Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt): evidence that the holotoxin is composed of three subunits: CdtA, CdtB, and CdtC

We have shown the Actinobacillus actinomycetemcomitans produces an immunosuppressive factor encoded by the cytolethal distending toxin (cdt)B gene, which is homologous to a family of Cdts expressed by several Gram-negative bacteria. We now report that the capacity for CdtB to induce G(2) arrest in Jurkat cells is greater in the presence of the other Cdt peptides: CdtA and CdtC. Plasmids containing the cdt operon were constructed and expressed in Escherichia coli; each plasmid contained a modified cdt gene that expressed a Cdt peptide containing a C-terminal His tag. All three Cdt peptides copurified with the His-tagged Cdt peptide. Each of the peptides associated with the complex was truncated; N-terminal amino acid analysis of CdtB and CdtC indicated that the truncation corresponds to cleavage of a previously described signal sequence. CdtA was present in two forms in crude extracts, 25 and 18 kDa; only the 18-kDa fragment copurified with the Cdt complexes. Cdt complexes were also immunoprecipitated from A. actinomycetemcomitans extracts using anti-CdtC mAb. Exposure of Jurkat cells to 40 pg resulted in >50% accumulation of G(2) cells. CdtB and CdtC were detected by immunofluorescence on the cell surface after 2-h exposure to the holotoxin. CdtA was not detected by immunofluorescence, but all three peptides were associated with Jurkat cells when analyzed by Western blot. These studies suggest that the active Cdt holotoxin is a heterotrimer composed of truncated CdtA, CdtB, and CdtC, and all three peptides appear to associate with lymphocytes.     

Functional studies of the recombinant subunits of a cytolethal distending holotoxin

Cytolethal distending toxin (CDT) is a multicomponent bacterial holotoxin that targets most eukarytotic cells causing distension and cell cycle arrest. A number of diverse pathogenic bacterial species associated with diarrhoea, chancroid, chronic hepatitis and periodontal disease produce a CDT. Synthesis of the holotoxin is directed by the expression of three genes, cdtA , cdtB and cdtC . Although the product of the CdtB gene was previously identified as a type I deoxyribonuclease, the functions of the cdtA and cdtC products have not been characterized. Using the periodontal pathogen, Actinobacillus actinomycetemcomitans , we demonstrate that the recombinant product of the CdtA gene binds to the surface of Chinese hamster ovary (CHO) cells. This protein did not induce distension or cytotoxicity when introduced into the cytosol using a lipid-based protein delivery system. Recombinant CdtB and CdtC proteins failed to bind to CHO cells. However, the delivery of either CdtB or CdtC into the cytosol resulted in the characteristic pattern of distension followed by cell death. Based on these results, it appears that the CdtA protein subunit alone is responsible for anchoring the holotoxin to the cell surface. The CdtC subunit, in concert with CdtB, contributes to the cytotoxic activities of the holotoxin. The specific mechanism of CdtC cytotoxicity is currently unknown.     

Haemophilus parasuis encodes two functional cytolethal distending toxins: CdtC contains an atypical cholesterol recognition/interaction region

Haemophilus parasuis is the causative agent of Glässer''s disease of pigs, a disease associated with fibrinous polyserositis, polyarthritis and meningitis. We report here H. parasuis encodes two copies of cytolethal distending toxins (Cdts), which these two Cdts showed the uniform toxin activity in vitro. We demonstrate that three Cdt peptides can form an active tripartite holotoxin that exhibits maximum cellular toxicity, and CdtA and CdtB form a more active toxin than CdtB and CdtC. Moreover, the cellular toxicity is associated with the binding of Cdt subunits to cells. Further analysis indicates that CdtC subunit contains an atypical cholesterol recognition/interaction amino acid consensus (CRAC) region. The mutation of CRAC site resulted in decreased cell toxicity. Finally, western blot analysis show all the 15 H. parasuis reference strains and 109 clinical isolates expressed CdtB subunit, indicating that Cdt is a conservative putative virulence factor for H. parasuis. This is the first report of the molecular and cellular basis of Cdt host interactions in H. parasuis.     

Cytolethal distending toxin: a bacterial bullet targeted to nucleus

Cytolethal distending toxin (Cdt) is a newly added member of bacterial protein toxins that hijack the control system of eukaryotic cells. Cdts are produced by several pathogenic bacteria causing chronic infectious diseases. They are composed of three subunits, CdtA, CdtB and CdtC, which together form a ternary complex. CdtB is the active component, and CdtA and CdtC are involved in delivering the CdtB into the cells. The sophisticated strategy of Cdt to control host cells is CdtB-mediated limited DNA damage of the host cell chromosome, which triggers the response of the cell cycle checkpoint and results in G2 arrest in the cells. Cdt also induces apoptotic cell death of lymphocytes, which may be relevant to onset or persistence of chronic infection by the producing bacteria. The study of this toxin is expected to provide us information on a novel strategy by which bacteria interact with host cells.     

Characterization of point mutations in the cdtA gene of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans

The Cdt is a family of gram-negative bacterial toxins that typically arrest eukaryotic cells in the G0/G1 or G2/M phase of the cell cycle. The toxin is a heterotrimer composed of the cdtA, cdtB and cdtC gene products. Although it has been shown that the CdtA protein subunit binds to cells in culture and in an enzyme-linked immunosorbent assay (CELISA) the precise mechanisms by which CdtA interacts with CdtB and CdtC has not yet been clarified. In this study we employed a random mutagenesis strategy to construct a library of point mutations in cdtA to assess the contribution of individual amino acids to binding activity and to the ability of the subunit to form biologically active holotoxin. Single unique amino acid substitutions in seven CdtA mutants resulted in reduced binding of the purified recombinant protein to Chinese hamster ovary cells and loss of binding to the fucose-containing glycoprotein, thyroglobulin. These mutations clustered at the 5'- and 3'-ends of the cdtA gene resulting in amino acid substitutions that resided outside of the aromatic patch region and a conserved region in CdtA homologues. Three of the amino acid substitutions, at positions S165N (mutA81), T41A (mutA121) and C178W (mutA221) resulted in gene products that formed holotoxin complexes that exhibited a 60% reduction (mutA81) or loss (mutA121, mutA221) of proliferation inhibition. A similar pattern was observed when these mutant holotoxins were tested for their ability to induce cell cycle arrest and to convert supercoiled DNA to relaxed and linear forms in vitro. The mutations in mutA81 and mutA221 disrupted holotoxin formation. The positions of the amino acid substitutions were mapped in the Haemophilus ducreyi Cdt crystal structure providing some insight into structure and function.     

Mechanisms of assembly and cellular interactions for the bacterial genotoxin CDT

Many bacterial pathogens that cause different illnesses employ the cytolethal distending toxin (CDT) to induce host cell DNA damage, leading to cell cycle arrest or apoptosis. CDT is a tripartite holotoxin that consists of a DNase I family nuclease (CdtB) bound to two ricin-like lectin domains (CdtA and CdtC). Through the use of structure-based mutagenesis, biochemical and cellular toxicity assays, we have examined several key structural elements of the CdtA and CdtC subunits for their importance to toxin assembly, cell surface binding, and activity. CdtA and CdtC possess N- and C-terminal nonglobular polypeptides that extensively interact with each other and CdtB, and we have determined the contribution of each to toxin stability and activity. We have also functionally characterized two key binding elements of the holotoxin revealed from its crystal structure. One is an aromatic cluster in CdtA, and the other is a long and deep groove that is formed at the interface of CdtA and CdtC. We demonstrate that mutations of the aromatic patch or groove residues impair toxin binding to HeLa cells and that cell surface binding is tightly correlated with intoxication of cultured cells. These results establish several structure-based hypotheses for the assembly and function of this toxin family.     

Reconstitution and purification of cytolethal distending toxin of Actinobacillus actinomycetemcomitans

Cytolethal distending toxin (CDT) has been found in various pathogenic bacterial species and causes a cell distending and a G2 arrest against eukaryotic cells. All the cdtABC genes, which encode CDT, are known to be required for the CDT activities although the CDT holotoxin structure has not been elucidated. We cloned the cdtABC genes of Actinobacillus actinomycetemcomitans and constructed an Escherichia coli expression system for them. We found that crude extracts from six deletion mutants (delta cdtA, delta cdtB, delta cdtC, delta cdtBC, delta cdtAC, and delta cdtAB) of recombinant E. coli, which showed very weak or no detectable CDT activities, restored the CDT activities when pre-mixing and pre-incubation of them were performed in combinations to contain all the CdtA, CdtB, and CdtC proteins. These results indicate that all the Cdt proteins are required for the CDT activities. We also found that the chimera CdtB protein, CdtB-intein-CBD (chitin binding domain) like CdtB protein itself assembled with CdtA and CdtC. The reconstituted CDT containing the chimera CdtB protein was specifically extracted by chitin beads and the only CDT portion was isolated from the chitin beads by a cleavage reaction of the intein. The purified reconstituted-CDT was found to consist of CdtA, CdtB, and CdtC proteins, and showed appreciable CDT activities, indicating that the CDT holotoxin structure is the CdtABC complex. To our knowledge, this is the first report succeeded in complete purification of an active CDT and may offer useful tools for elucidation of the toxic mechanism of CDT.     

Dynamics and assembly of the cytolethal distending toxin

The cytolethal distending toxin (CDT) is a widespread bacterial toxin that consists of an active subunit CdtB with nuclease activity and two ricin-like lectin domains, CdtA and CdtC, that are involved in the delivery of CdtB into the host cell. The three subunits form a tripartite complex that is required to achieve the fully active holotoxin. In the present study we investigate the assembly and dynamic properties of the CDT holotoxin using molecular dynamics simulations and binding free energy calculations. The results have revealed that CdtB likely adopts a different conformation in the unbound state with a closed DNA binding site. The two characterized structural elements of the aromatic patch and groove on the CdtA and CdtC protein surfaces exhibit high mobility, and free energy calculations show that the heterodimeric complex CdtA-CdtC, as well as the CdtA-CdtB and CdtB-CdtC sub-complexes are less energetically stable as compared to the binding in the tripartite complex. Analysis of the dynamical cross-correlation map reveals information on the correlated motions and long-range interplay among the CDT subunits associated with complex formation. Finally, the estimated binding free energies of subunit interactions are presented, together with the free energy decomposition to determine the contributions of residues for both binding partners, providing insight into the protein-protein interactions in the CDT holotoxin.     

Differences in crystal and solution structures of the cytolethal distending toxin B subunit: Relevance to nuclear translocation and functional activation

Cytolethal distending toxin (CDT) induces cell cycle arrest and apoptosis in eukaryotic cells, which are mediated by the DNA-damaging CdtB subunit. Here we report the first x-ray structure of an isolated CdtB subunit (Escherichia coli-II CdtB, EcCdtB). In conjunction with previous structural and biochemical observations, active site structural comparisons between free and holotoxin-assembled CdtBs suggested that CDT intoxication is contingent upon holotoxin disassembly. Solution NMR structural and 15N relaxation studies of free EcCdtB revealed disorder in the interface with the CdtA and CdtC subunits (residues Gly233-Asp242). Residues Leu186-Thr209 of EcCdtB, which encompasses tandem arginine residues essential for nuclear translocation and intoxication, were also disordered in solution. In stark contrast, nearly identical well defined alpha-helix and beta-strand secondary structures were observed in this region of the free and holotoxin CdtB crystallographic models, suggesting that distinct changes in structural ordering characterize subunit disassembly and nuclear localization factor binding functions.     

Cytolethal distending toxin: limited damage as a strategy to modulate cellular functions

The coevolution of bacterial pathogens and their hosts has contributed to the development of very complex and sophisticated functional pathogen--host interfaces. Thus, well-adapted pathogens have evolved a variety of strategies to manipulate host cell functions precisely. For example, a group of unrelated Gram-negative pathogenic bacteria have evolved a toxin, known as cytolethal distending toxin (CDT), that has the ability to control cell cycle progression in eukaryotic cells. Recent studies have identified CdtB as the active subunit of the CDT holotoxin. Through its nuclease activity, CdtB causes limited DNA damage, thereby triggering the DNA-damage response that ultimately results in the observed arrest of the cell cycle. In addition, it has been established that CDT is a tripartite AB toxin in which CdtB is the active 'A' subunit and CdtA and CdtC constitute the heterodimeric 'B' subunit required for the delivery of CdtB into the target cell. The mechanism of action of CDT suggests that the infliction of limited damage could be a strategy used by pathogenic bacteria to modulate host cell functions.     

beta-Helix is a likely core structure of yeast prion Sup35 amyloid fibers

Helicobacter hepaticus, a causal agent of hepatocarcinoma in mice, exhibits a cytolethal distending toxin activity. The three subunits of this holotoxin, CdtA, CdtB, and CdtC, and three CdtB mutants were produced as recombinant histidine-tagged proteins by using an in vitro cell-free protein expression system. We found that the presence of the three H. hepaticus Cdt subunits is required for cellular toxicity and that only a C-terminal CdtB mutation abolishes the activity of the complex. In vitro, H. hepaticus CdtB exhibits a DNase activity which is also abolished by this C-terminal CdtB mutation. These results suggest that the effect of H. hepaticus CDT probably involves the DNase activity of CdtB.     

Cytolethal distending toxin B as a cell-killing component of tumor-targeted anthrax toxin fusion proteins

Cytolethal distending toxin (Cdt) is produced by Gram-negative bacteria of several species. It is composed of three subunits, CdtA, CdtB, and CdtC, with CdtB being the catalytic subunit. We fused CdtB from Haemophilus ducreyi to the N-terminal 255 amino acids of Bacillus anthracis toxin lethal factor (LFn) to design a novel, potentially potent antitumor drug. As a result of this fusion, CdtB was transported into the cytosol of targeted cells via the efficient delivery mechanism of anthrax toxin. The fusion protein efficiently killed various human tumor cell lines by first inducing a complete cell cycle arrest in the G2/M phase, followed by induction of apoptosis. The fusion protein showed very low toxicity in mouse experiments and impressive antitumor effects in a Lewis Lung carcinoma model, with a 90% cure rate. This study demonstrates that efficient drug delivery by a modified anthrax toxin system combined with the enzymatic activity of CdtB has great potential as anticancer treatment and should be considered for the development of novel anticancer drugs.     

Distinct Roles for CdtA and CdtC during Intoxication by Cytolethal Distending Toxins

Cytolethal distending toxins (CDTs) are heterotrimeric protein exotoxins produced by a diverse array of Gram-negative pathogens. The enzymatic subunit, CdtB, possesses DNase and phosphatidylinositol 3-4-5 trisphosphate phosphatase activities that induce host cell cycle arrest, cellular distension and apoptosis. To exert cyclomodulatory and cytotoxic effects CDTs must be taken up from the host cell surface and transported intracellularly in a manner that ultimately results in localization of CdtB to the nucleus. However, the molecular details and mechanism by which CDTs bind to host cells and exploit existing uptake and transport pathways to gain access to the nucleus are poorly understood. Here, we report that CdtA and CdtC subunits of CDTs derived from Haemophilus ducreyi (Hd-CDT) and enteropathogenic E. coli (Ec-CDT) are independently sufficient to support intoxication by their respective CdtB subunits. CdtA supported CdtB-mediated killing of T-cells and epithelial cells that was nearly as efficient as that observed with holotoxin. In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type. Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response. Finally, host cellular cholesterol was found to influence sensitivity to intoxication mediated by Ec-CdtA, revealing a role for cholesterol or cholesterol-rich membrane domains in intoxication mediated by this subunit. In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways.     

Deletion and purification studies to elucidate the structure of the Actinobacillus actinomycetemcomitans cytolethal distending toxin

Cytolethal distending toxin (CDT) is one of the exotoxins produced by Actinobacillus actinomycetemcomitans, an agent of localized aggressive periodontitis. We constructed N-terminal deletion mutants of CdtA using an Escherichia coli expression system and found that ADelta19-47, with a deletion from Asn-19 to Pro-47, showed comparable CDT activity but no apparent heterogeneity of CdtA. The wild-type CDT (wtCDT) and the mutant CDT (ADelta19-47CDT) were purified to homogeneity by introducing a histidine tag into the C-terminal end of CdtB. Both purified wtCDT and purified ADelta19-47CDT showed strong CDT activity and a tripartite structure composed of CdtA (subunit A), 31 kDa CdtB (subunit B), and 18.5 kDa CdtC (subunit C) in nearly a 1:1:1 stoichiometry. Importantly, subunit A was identified as heterogeneous with three CdtA variants in wtCDT, but homogeneous in ADelta19-47CDT. Purified CDTs also showed high stability that was absolutely dependent on the presence of sucrose in the buffer. In conclusion, the region from the Asn-19 to Pro-47 of CdtA contributes to the heterogeneous production of CdtA, but is dispensable for the toxin activity. Furthermore, this study describes an effective protocol for the purification of a native rather than reconstituted CDT, and clarifies the subunit composition of the active CDT holotoxin.     

Characterization of Putative Cholesterol Recognition/Interaction Amino Acid Consensus-Like Motif of Campylobacter jejuni Cytolethal Distending Toxin C

Cytolethal distending toxin (CDT) produced by Campylobacter jejuni comprises a heterotrimeric complex formed by CdtA, CdtB, and CdtC. Among these toxin subunits, CdtA and CdtC function as essential proteins that mediate toxin binding to cytoplasmic membranes followed by delivery of CdtB into the nucleus. The binding of CdtA/CdtC to the cell surface is mediated by cholesterol, a major component in lipid rafts. Although the putative cholesterol recognition/interaction amino acid consensus (CRAC) domain of CDT has been reported from several bacterial pathogens, the protein regions contributing to CDT binding to cholesterol in C. jejuni remain unclear. Here, we selected a potential CRAC-like region present in the CdtC from C. jejuni for analysis. Molecular modeling showed that the predicted functional domain had the shape of a hydrophobic groove, facilitating cholesterol localization to this domain. Mutation of a tyrosine residue in the CRAC-like region decreased direct binding of CdtC to cholesterol rather than toxin intermolecular interactions and led to impaired CDT intoxication. These results provide a molecular link between C. jejuni CdtC and membrane-lipid rafts through the CRAC-like region, which contributes to toxin recognition and interaction with cholesterol.     

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.     

Inhibition of protein tyrosine phosphatases suppresses P-selectin exocytosis in activated human platelets

The cytolethal distending toxin (CDT) of Haemophilus ducreyi is encoded by the cdtABC genes, but the composition of active CDT is not known. Both immunoaffinity and metal affinity chromatographic methods were used to purify H. ducreyi CDT from recombinant Escherichia coli strains bearing wild-type or mutated H. ducreyi cdtABC genes. Both affinity-purified preparations contained CdtA, CdtB, and CdtC proteins. These purification efforts also revealed that the formation of a noncovalent CdtB-CdtC complex and production of a fully active CDT complex required the presence of a functional CdtA protein. When purified recombinant CdtB and CdtC proteins were mixed, only very slight CDT activity was detected. In contrast, when a bacterial cell extract containing CdtA was mixed with purified preparations of both CdtB and CdtC, full CDT activity was reconstituted in vitro. These results indicate that CdtA is essential for normal H. ducreyi CDT activity and that CdtA likely modifies or alters either CdtB or CdtC or both to form the active CDT complex.     

DNase I homologous residues in CdtB are critical for cytolethal distending toxin-mediated cell cycle arrest

Cytolethal distending toxins (CDTs) block cell division by arresting the eukaryotic cell cycle at G2/M. Although previously not recognized in standard BLAST searches, a position-specific iterated (PSI) BLAST search of the protein data bank using CDT polypeptides as query sequences indicated that CdtB bears significant position-specific homology to type I mammalian DNases. The PSIBLAST sequence alignment reveals that residues of DNase I involved in phosphodiester bond hydrolysis (His134 and His252) are conserved in CdtB as well as their respective hydrogen bond pairs (Glu78 and Asp212). CdtB also contains a pentapeptide motif found in all DNase I enzymes. Further, crude CDT preparations possess detectable DNase activity not associated with identical preparations from control cells. Five CdtB mutations in amino acids corresponding to DNase I active site residues were prepared and expressed together with wild-type CdtA and CdtC polypeptides. Mutation in four of the five DNase-specific active site residues resulted in CDT preparations that lacked DNase activity and failed to induce cellular distension or arrest division of HeLa cells. The fifth mutation, Glu86 (Glu78 in DNase I), retained the ability to induce a moderate level of cell cycle arrest and displayed reduced DNase activity relative to wild-type CDT. Together, these data suggest that the CDT holotoxin has intrinsic DNase activity that is associated with the CdtB polypeptide and that this DNase activity may be responsible for the CDT-induced cell cycle arrest.     

Cholesterol-rich membrane microdomains mediate cell cycle arrest induced by Actinobacillus actinomycetemcomitans cytolethal-distending toxin

We have previously shown that Actinobacillus actinomycetemcomitans cytolethal-distending toxin (Cdt) is a potent immunosuppressive agent that induces G2/M arrest in human lymphocytes. In this study, we explored the possibility that Cdt-mediated immunotoxicity involves lipid membrane microdomains. We first determined that following treatment of Jurkat cells with Cdt holotoxin all three Cdt subunits localize to these microdomains. Laser confocal microscopy was employed to colocalize the subunits with GM1-enriched membrane regions which are characteristic of membrane rafts. Western blot analysis of isolated lipid rafts also demonstrated the presence of Cdt peptides. Cholesterol depletion, using methyl beta-cyclodextrin, protected cells from the ability of the Cdt holotoxin to induce G2 arrest. Moreover, cholesterol depletion reduced the ability of the toxin to associate with Jurkat cells. Thus, lipid raft integrity is vital to the action of Cdt on host cells. The implications of our observations with respect to Cdt mode of action are discussed.     

The toxicity of the Aggregatibacter actinomycetemcomitans cytolethal distending toxin correlates with its phosphatidylinositol‐3,4,5‐triphosphate phosphatase activity

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.