Genetic constructions,hyperexpressing recombinant fragments of cytolethal distending toxin of Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is a gram negative bacterium and an infectious agent of human diseases with severe oral and extra oral manifestations. One of the major virulence factors in this microorganism is cytolethal distending toxin (CDT). This toxin consists of three subunits—A, B, and C and is able to disrupt cell cycle by intrinsic DNAse activity of its B-subunit. Due to the fact that this protein can represent an important component of diagnostic, prophylactic and therapeutic preparations, production of CDT subunits in preparative quantities is of considerable practical importance. In the current study we demonstrated that deletion of NH₂-terminal regions from the molecules of CDT-A, -B, or -C resulted in 20–400-fold increase in production of the corresponding subunits. These truncated molecules were used as immunogens to raise monospecific sera, which were shown in western blot to react specifically with the homologous subunits of cytolethal distending toxin.     

Blockade of the PI‐3K signalling pathway by the Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces macrophages to synthesize and secrete pro‐inflammatory cytokines

The Aggregatibactor actinomycetemcomitans cytolethal distending toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes; these toxic effects are due to the active subunit, CdtB, which functions as a phosphatidylinositol‐3,4,5‐triphosphate (PIP3) phosphatase. We now extend our investigation and demonstrate that Cdt is able to perturb human macrophage function. THP‐1‐ and monocyte‐derived macrophages were found not to be susceptible to Cdt‐induced apoptosis. Nonetheless, the toxin was capable of binding to macrophages and perturbing PI‐3K signalling resulting in decreased PIP3 levels and reduced phosphorylation of Akt and GSK3β; these changes were accompanied by concomitant alterations in kinase activity. Exposure of monocytes and macrophages to Cdt resulted in pro‐inflammatory cytokine production including increased expression and release of IL‐1β, TNFα and IL‐6. Furthermore, treatment of cells with either TLR‐2, ‐3 or ‐4 agonists in the presence of Cdt resulted in an augmented pro‐inflammatory response relative to agonist alone. GSK3β inhibitors blocked the Cdt‐induced pro‐inflammatory cytokine response suggesting a pivotal role for PI‐3K blockade, concomitant decrease in GSK3β phosphorylation and increased kinase activity. Collectively, these studies provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt‐producing organisms.     

A novel mode of action for a microbial-derived immunotoxin: the cytolethal distending toxin subunit B exhibits phosphatidylinositol 3,4,5-triphosphate phosphatase activity

The Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt) is a potent immunotoxin that induces G(2) arrest in human lymphocytes. We now show that the CdtB subunit exhibits phosphatidylinositol (PI)-3,4,5-triphosphate phosphatase activity. Breakdown product analysis indicates that CdtB hydrolyzes PI-3,4,5-P(3) to PI-3,4-P(2) and therefore functions in a manner similar to phosphatidylinositol 5-phosphatases. Conserved amino acids critical to catalysis in this family of enzymes were mutated in the cdtB gene. The mutant proteins exhibit reduced phosphatase activity along with decreased ability to induce G(2) arrest. Consistent with this activity, Cdt induces time-dependent reduction of PI-3,4,5-P(3) in Jurkat cells. Lymphoid cells with defects in SHIP1 and/or ptase and tensin homolog deleted on chromosome 10 (PTEN) (such as Jurkat, CEM, Molt) and, concomitantly, elevated PI-3,4,5-P(3) levels were more sensitive to the toxin than HUT78 cells which contain functional levels of both enzymes and low levels of PI-3,4,5-P(3). Finally, reduction of Jurkat cell PI-3,4,5-P(3) synthesis using the PI3K inhibitors, wortmannin and LY290004, protects cells from toxin-induced cell cycle arrest. Collectively, these studies show that the CdtB not only exhibits PI-3,4,5-P(3) phosphatase activity, but also that toxicity in lymphocytes is related to this activity.     

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.     

Induction of cell cycle arrest in lymphocytes by Actinobacillus actinomycetemcomitans cytolethal distending toxin requires three subunits for maximum activity

We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor encoded by the cytolethal distending toxin (cdt)B gene. In this study, we used rCdt peptides to study the contribution of each subunit to toxin activity. As previously reported, CdtB is the only Cdt subunit that is capable of inducing cell cycle arrest by itself. Although CdtA and CdtC do not exhibit activity alone, each subunit is able to significantly enhance the ability of CdtB to induce G2 arrest in Jurkat cells; these effects were dependent upon protein concentration. Moreover, the combined addition of both CdtA and CdtC increased the ED50 for CdtB >7000-fold. In another series of experiments, we demonstrate that the three Cdt peptides are able to form a functional toxin unit on the cell surface. However, these interactions first require that a complex forms between the CdtA and CdtC subunits, indicating that these peptides are required for interaction between the cell and the holotoxin. This conclusion is further supported by experiments in which both Jurkat cells and normal human lymphocytes were protected from Cdt holotoxin-induced G2 arrest by pre-exposure to CdtA and CdtC. Finally, we have used optical biosensor technology to show that CdtA and CdtC have a strong affinity for one another (10(-7) M). Furthermore, although CdtB is unable to bind to either CdtA or CdtC alone, it is capable of forming a stable complex with CdtA/CdtC. The implications of our results with respect to the function and structure of the Cdt holotoxin are discussed.     

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.     

Induction of apoptosis in human T cells by Actinobacillus actinomycetemcomitans cytolethal distending toxin is a consequence of G2 arrest of the cell cycle.

We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor that is encoded by the cdtB gene, which is homologous to a family of cytolethal distending toxins (Cdt) expressed by several Gram-negative bacteria. Moreover, we have shown that CdtB impairs lymphocyte function by inducing G(2) arrest of the cell cycle. We now report that both CdtB as well as an extract prepared from an Escherichia coli strain that expresses all three of the A. actinomycetemcomitans cdt genes (rCdtABC) induce apoptosis. Pretreatment of lymphocytes with either CdtB or rCdtABC leads to DNA fragmentation in activated lymphocytes at 72 and 96 h. No DNA fragmentation was induced in nonactivated cells. Flow cytometric analysis of the Cdt-treated lymphocytes demonstrates a reduction in cell size and an increase in nuclear condensation. Mitochondrial function was also perturbed in cells pretreated with either CdtB or rCdtABC. An increase in the expression of the mitochondria Ag, Apo 2.7, was observed along with evidence of the development of a mitochondrial permeability transition state; this includes a decrease in the transmembrane potential and elevated generation of reactive oxygen species. Activation of the caspase cascade, which is an important biochemical feature of the apoptotic process, was also observed in Cdt-treated lymphocytes. Overexpression of the bcl-2 gene in the human B lymphoblastoid cell line, JY, led to a decrease in Cdt-induced apoptosis. Interestingly, Bcl-2 overexpression did not block Cdt-induced G(2) arrest. The implications of our results with respect to the immunosuppressive functions of Cdt proteins are discussed.     

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.     

The signaling lipid phosphatidylinositol‐3,5‐bisphosphate targets plant CLC‐a anion/H+ exchange activity

Phosphatidylinositol‐3,5‐bisphosphate (PI(3,5)P₂) is a low‐abundance signaling lipid associated with endo‐lysosomal and vacuolar membranes in eukaryotic cells. Recent studies on Arabidopsis indicated a critical role of PI(3,5)P₂ in vacuolar acidification and morphology during ABA‐induced stomatal closure, but the molecular targets in plant cells remained unknown. By using patch‐clamp recordings on Arabidopsis vacuoles, we show here that PI(3,5)P₂ does not affect the activity of vacuolar H⁺‐pyrophosphatase or vacuolar H⁺‐ATPase. Instead, PI(3,5)P₂ at low nanomolar concentrations inhibited an inwardly rectifying conductance, which appeared upon vacuolar acidification elicited by prolonged H⁺ pumping activity. We provide evidence that this novel conductance is mediated by chloride channel a (CLC‐a), a member of the anion/H⁺ exchanger family formerly implicated in stomatal movements in Arabidopsis. H⁺‐dependent currents were absent in clc‐a knock‐out vacuoles, and canonical CLC‐a‐dependent nitrate/H⁺ antiport was inhibited by low concentrations of PI(3,5)P₂. Finally, using the pH indicator probe BCECF, we show that CLC‐a inhibition contributes to vacuolar acidification. These data provide a mechanistic explanation for the essential role of PI(3,5)P₂ and advance our knowledge about the regulation of vacuolar ion transport.     

Expression of the cytolethal distending toxin (Cdt) operon in Actinobacillus actinomycetemcomitans: evidence that the CdtB protein is responsible for G2 arrest of the cell cycle in human T cells

We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor that is encoded by the cdtB gene, which is homologous to a family of cytolethal distending toxins (Cdt) expressed by several gram-negative bacteria. In this study, we report that the cdt locus in A. actinomycetemcomitans is composed of five open reading frames, designated orf1, orf2, cdtA, cdtB, and cdtC. The deduced amino acid sequences of the five open reading frames are highly conserved among A. actinomycetemcomitans strains 652, Y4, 29522, and HK1651. There is also strong homology with the Cdt proteins of Haemophilus ducreyi (87-91%), but only partial homology with that of Campylobacter jejuni and Escherichia coli (29-48%). Analysis of A. actinomycetemcomitans mRNA by RT-PCR suggests that the two small open reading frames upstream of cdtA are coexpressed with cdtA, cdtB, and cdtC. We next utilized a series of plasmids that express various combinations of the cdt genes to determine their requirement for expression of immunoinhibitory activity. Cell extracts of E. coli transformed with each of the plasmids were tested for their capacity to induce G2 arrest in the cell cycle of PHA-activated human T cells. These experiments suggest that expression of cdtB alone is sufficient to induce G2 arrest in human T cells, but do not exclude the possibility that cdtC also contributes to cell cycle arrest. The implications of our results with respect to the function of the individual Cdt proteins are discussed.     

Chemiluminescence enhancement of 1,2‐di[3,4,5‐tri(3,4,5‐trihydroxybenzoyloxy)benzoyloxy] benzene in the presence of quaternary ammonium ions

The chemiluminescence intensity of 1,2‐di[3,4,5‐tri(3,4,5‐trihydroxybenzoyloxy)benzoyloxy] benzene increased in the presence of quaternary ammonium ions, such as acetylcholine chloride, choline chloride or benzyltrimethylammonium chloride. The complex of 1,2‐di[3,4,5‐tri(3,4,5‐trihydroxybenzoyloxy)benzoyloxy] benzene with acetylcholine chloride, choline chloride or benzyltrimethylammonium chloride was investigated by ¹H‐NMR spectroscopy. The structure of the complex formed from 1,2‐di[3,4,5‐tri(3,4,5‐trihydroxybenzoyloxy)benzoyloxy] benzene with choline chloride was described by an ab initio quantum chemical calculation. Copyright © 2010 John Wiley & Sons, Ltd.     

The SH3 domain of postsynaptic density 95 mediates inflammatory pain through phosphatidylinositol‐3‐kinase recruitment

Sensitization to inflammatory pain is a pathological form of neuronal plasticity that is poorly understood and treated. Here we examine the role of the SH3 domain of postsynaptic density 95 (PSD95) by using mice that carry a single amino‐acid substitution in the polyproline‐binding site. Testing multiple forms of plasticity we found sensitization to inflammation was specifically attenuated. The inflammatory response required recruitment of phosphatidylinositol‐3‐kinase‐C2α to the SH3‐binding site of PSD95. In wild‐type mice, wortmannin or peptide competition attenuated the sensitization. These results show that different types of behavioural plasticity are mediated by specific domains of PSD95 and suggest novel therapeutic avenues for reducing inflammatory pain.     

The effects of extracellular adenosine 5′‐triphosphate on the tobacco proteome

Extracellular adenosine 5′‐triphosphate (eATP) is emerging as an important plant signalling compound capable of mobilising intracellular second messengers such as Ca²⁺, nitric oxide, and reactive oxygen species. However, the downstream molecular targets and the spectrum of physiological processes that eATP regulates are largely unknown. We used exogenous ATP and a non‐hydrolysable analogue as probes to identify the molecular and physiological effects of eATP‐mediated signalling in tobacco. 2‐DE coupled with MS/MS analysis revealed differential protein expression in response to perturbation of eATP signalling. These proteins are in several functional classes that included photosynthesis, mitochondrial ATP synthesis, and defence against oxidative stress, but the biggest response was in the pathogen defence‐related proteins. Consistent with this, impairment of eATP signalling induced resistance against the bacterial pathogen Erwinia carotovora subsp. carotovora. In addition, disease resistance activated by a fungal pathogen elicitor (xylanase from Trichoderma viride) was concomitant with eATP depletion. These results reveal several previously unknown putative molecular targets of eATP signalling, which pinpoint eATP as an important hub at which regulatory signals of some major primary metabolic pathways and defence responses are integrated.     

Chronic exposure to the cytolethal distending toxins of Gram‐negative bacteria promotes genomic instability and altered DNA damage response

Epidemiological evidence links chronic bacterial infections to the increased incidence of certain types of cancer but the molecular mechanisms by which bacteria contribute to tumour initiation and progression are still poorly characterized. Here we show that chronic exposure to the genotoxin cytolethal distending toxin (CDT) of Gram‐negative bacteria promotes genomic instability and acquisition of phenotypic properties of malignancy in fibroblasts and colon epithelial cells. Cells grown for more than 30 weeks in the presence of sublethal doses of CDT showed increased mutation frequency, and accumulation of chromatin and chromosomal aberrations in the absence of significant alterations of cell cycle distribution, decreased viability or senescence. Cell survival was dependent on sustained activity of the p38 MAP kinase. The ongoing genomic instability was associated with impaired activation of the DNA damage response and failure to efficiently activate cell cycle checkpoints upon exposure to genotoxic stress. Independently selected sublines showedenhanced anchorage‐independent growth as assessed by the formation of colonies in semisolid agarose. These findings support the notion that chronic infection by CDT‐producing bacteria may promote malignant transformation, and point to the impairment of cellular control mechanisms associated with the detection and repair of DNA damage as critical events in the process.