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Actinobacillus actinomycetemcomitans, a gram-negative coccobacillus found in the human oral cavity, has been implicated in the pathogenesis of certain forms of periodontal disease and also in several systemic diseases, such as endocarditis, meningitis, and osteomyelitis (32, 38). This organism has the usual panoply of putative virulence factors, such as lipopolysaccharide (LPS) (6, 19, 43). It also secretes a number of unusual proteins, including a leukocyte-specific leukotoxin with proapoptotic activity (15), a chaperonin 60 with osteolytic activity (14, 34), and various inhibitors of cell cycle progression (21, 39, 40).

There is growing evidence that microorganisms have evolved a range of mechanisms to evade both the innate and acquired host immune response (7). It has been proposed that virulence factors acting to impair host defense mechanisms play significant roles in the pathology of infections with A. actinomycetemcomitans (29). One recently discovered class of bacterial toxin, called cytolethal distending toxin (CDT), has been isolated from a range of pathogenic bacteria, including Escherichia coli, Campylobacter species, Shigella species, Haemophilus ducreyi, and Helicobacter species (1, 22, 23, 24, 28, 41, 45). This toxin was originally discovered as a factor which induced distension of CHO cells (reviewed in reference 24). However, it is now clear that the biological activities of CDT, such as distension, actin rearrangement, and apoptosis, depend on the type of cell being studied (24). Thus, mouse Y-1 adrenal cells and NIH 3T3 fibroblasts are not affected by CDT treatment (2, 13). CDT is able to induce growth arrest at the G₂/M phase in epithelial cells and apoptosis of cultured B-cell lines in an ataxia telangiectasia-mutated kinase-dependent manner (3). Recent studies suggest that degradation of chromosomal DNA by CdtB, which possesses DNase I motifs, is responsible for the blocking of the cell cycle-dependent dephosphorylation of Cdc2, the catalytic subunit of cyclin B, followed by arrest of sensitive cells in the G₂/M phase (5, 17).

A. actinomycetemcomitans produces CDT, which is encoded by three genes, cdtA, cdtB, and cdtC, located on the bacterial chromosome in tandem to form an operon with two other genes that have not yet been characterized (18, 31, 35). All Cdt proteins have signal peptides and can be isolated in the culture supernatant (18, 31, 35). Shenker and coworkers have reported that the A. actinomycetemcomitans CdtB is the active toxic component able to block the proliferation of T lymphocytes and thus induce immunosuppression (30, 31). In contrast, it has been reported that a CdtC-deficient mutant of H. ducreyi lacked cytotoxicity (33) and that a monoclonal antibody to H. ducreyi CdtC neutralized CDT cytotoxicity (1). Thus, the roles of the various Cdt proteins are still unclear.

In addition to inhibiting cell cycle progression, it has recently been reported that Campylobacter jejuni CDT directly mediated the release of interleukin-8 (IL-8) from intestinal epithelial cells (11). This suggested the possibility that CDT may play a dual role and that different components of this toxin could play different roles in inducing or inhibiting immune responses. In this report, we have expressed each A. actinomycetemcomitans cdt gene product independently using an E. coli expression system, purified each toxin "subunit," and assessed its capacity to stimulate the release of cytokines from human peripheral blood mononuclear cells (PBMCs).

Bacterial strains and growth conditions. A. actinomycetemcomitans Y4 (serotype b; ATCC 43718) was grown on brain heart infusion agar (Oxoid, Hampshire, United Kingdom) supplemented with 5% (vol/vol) horse blood at 37°C for 2 days in an atmosphere of 5% CO₂, harvested from the plates with sterile saline, and centrifuged at 3,000 × g for 20 min (10). E. coli strains TOP10 (Invitrogen, Leek, The Netherlands) and HMS174(DE3) (Novagen, Nottingham, United Kingdom) were used in this study. E. coli was routinely grown in Luria-Bertani (LB) broth.

Cells and culture conditions. The human epithelial cell line HEp-2 was grown in Dulbecco's minimal essential medium (GIBCO, Paisley, United Kingdom) containing L-glutamine, 10% fetal calf serum, streptomycin (100 µg/ml), and penicillin (100 IU/ml) in an atmosphere containing 5% CO₂. PBMCs were prepared from buffy coat blood by density gradient centrifugation as previously described (36).

Cloning of cdt genes into an N-terminal polyhistidine expression vector. The oligonucleotides 5'-GGATCCTGTTCGTCAAATCAACGA and 5'-CTGCAGTTAATTAACCGCTGTTGC were designed to amplify the 625-bp cdtA gene. The oligonucleotides 5'-GGATCCAACTTGAGTGATTTCAAA and 5'-CTGCAGTTAGCGATCATGAACAAA were designed to amplify the 785-bp cdtB gene. The oligonucleotides 5'-GGATCCCATGCAGAATCAAATCCT and 5'-CTGCAGTTAGCTACCCTGATTTCT were designed to amplify the 506-bp cdtC gene. These primers were based on the sequence data for the cdt genes reported by Sugai and coworkers (35) and were designed to amplify each cdt gene without the DNA encoding the N-terminal signal peptide and also contained recognition sequences for restriction enzymes BamHI and PstI (underlined). Chromosomal DNA from A. actinomycetemcomitans was used as the template for PCR. The PCR fragments were initially cloned into pCR4-TOPO (Invitrogen) and transformed into E. coli TOP10. The cdt genes were cut from the pCR4-TOPO on BamHI-NotI fragments and ligated to BamHI-NotI-digested pET-28a(+) (Novagen). The ligation mixtures were transformed into E. coli HMS174(DE3), and transformants were selected by growing at 30°C on LB agar containing kanamycin (30 µg/ml).

Expression of cdt genes and purification of recombinant proteins. For gene expression, positive clones were grown overnight in LB broth containing kanamycin (30 µg/ml) and rifampin (200 µg/ml), diluted 1:20 in fresh broth, and incubated for a further 2 h at 37°C. Gene expression was induced with 1 mM isopropyl--D-thiogalactopyranoside (IPTG) for 6 h at 30°C. Cells were harvested by centrifugation at 6,000 × g for 20 min and were then resuspended and lysed for 10 min with bacterial permeabilizing reagent (B-PER) protein extraction reagent (Pierce & Warriner Ltd., Cheshire, United Kingdom). The expressed proteins were contained in inclusion bodies. Purification of these inclusion bodies was performed as described by the manufacturer of the B-PER reagent. Briefly, lysates were centrifuged and pellets were resuspended with the same volume of B-PER-containing lysozyme (100 µg/ml), and the lysates were then incubated for a further 5 min at room temperature. A 10× volume of 1:10-diluted B-PER was added to the lysates, and the inclusion bodies were collected by centrifugation at 15,000 × g for 20 min. After being washed twice with the same volume of 1:10-diluted B-PER, the pellets were treated with 8 M urea containing 300 mM NaCl and 100 mM sodium phosphate buffer (pH 8.0). The recombinant proteins were purified using Ni-nitrilotriacetic acid-agarose columns under denaturing conditions as specified by the manufacturer (Qiagen Ltd.), except that after lysates were loaded onto the column, an additional column wash, consisting of 2.5 mg of polymyxin B/ml in wash buffer, was performed to remove contaminating LPS. The refolding of denatured proteins was performed as described by Takemura et al. (37).

Analysis of cell cycle. To measure the cell cycle arrest induced by the Cdt proteins, HEp-2 cells at a density of 2 × 10⁵ cells/ml were cultured in Dulbecco's minimal essential medium with 40, 200, or 1,000 ng of recombinant Cdt (rCdt) protein/ml for 1 to 4 days. These concentrations were determined by initial dose-ranging experiments to give a good dose-response relationship between toxin concentration and cell cycle inhibition. At the end of the culture period the HEp-2 cells were washed and fixed for 60 min with 80% cold ethanol. After washing, the cells were stained in the dark at 4°C for 1 h with propidium iodide (10 µg/ml) in phosphate-buffered saline containing RNase (1 mg/ml). The data from 2 × 10⁴ cells were collected on a FACScan flow cytometer (Becton Dickinson). Cell cycle analysis was performed using CellQuest. All experiments examining cell cycle inhibition and cytokine induction were repeated a minimum of three times and gave consistent results.

Cytotoxic assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viable-cell assay was performed as previously described (20). Briefly, HEp-2 cells were plated into 96-well plates at a concentration of 2 × 10⁴ cells/well. Various concentrations of the rCdt proteins were added, and the HEp-2 cells were cultured for a further 3 to 6 days. At the end of the culture period a stock MTT solution (20 µg/well) was added to the wells, and the plate contents were incubated for a final 4 h. Acid-isopropanol (100 µl of 0.04 N HCl in isopropanol) was added to cells to elute the red formazan, and the solution was mixed thoroughly. The concentration of formazan in solution in each well was determined on a Dynex plate reader by using a test wavelength of 570 nm and a reference wavelength of 620 nm.

Assay of cytokine production by PBMCs. Human PBMCs were prepared from normal donor blood by density gradient centrifugation and differential adherence as described by Tabona et al. (36) and were plated at a density of 2.0 × 10⁶ cells/ml in 24-well plates and stimulated with a graded concentration of rCdt proteins. Initial dose-ranging studies were done to define the most sensible toxin protein concentrations for obtaining good dose-response relationships. To control for LPS contamination in the recombinant proteins, polymyxin B (2 µg/ml) was added to each well, the contents of which were then incubated for 20 h. Cytokine synthesis was determined by two-site enzyme-linked immunosorbent assay. The coating and detection antibodies for IL-10, gamma interferon (IFN-), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were from Pharmingen (Oxford, United Kingdom), and those for IL-12 were from BioSource (Watford, United Kingdom). Assays for IL-1, IL-6, IL-8, and tumor necrosis factor alpha (TNF-) and all cytokine standards were from the National Institute for Biological Standards and Control. Cell supernatants were assayed for the presence of all cytokines using the enzyme-linked immunosorbent assay as previously described (11, 36, 42). In some experiments, cells were stimulated with E. coli LPS (DIFCO) at a concentration of 10 ng/ml and the anti-CD14 antibody MY4 was used as to control for the role of CD14 in cell stimulation.

Cloning and expression of cdt genes. The cdtA, cdtB, and cdtC genes encoding the mature proteins without signal peptides were amplified by PCR from chromosomal DNA of A. actinomycetemcomitans and were inserted into the pET-28a(+) N-terminal polyhistidine-tagged fusion vector. These plasmid constructss were introduced into E. coli HMS174(DE3). All Cdt protein products were found in inclusion bodies and were refolded in arginine-containing buffer after being dissolved in 8 M urea. These proteins were then dialyzed against phosphate-buffered saline to remove low-molecular-weight reagents, including urea and arginine. All rCdt proteins (rCdtA, rCdtB and rCdtC) were homogenous, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and migrated with molecular masses of about 26, 32, and 22 kDa, respectively.

Ability of rCdt proteins to arrest cell cycle. To determine the contribution of each CDT component in generating biological activity, independently expressed and purified rCdt proteins were analyzed for their effects on cell cycle progression. Preliminary dose-ranging experiments found that exposure of cells to Cdt proteins in the range of 40 to 1,000 ng/ml produced a satisfactory dose-response relationship. As shown in Fig. 1, in control cultures of HEp-2 cells exposed to medium alone, 78% of the cells were in the G₀/G₁ phase of the cell cycle with a 2n DNA content. None of the rCdt proteins, when added alone, could induce cell cycle arrest in HEp-2 cells. Treatment of HEp-2 cells with a Cdt "complex" containing all three recombinant proteins resulted in 74% of the cells being present in the peak of propidium iodide fluorescence, consisting of cells in G₂/M phase. Cultures exposed to Cdt complexes containing rCdtA and rCdtB or containing rCdtA and rCdtC did not demonstrate cell cycle arrest. However, treatment of cells with rCdtB and rCdtC did produce cell cycle arrest.

View larger version (29K): [in this window] [in a new window]   FIG. 1.   Effect of multiple Cdt complexes on cultured HEp-2 cells. HEp-2 cells were incubated for 3 days in the presence of 1.0 (A), 0.2 (B), or 0.04 (C) µg of combined rCdtA, -B, and -C/ml and were heat treated (100°C, 20 min) with 1.0 µg of combined rCdtA, -B, and -C/ml (D). Likewise, cells were incubated with 1.0-µg/ml rCdtA and -B (E), rCdtA and -C (F), rCdtB and -C (G), rCdtA alone (H), rCdtB alone (I), rCdtC alone (J), and medium alone (K). The cells were then analyzed for cell cycle distribution following staining with propidium iodide. The percentages of cells in G0, G1, S, and G2/M phases of the cell cycle are shown. At least 15,000 cells were analyzed per sample.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Effect of multiple Cdt complexes on cell viability. HEp-2 cells were incubated for 3 days in the presence of 1.0 () and 10.0 () µg of the multiple complexes of the Cdt proteins per ml. The cytotoxicity of the toxins was then assessed by a colorimetric cytotoxicity assay as described in Materials and Methods. The data are the means and standard deviations of three replicate cultures.

Capacity of Cdts to stimulate cytokine production. To investigate the capacity of CDT to stimulate cytokine synthesis, various combinations of the recombinant proteins were incubated with human PBMCs and the release of IL-1, IL-6, IL-8, IL-10, IL-12, TNF-, IFN-, and GM-CSF was measured. To confirm that the effects seen were not due to LPS contamination, the rCdt proteins were heated to 100°C for 20 min or the anti-CD14 monoclonal antibody MY4 was added at a concentration known to block the action of nanogram levels of LPS as defined by Tabona et al. (36). Heat treatment completely abolished the cytokine-inducing activity of the CDTs, but the anti-CD14 monoclonal antibody, while able to block LPS, was unable to inhibit the activity of individual or combined rCdt proteins. The effect of these treatments on the production of IL-8 is shown in Fig. 3. Similar results were also found when media supporting the CDT-stimulated cells were assayed for IL-1, IL-6, and IFN- (not shown).

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Effect of addition of MY4, an anti-human CD14 antibody (CD14), or of heat treatment on the capacity of rCdt to induce the secretion of IL-8 from human PBMCs. PBMCs were cultured with 10 ng of LPS/ml or 1 µg of CdtA, -B, and -C/ml in the presence or absence of anti-CD14 antibody. Heat treatment was performed at 100°C for 20 min. The results are expressed as the mean and standard deviation of three replicate cultures.