This review summarizes the current status and recent advances in our understanding of the role that the cytolethal distending toxin (Cdt) plays as a virulence factor in promoting disease by toxin-producing pathogens. A major focus of this review is on the relationship between structure and function of the individual subunits that comprise the AB2 Cdt holotoxin. In particular, we concentrate on the molecular mechanisms that characterize this toxin and which account for the ability of Cdt to intoxicate multiple cell types by utilizing a ubiquitous binding partner on the cell membrane. Furthermore, we propose a paradigm shift for the molecular mode of action by which the active Cdt subunit, CdtB, is able to block a key signaling cascade and thereby lead to outcomes based upon programming and the role of the phosphatidylinositol 3-kinase (PI-3K) in a variety of cells. Based upon the collective Cdt literature, we now propose that Cdt is a unique and potent virulence factor capable of acting as a tri-perditious toxin that impairs host defenses by: (1) disrupting epithelial barriers; (2) suppressing acquired immunity; (3) promoting pro-inflammatory responses. Thus, Cdt plays a key role in facilitating the early stages of infection and the later stages of disease progression by contributing to persistence and impairing host elimination.

The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes and other cell types. We have shown that the active subunit, CdtB, exhibits phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase activity, leading us to propose that Cdt toxicity is the result of PIP3 depletion and perturbation of phosphatidylinositol-3-kinase (PI-3K)/PIP3/Akt signalling. To further explore this relationship, we have focused our analysis on identifying residues that comprise the catalytic pocket and are critical to substrate binding rather than catalysis. In this context, we have generated several CdtB mutants and demonstrate that, in each instance, the ability of the toxin to induce cell cycle arrest correlates with retention of phosphatase activity. We have also assessed the effect of Cdt on downstream components of the PI-3K signalling pathway. In addition to depletion of intracellular concentrations of PIP3, toxin-treated lymphocytes exhibit decreases in pAkt and pGSK3β. Further analysis indicates that toxin-treated cells exhibit a concomitant loss in Akt activity and increase in GSK3β kinase activity consistent with observed changes in their phosphorylation status. We demonstrate that cell susceptibility to Cdt is dependent upon dephosphorylation and concomitant activation of GSK3β. Finally, we demonstrate that, in addition to lymphocytes, HeLa cells exposed to a CdtB mutant that retains phosphatase activity and not DNase activity undergo G2 arrest in the absence of H2AX phosphorylation. Our results provide further insight into the mode of action by which Cdt may function as an immunotoxin and induce cell cycle arrest in target cells such as lymphocytes.

The cytolethal distending toxin (Cdt) is produced from a number of bacteria capable of causing infection and inflammatory disease. Our previous studies with Actinobacillus actinomycetemcomitans Cdt demonstrate not only that the active toxin subunit functions as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase but also that macrophages exposed to the toxin were stimulated to produce proinflammatory cytokines. We now demonstrate that the Cdt-induced proinflammatory response involves the activation of the NLRP3 inflammasome. Specific inhibitors and short hairpin RNA (shRNA) were employed to demonstrate requirements for NLRP3 and ASC as well as caspase-1. Furthermore, Cdt-mediated inflammasome activation is dependent upon upstream signals, including reactive oxygen species (ROS) generation and Cdt-induced increases in extracellular ATP levels. Increases in extracellular ATP levels contribute to the activation of the P2X 7 purinergic receptor, leading to K ؉ efflux. The relationship between the abilities of the active toxin subunit CdtB to function as a lipid phosphatase, activate the NLRP3 inflammasome, and induce a proinflammatory cytokine response is discussed. These studies provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as Aggregatibacter actinomycetemcomitans.T he cytolethal distending toxins (Cdts) are a family of heatlabile protein cytotoxins produced by several bacterial species, including Campylobacter jejuni, Shigella species, Haemophilus ducreyi, Aggregatibacter actinomycetemcomitans, and diarrheal disease-causing enteropathogens such as some Escherichia coli isolates (1-7). There is clear evidence that Cdts are encoded by three genes, designated cdtA, cdtB, and cdtC, which are arranged as an apparent operon (7-12). These three genes specify three polypeptides, designated CdtA, CdtB, and CdtC, with apparent molecular masses of 28, 32, and 20 kDa, respectively, that form a heterotrimeric holotoxin. Several cell lines and cell types have been shown to be sensitive to Cdt-induced cell cycle arrest and cell death via apoptosis; these include human lymphoid cells, fibroblasts, human embryonic intestinal epithelial cells, a human colon carcinoma cell line, and human keratinocytes, among others (7,11,12). There is considerable agreement that the heterotrimeric holotoxin functions as an AB 2 toxin, where CdtB is the active (A) unit and the complex of CdtA and CdtC comprises the binding (B) unit (13)(14)(15). In this regard, we have shown that CdtA and CdtC are required for the toxin to associate with lipid microdomains within lymphocyte membranes and that Cdt-mediated toxicity is dependent upon the integrity of these lipid domains (16). Furthermore, we have demonstrated that the CdtC subunit contains a cholesterol recognition sequence that is required for interactions with membrane cholesterol (16,17).The active Cdt subunit CdtB exhibits enzymatic activity, enabling it to degrade the signaling lipid phosphatidylino...

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Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles ( A ggregatibacter actinomycetemcomitans is a Gram-negative organism that is associated with aggressive forms of periodontitis and other systemic infections (1-5). Periodontitis is a chronic infectious inflammatory disorder that ultimately leads to the destruction of tooth-supporting tissue. While the exact nature of the pathogenesis of periodontal disease and the contribution of bacteria to this process are not known, it is becoming increasingly clear that A. actinomycetemcomitans produces several potential virulence factors; these include adhesins and fimbria which have been shown to contribute to colonization of the human oral cavity as well as two exotoxins, cytolethal distending toxin (Cdt) and leukotoxin, both of which are capable of killing and/or altering the function of host immune cells (4,(6)(7)(8).The Cdts are a family of heat-labile protein cytotoxins produced by several additional bacterial species, including Campylobacter jejuni, Shigella species, Haemophilus ducreyi, and diarrheal disease-causing enteropathogens such as some Escherichia coli isolates (9-15). There is clear evidence that Cdts are encoded by three genes, designated cdtA, cdtB, and cdtC, which are arranged as an apparent operon (7,(15)(16)(17)(18)(19)(20). The Cdt holotoxin consists of three subunits, CdtA, CdtB, and CdtC, that form a heterotrimeric complex. Furthermore, there is considerable agreement among investigators that, regardless of the microbial source of Cdt, the heterotrimeric holotoxin functions as an AB 2 toxin where CdtB is the active (A) unit and the complex of CdtA and CdtC comprises the binding (B) unit (18,21,22). Indeed, several investigators have demonstrated that the internalization of CdtB requires the presence of both CdtA and CdtC (21,23,24).While several cell types and cell lines have been shown to be susceptible to the toxic actions of Cdt, tropism for specific cells and/or tissue remains to be identified. In this regard, we have demonstrated that lymphocytes in vitro are most susceptible, requiring very low concentrations of Cdt (picograms/milliliter) to induce cell cycle arrest and apoptosis versus other cell types that typically require as much as microgram quantities (25). Typically, susceptibility to bacterial toxins is dependent upon the expression of specific receptors or moieties that enable the toxin to preferentially associate with target host cells. Structural analysis of CdtA and CdtC identified ricin-like lectin domains, suggesting that these units interact wi...