB), these lesions will block RF progression, resulting in ssDNA accumulation. ssDNA is then coated by RPA, leading to ATR activation. Then, the RF collapses, resulting in DSB formation and ATM pathway activation. RAD51 is recruited in order to restart the RF through HR repair.