Haemophilus Ducreyi Cytolethal Distending Toxin Research Articles

The fate of the CdtA subunit from the Haemophilus ducreyi cytolethal distending toxin.

Cytolethal distending toxin (CDT) is a virulence factor produced by many Gram-negative bacteria, including Haemophilus ducreyi, the causative agent of genital cancroid. CDT is a heterotrimeric toxin consisting of a cell-binding domain (CdtA + CdtC) and a catalytic domain (CdtB) that has DNase activity. After binding to the host plasma membrane, CDT undergoes endocytosis and travels to the endoplasmic reticulum (ER). Only then does CdtB move into the nucleus, causing DNA damage that induces cell-cycle arrest and apoptosis. The previous CDT trafficking model suggested that CdtA remains on the plasma membrane while the CdtB/CdtC heterodimer is transported inside the cell. This model is based on experiments that were unable to detect CdtA inside the host cell. Here, we examine this model and demonstrate that CDT is internalized as an intact holotoxin. Furthermore, the acidification of the endosomes induces CdtA release from the CdtB/CdtC heterodimer. Using a cell-based ELISA, we report that CdtA facilitates CDT binding to the plasma membrane and demonstrate that nearly the entire pool of surface-bound toxin is internalized from the plasma membrane within 20 minutes. As determined by Western blot, all of internalized CdtA and most of internalized CdtB and CdtC are rapidly degraded in the lysosomes. Acidic conditions found in the early endosomes (pH 6.0-6.3) led to a loss of protein structure in CdtA and its release from the CDT holotoxin as determined by circular dichroism and surface plasmon resonance, respectively. These results demonstrate that CDT is internalized as an intact holotoxin, with the acidic environment of endosomes triggering the separation of CdtA from the CdtB/CdtC heterodimer.

Acid-induced disassembly of the Haemophilus ducreyi cytolethal distending toxin

The cytolethal distending toxins (CDTs) produced by many Gram-negative pathogens are tripartite genotoxins with a single catalytic subunit (CdtB) and two cell-binding subunits (CdtA + CdtC). CDT moves by vesicle carriers from the cell surface to the endosomes and through the Golgi apparatus en route to the endoplasmic reticulum (ER). CdtA dissociates from the rest of the toxin before reaching the Golgi apparatus, and CdtB separates from CdtC in the ER. The free CdtB subunit, which is only active after holotoxin disassembly, then crosses the ER membrane and enters the nucleus where it generates DNA breaks. We hypothesized that the acidified lumen of the endosomes is responsible for separating CdtA from the CdtB/CdtC heterodimer. To test this prediction, possible acid-induced disruptions to the CDT holotoxin were monitored by size exclusion chromatography and surface plasmon resonance. We found that CDT could not efficiently assemble from its individual subunits at the early endosome pH of 6.3. Partial disassembly of the CDT holotoxin also occurred at pH 6.3, with complete separation of CdtA from an intact CdtB/CdtC heterodimer occurring at both pH 6.0 and the late endosome pH of 5.6. Acidification caused the precipitation of CdtA at pH 6.5 and below, but neither CdtB nor CdtC were affected by a pH as low as 5.2. Circular dichroism further showed that the individual CdtB subunit adopts a different secondary structure as compared to its structure in the holotoxin. We conclude the first stage of CDT disassembly occurs in the early endosomes, where an acid-induced alteration to CdtA releases it from the CdtB/CdtC heterodimer.

Endocytosis of the CdtA subunit from the Haemophilus ducreyi cytolethal distending toxin.

Many Gram-negative pathogens produce a cytolethal distending toxin (CDT) with two cell-binding subunits (CdtA + CdtC) and a catalytic CdtB subunit. After adhesion to the plasma membrane of a target cell, CDT moves by retrograde transport to endoplasmic reticulum. CdtB then enters the nucleus where it generates DNA breaks that lead to cell cycle arrest and apoptosis or senescence. CdtA anchors the CDT holotoxin to the plasma membrane and is thought to remain on the cell surface after endocytosis of the CdtB/CdtC heterodimer. Here, we re-examined the potential endocytosis and intracellular transport of CdtA from the Haemophilus ducreyi CDT. We recorded the endocytosis of holotoxin-associated CdtA with a cell-based enzyme-linked immunoabsorbent assay (CELISA) and visualised its presence in the early endosomes by confocal microscopy 10min after CDT binding to the cell surface. Western blot analysis documented the rapid degradation of internalised CdtA. Most of internalised CdtB and CdtC were degraded as well. The rapid rate of CDT internalisation and turnover, which could explain why CdtA endocytosis was not detected in previous studies, suggests only a minor pool of cell-associated CdtB reaches the nucleus. Our work demonstrates that CDT is internalised as an intact holotoxin and identifies the endosomes as the site of CdtA dissociation from CdtB/CdtC. TAKE AWAYS: During the endocytosis of CDT, CdtA is thought to remain at the cell surface. A cell-based ELISA documented the rapid endocytosis of CdtA. CdtA was visualised in the early endosomes by confocal microscopy. Intracellular CdtA was rapidly degraded, along with most of CdtB and CdtC.

Detoxified Haemophilus ducreyi cytolethal distending toxin and induction of toxin specific antibodies in the genital tract

Haemophilus ducreyi causes genital ulceration (chancroid), a sexually transmitted infection and still an important factor which contributes to the spread of HIV in developing countries. The bacterium produces a cytolethal distending toxin (HdCDT) causing cell cycle arrest and apoptosis/necrosis of human cells and contributes to the aggravation of ulcers. The aim of the study was to induce toxin-neutralizing antibodies in the genital tract of mice. Repeated subcutaneous (sc) immunisations with 5–10 μg active HdCDT induced low levels of serum anti-HdCDT IgG without neutralizing capacity. High levels of specific IgG1 antibodies in serum and genital tract were generated after sc immunisations with 10 μg formaldehyde detoxified HdCDT toxoid alone and the addition of aluminium salts or RIBI (based on the lipid A moiety) as adjuvant further increased the level of serum antibodies. A high correlation was found between elevated levels of anti-HdCDT IgG in sera, the level of neutralizing activity and the antibody level in genital tract ( r = 0.8). Thus, induction of high antibody levels specific to HdCDT in the genital tissue can be achieved by parenteral immunisation with the toxoid. The HdCDT toxoid can be considered as a candidate component in vaccine against chancroid.

Toxic activity of the CdtB component of Haemophilus ducreyi cytolethal distending toxin expressed from an adenovirus 5 vector

The Haemophilus ducreyi cytolethal distending toxin (HdCDT) catalytic subunit CdtB has DNase-like activity and mediates DNA damage after its delivery into target cells. We constructed a replication-deficient adenovirus type 5 (Ad5) vector expressing CdtB and investigated the toxic properties of this vector on HeLa cells. Ad5CdtB caused loss of cell viability, morphologic changes, and cell cycle arrest, findings similar to HdCDT intoxication. This confirmed that CdtB is responsible for the toxicity of the holotoxin when expressed in cells following transduction by an adenoviral vector, and indicated a possible potential of this novel strategy in studies of activity of intracellular products and in gene therapy of cancer.

Bacterial intoxication evokes cellular senescence with persistent DNA damage and cytokine signalling

Cytolethal distending toxins (CDTs) are proteins produced and secreted by facultative pathogenic strains of Gram-negative bacteria with potentially genotoxic effects. Mammalian cells exposed to CDTs undergo cell type-dependent cell-cycle arrest or apoptosis; however, the cell fate responses to such intoxication are mechanistically incompletely understood. Here we show that both normal and cancer cells (BJ, IMR-90 and WI-38 fibroblasts, HeLa and U2-OS cell lines) that survive the acute phase of intoxication by Haemophilus ducreyi CDT possess the hallmarks of cellular senescence. This characteristic phenotype included persistently activated DNA damage signalling (detected as 53BP1/γH2AX+ foci), enhanced senescence-associated β-galactosidase activity, expansion of promyelocytic leukaemia nuclear compartments and induced expression of several cytokines (especially interleukins IL-6, IL-8 and IL-24), overall features shared by cells undergoing replicative or premature cellular senescence. We conclude that analogous to oncogenic, oxidative and replicative stresses, bacterial intoxication represents another pathophysiological stimulus that induces premature senescence, an intrinsic cellular response that may mechanistically underlie the ‘distended’ morphology evoked by CDTs. Finally, the activation of the two anticancer barriers, apoptosis and cellular senescence, together with evidence of chromosomal aberrations (micronucleation) reported here, support the emerging genotoxic and potentially oncogenic effects of this group of bacterial toxins, and warrant further investigation of their role(s) in human disease.

A novel mode of translocation for cytolethal distending toxin

Thermal instability in the toxin catalytic subunit may be a common property of toxins that exit the endoplasmic reticulum (ER) by exploiting the mechanism of ER-associated degradation (ERAD). The Haemophilus ducreyi cytolethal distending toxin (HdCDT) does not utilize ERAD to exit the ER, so we predicted the structural properties of its catalytic subunit (HdCdtB) would differ from other ER-translocating toxins. Here, we document the heat-stable properties of HdCdtB which distinguish it from other ER-translocating toxins. Cell-based assays further suggested that HdCdtB does not unfold before exiting the ER and that it may move directly from the ER lumen to the nucleoplasm. These observations suggest a novel mode of ER exit for HdCdtB.

In vivovirulence properties of bacterial cytolethal-distending toxin

Multiple pathogenic Gram-negative bacteria produce cytolethal-distending toxins (CDTs). CDT is typically composed of three subunits: the catalytic subunit CdtB has DNase I-like activity, whereas CdtA and CdtC are binding proteins for delivering CdtB into target cells. Translocation of CdtB to the nucleus induces genotoxic effects on host DNA, triggering DNA repair cascades that lead to cell cycle arrest and eventual cell death. Several lines of evidence indicate that this toxin contributes to the pathogenicity of CDT-producing bacteria in vivo. Helicobacter hepaticus and Campylobacter jejuni CDTs are essential for persistent infection of the gastrointestinal tract and increase the severity of mucosal inflammation or liver disease in susceptible mouse strains. Haemophilus ducreyi CDT may contribute to the pathogenesis of chancroid in rabbits. Recently, H. hepaticus CDT has been shown to play a crucial role in promoting the progression of infectious hepatitis to pre-malignant, dysplastic lesions via activation of a pro-inflammatory NF-kappaB pathway and increased proliferation of hepatocytes, providing the first evidence that CDT has carcinogenic potential in vivo. Thus, both in vitro and in vivo data indicate that CDT is a bacterial virulence factor.

Formaldehyde treatment increases the immunogenicity and decreases the toxicity of Haemophilus ducreyi cytolethal distending toxin

Haemophilus ducreyi cytolethal distending toxin (HdCDT) is a tripartite AB toxin, which causes DNA damage in affected cells. We investigated the effects of formaldehyde on the chemical, biological, and immunological properties of the HdCDT complex, which was purified by immobilizing the glutathione S-transferase (GST)-CdtB fusion protein, followed by binding of the CdtA and CdtC recombinant proteins. The HdCDT was treated with increasing concentrations of formaldehyde in the presence of lysine. The treatment of HdCDT at 1 and 0.1 mg protein/ml with 320 and 80 mM of formaldehyde, respectively, resulted in the complete abrogation of cytotoxic activity, loss of DNase activity, and loss of binding capacity to HeLa cells. The toxoid showed protein bands of 75–150 kDa in SDS-PAGE, composed of the three cross-linked CDT components detected by immunoblotting. Three doses of 10 μg protein/mouse of the formaldehyde-treated HdCDT elicited toxin-neutralizing antibodies at titers about 200 times higher than those elicited by the native toxin. The described methodology may be applied to produce immunogenic toxoids from other CDTs, which might be used as candidate components in vaccines against CDT-producing bacteria, including H. ducreyi.

Variation of loop sequence alters stability of cytolethal distending toxin (CDT): Crystal structure of CDT from Actinobacillus actinomycetemcomitans

Cytolethal distending toxin (CDT) secreted by Actinobacillus actinomycetemcomitans induces cell cycle arrest of cultured cells in the G2 phase. The crystal structure of the natural form of A. actinomycetemcomitans DCT (aCDT) has been determined at 2.4 A resolution. aCDT is a heterotrimer consisting of the three subunits, aCdtA, aCdtB, and aCdtC. Two crystallographically independent aCDTs form a dimer through interactions of the aCdtB subunits. The primary structure of aCDT has 94.3% identity with that of Haemophilus ducreyi CDT (hCDT), and the structure of aCDT is quite similar to that of hCDT reconstituted from the three subunits determined recently. However, the molecular packings in the crystal structures of aCDT and hCDT are quite different. A careful analysis of molecular packing suggests that variation of the amino acid residues in a nonconserved loop (181TSSPSSPERRGY192 of aCdtB and 181NSSSSPPERRVY192 of hCdtB) is responsible for the different oligomerization of very similar CDTs. The loop of aCdtB has a conformation to form a dimer, while the loop conformation of hCdtB prevents hCDT from forming a dimer. Although dimerization of aCDT does not affect toxic activity, it changes the stability of protein. aCDT rapidly aggregates and loses toxic activity in the absence of sucrose in a buffered solution, while hCDT is stable and retains toxic activity. Another analysis of crystal structures of aCDT and hCDT suggests that the receptor contact area is in the deep groove between CdtA and CdtC, and the characteristic "aromatic patch" on CdtA.

Comparative structure–function analysis of cytolethal distending toxins

Cytolethal distending toxins (CDTs) constitute a family of bacterial proteins that enter eukaryotic cells with genotoxic activity leading to cell cycle arrest and apoptosis. CDTs are widespread, having been found in a variety of Gram-negative pathogens with a broad tissue tropism. The recently determined crystal structure of the Haemophilus ducreyi CDT provides a powerful starting point for analysis of the structure and function in this toxin family. In this study, we apply comparative modeling and structural analysis to extend the experimental structural information to multiple CDT toxins from a diverse species. Analysis of structurally and functionally important residues in the active subunit, CdtB, and putative cell delivery elements, CdtA and CdtC, begins to establish the fundamental, mechanistic elements of this unique holotoxin. The results reveal that key structural features with important functional consequences are highly conserved across different CDTs, providing a blueprint for directed examination of functional hypotheses in a variety of pathogenic contexts.

Cellular internalization of cytolethal distending toxin: a new end to a known pathway

The cytolethal distending toxins (CDTs) are unique in their ability to induce DNA damage, activate checkpoint responses and cause cell cycle arrest or apoptosis in intoxicated cells. However, little is known about their cellular internalization pathway. We demonstrate that binding of the Haemophilus ducreyi CDT (HdCDT) on the plasma membrane of sensitive cells was abolished by cholesterol extraction with methyl-beta-cyclodextrin. The toxin was internalized via the Golgi complex, and retrogradely transported to the endoplasmic reticulum (ER), as assessed by N-linked glycosylation. Further translocation from the ER did not require the ER-associated degradation (ERAD) pathway, and was Derlin-1 independent. The genotoxic activity of HdCDT was dependent on its internalization and its DNase activity, as induction of DNA double-stranded breaks was prevented in Brefeldin A-treated cells and in cells exposed to a catalytically inactive toxin. Our data contribute to a better understanding of the CDT mode of action and highlight two important aspects of the biology of this bacterial toxin family: (i) HdCDT translocation from the ER to the nucleus does not involve the classical pathways followed by other retrogradely transported toxins and (ii) toxin internalization is crucial for execution of its genotoxic activity.

Induction of apoptosis/necrosis in various human cell lineages by Haemophilus ducreyi cytolethal distending toxin

We investigated the impact of highly purified Haemophilus ducreyi cytolethal distending toxin (HdCDT) on the apoptosis and necrosis of various human cells; including myeloid cells, epithelial cells, keratinocytes, and primary fibroblasts. The levels of apoptosis and necrosis induced in these cells were compared to those induced by HdCDT in human T cells and in the Jurkat T cell line. Levels of caspase-3 activity were measured, and membrane changes like phosphatidylserine (PS) translocation was evaluated after double-staining with the fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI) using flow cytometry. HdCDT induced various degrees of apoptosis and necrosis in dose- and time-dependent manners in cells of various lineages. Early and late apoptosis (annexin V-stained cells) were induced in more than 90% of T cells and monocytes after treatment with 100 ng/ml HdCDT for 24 and 48 h, respectively. The corresponding numbers for epithelial cells, keratinocytes, and fibroblasts were 26–32% after treatment with 100 ng/ml HdCDT for 48 h. HdCDT appears to eliminate effectively by inducing apoptosis those cells that are involved in immune responses. Epithelial cells, keratinocytes and fibroblasts, which are important for the healing of chancroid ulcers, are eliminated by apoptosis or necrosis after contact with HdCDT, albeit slower and to a lesser extent than T cells.

The Haemophilus ducreyi cytolethal distending toxin induces DNA double-strand breaks and promotes ATM-dependent activation of RhoA.

Among bacterial protein toxins, the cytolethal distending toxins (CDTs) are unique in their ability to activate the DNA damage checkpoint responses, causing cell cycle arrest or apoptosis in intoxicated cells. We provide direct evidence that natural intoxication of cells with the Haemophilus ducreyi CDT (HdCDT) holotoxin induces DNA double-strand breaks similarly to ionizing radiation. Upon DNA damage, epithelial cells and fibroblasts promote the formation of actin stress fibres via activation of the small GTPase RhoA. This phenomenon is not toxin specific, but is part of the ATM-induced cellular responses to genotoxic stresses, including ionizing radiation. Activation of RhoA is associated with prolonged cell survival, as HdCDT-treated epithelial cells expressing a dominant-negative form of RhoA detach and consequently die faster than cells expressing a functional RhoA. Our data highlight several novel aspects of CDT biology: (i) we show that a member of the CDT family causes DNA double-strand breaks in naturally intoxicated cells, acting as a true genotoxic agent; and (ii) we disclose the existence of a novel signalling pathway for intracellularly triggered activation of the RhoA GTPase via the ATM kinase in response to DNA damage, possibly required to prolong cell survival.

Toxicity and immunogenicity of purified Haemophilus ducreyi cytolethal distending toxin in a rabbit model

The cytolethal distending toxin of Haemophilus ducreyi (HdCDT) is a three-component toxin that induces the arrest of the mammalian cell cycle in the G2 phase. All of the individual gene products, CdtA, CdtB and CdtC, are required for toxic activity on cultured mammalian cells. The CdtB component alone exerts nuclease activity. The individual HdCDT components were purified by affinity chromatography or ion-exchange chromatography followed by gel-filtration. HdCDT was reconstituted and purified by the immobilization of a GST-CdtB fusion on a GSTrap column and the subsequent addition of cell sonicates from Escherichia coli recombinants that produced CdtA and CdtC. The purified HdCDT preparation contained all three CDT proteins, as detected by immuno-blotting, and had high cytotoxic activity (10 6 CPU/ml). Immunization of rabbits with the HdCDT complex and with the individual CdtA, CdtB and CdtC proteins elicited high titres of antibodies, as detected by ELISA. All of the immune sera had toxin-neutralizing activities. The pathological effects of the HdCDT complex were investigated in rabbits, since the proliferation of two rabbit cell lines, SIRC and RK-13, was inhibited by HdCDT. Intradermal injection of HdCDT (1, 10, 50 and 100 μg protein) into naive rabbits resulted in dose-dependent skin reactions (erythema) about 24 h after injection. Similar effects were not observed when the individual HdCDT proteins were injected. HdCDT injection into immune rabbits resulted in dose-dependent skin responses that were characterized by both erythema and oedema. Histological evaluation of the 24-h lesions in naive rabbits that were injected with HdCDT, revealed moderate levels of inflammatory cells, which were mainly granulocytes and macrophages, and dilatation of blood vessels. The skin reactions in HdCDT-injected immunized rabbits showed pronounced vascular changes and extensive infiltration of inflammatory cells, including eosinophils. All of the pathological changes healed after 3 days. In conclusion, purified HdCDT holotoxin is a complex of all three CDT proteins and all three components induce neutralizing antibodies when injected in rabbits. HdCDT causes dose-dependent pathologic skin reactions in both naive and immune rabbits, which is characterized by increased inflammatory responsiveness after each immunization.

The role of different protein components from the Haemophilus ducreyi cytolethal distending toxin in the generation of cell toxicity

Cytolethal distending toxin of Haemophilus ducreyi (HdCDT) is a multicomponent toxin, encoded by an operon consisting of three genes, cdtABC. To investigate the role of the individual products in generation of toxicity, recombinant plasmids were constructed allowing expression of each of the genes individually or in different combinations in Escherichia coli and Vibrio cholerae. Expression of all three genes (cdtABC) was necessary to generate toxicity on cells, and no activity was obtained using combinations in which only one or two of the genes were expressed. Of the individual gene products, the CdtA was shown to exist in two forms with an MW of 23 and 17 kDa, respectively. The CdtB protein alone resulted in DNase activity. CdtC purified from both toxic and non-toxic extracts (from strains expressingcdtCAB and cdtC, respectively) had a molecular weight of about 20 kDa and reacted with a CdtC-specific monoclonal antibody. However, the protein isoelectric point (pI) of CdtC from toxic preparations was about 1.5 pH units more basic than from non-toxic ones. Both forms were immunogenic giving rise to toxin-neutralizing antibodies. Toxicity was reconstructed by combining non-toxic cell sonicates from E. coli, expressing CdtA, CdtB and CdtC proteins individually. Only combinations including all three products gave toxicity, indicating that all are actively involved in the generation of toxic activity on cells. The reconstruction resulted in a 1.5 pH unit shift in the PI of CdtC, making it identical to that of the protein isolated from bacteria expressing cdtABC. The results showed that the CdtB component produces DNase activity, but cell toxicity depends on the involvement of the other two components of CDT and is associated with absorption of all three proteins by HEp-2 cells.

The impact of Haemophilus ducreyi cytolethal distending toxin on cells involved in immune response

The Haemophilus ducreyi cytolethal distending toxin (HdCDT) induces cell cycle arrest and thereby inhibits cell proliferation of many cultured mammalian cell-lines. We investigated the effect of HdCDT on circulating human hematopoietic cells, including T- and B-cells, monocytes and polymorphonuclear cells (PMN). Lymphocytes were stimulated with T- and B-cell specific mitogens, whereas monocytes and PMN with endotoxin. HdCDT inhibited the mitogen-induced proliferation of T-cells in a dose-dependent manner as assayed by [3H]-thymidine incorporation and MTT assays. Similarly to T-cells, HdCDT also inhibited the proliferation of B-cells and consequently the immunoglobulin production, measured by ELISPOT and ELISA assays. In contrast, the HdCDT did not affect monocytes or PMN, as measured by MTT assay. The TNF-α production by monocytes and the phagocytic ability of PMN were neither affected. The monocytic cell line THP-1 was, however, sensitive to the toxin, seen as a reduction of proliferation and viability after exposure to HdCDT. In conclusion, exposure to HdCDT significantly affects the proliferation and other biological activities of stimulated human T- and B-cells, while circulating monocytes and PMN are not sensitive to HdCDT. The sensitivity of cells of the acquired immune system to HdCDT may hamper specific host response to H. ducreyi and contribute to persistence of chancroid lesions.

The Haemophilus ducreyi Cytolethal Distending Toxin Induces Cell Cycle Arrest and Apoptosis via the DNA Damage Checkpoint Pathways

The cytolethal distending toxins (CDTs) induce cell cycle arrest by a mechanism still not well characterized. We demonstrate that the effect of the Haemophilus ducreyi CDT (HdCDT) is cell type-specific: B cell lines underwent apoptosis, epithelial cells and keratinocytes arrested exclusively in G(2), whereas normal fibroblasts arrested both in G(1) and G(2). We studied normal keratinocytes and fibroblasts, which are relevant for understanding the pathogenicity of H. ducreyi. The response to HdCDT resembles the checkpoint response activated by ionizing radiation. Both responses were characterized by an early induction of the p53 gene and the cyclin-dependent kinase inhibitor p21 in fibroblasts, and activation of the chk2 kinase in epithelial cells. In the Ataxia Telangiectasia-mutated gene (ATM)-deficient lymphoblastoid cell lines, intoxication was significantly delayed compared with ATM wild type cells, and was associated with a slower kinetic of p53 stabilization, suggesting that the early response to HdCDT is ATM-dependent. Activation of ATM-dependent pathways was further confirmed by the ability of caffeine to partially override the HdCDT-mediated cell cycle arrest. Our data shed new light on the mechanism of action of this novel family of bacterial toxins, limiting the target candidates to DNA or molecules directly involved in activation of checkpoint responses.

Cytolethal distending toxins and activation of DNA damage-dependent checkpoint responses

Abstract Cytolethal distending toxins (CDTs) are unique among bacterial protein toxins in their ability to cause DNA damage, due to their functional similarity to the mammalian deoxyribonuclease I (DNase I). The cellular response to CDT intoxication is characterised by activation of DNA damage-induced checkpoint responses, and the final outcome is cell type dependent. Cells of epithelial origin and normal keratinocytes are arrested in the G2 phase of the cell cycle, normal fibroblasts are also arrested in G1, while B cells die of apoptosis. CDTs are encoded by three linked genes ( cdtA, cdtB and cdtC), and CdtB is the toxin subunit which possesses the DNase I-like activity. All the three genes have to be present in the bacterium in order to produce an active cytotoxin, however cytotoxic Haemophilus ducreyi CDT, purified from a CdtABC recombinant E. coli strain, contains the CdtB and CdtC subunits, suggesting that they constitute the holotoxin and that CdtC may be required for CdtB internalization. The role of the CdtA subunit is currently unknown, but it might modify and therefore activate CdtC. This review will focus on the cellular responses induced by CDTs in mammalian cells.