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Departments of
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Pathology and
Biochemistry, University of Pennsylvania School of Dental Medicine, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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25–35 kDa. We recently reported that an immunosuppressive factor (ISF) produced by A. actinomycetemcomitans is a member of the family of Cdts. N-terminal amino acid analysis of purified A. actinomycetemcomitans ISF indicates 98% identity with the CdtB toxin of H. ducreyi (5). Subsequently, the entire gene encoding ISF was isolated and shown to be 95% identical with the CdtB protein of H. ducreyi. Moreover, both the purified ISF and rCdtB are capable of inducing G2 arrest in the cell cycle of mitogen-activated human T cells (11). However, it should be emphasized that Cdt-treated lymphocytes do not exhibit the morphologic alterations that are commonly observed with cell lines such as HeLa cells. Also, human lymphocytes are significantly more sensitive to the toxin than HeLa cells, which are often used as a target cell to define the action of the Cdts (7). These observations have led us to propose that lymphocytes may be primary targets for A. actinomycetemcomitans CdtB and possibly for other Cdt family members as well.
In previous studies, we have shown that G2 arrest induced by CdtB in human T cells appears to be related to the failure to activate the cyclin-dependent kinase, cdk1. Moreover, the accumulation of cells in the G2 phase of the cell cycle reaches a maximum at 72 h and declines thereafter. The subsequent decline in the population of G2 cells led us to question the fate of Cdt-treated lymphocytes. Does exposure to CdtB result in a transient arrest in the cell cycle, or alternatively, does it lead to cell death? We now report that treatment of human T cells with either CdtB or an extract prepared from an E. coli strain that expresses all three of the cdt genes results in irreversible cell cycle arrest that culminates in morphologic and biochemical alterations consistent with apoptotic cell death. These include: DNA fragmentation, decreased cell size, mitochondrial perturbation, and caspase activation. Furthermore, overexpression of the antiapoptotic protein, Bcl-2, prevents Cdt-induced apoptosis, but does not block G2 arrest.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CdtB was prepared from A. actinomycetemcomitans strain 652, as we previously described (12). Briefly, the bacteria were grown for 48 h at 37°C in PYG medium containing 0.4% sodium bicarbonate. Harvested organisms were washed with PBS and extracted in 50 mM Tris buffer, pH 8, containing 10 mM NaCl, 5 mM EDTA, 0.1 mM PMSF, lysozyme (11 µg/ml), and DNase (0.5 µg/ml). Following one cycle of freeze-thawing, debris and remaining bacterial cells were removed by centrifugation at 10,000 x g, and the supernatant was ultracentrifuged for 60 min at 100,000 x g. The CdtB peptide was then purified to homogeneity by sequential fractionation by ion exchange, gel filtration, and chromatofocusing chromatography, as previously described (12).
We also used rCdt for these studies. The rCdt was generated from a
plasmid, pUCAacdt3, which contains and expresses the cdtA,
cdtB, and cdtC genes (11). pUCAacdt3
as well as pUC19 (control) were transformed into E. coli
DH5
; 100-ml cultures were grown in Luria-Bertani medium
supplemented with 100 µg/ml ampicillin to an
OD610 of 0.4. The cell pellets were sonicated
following a wash in 50 mM Tris (pH 8) and centrifuged at 10,000 x
g; the supernatant that contains all three Cdt peptides was
designated rCdtABC and used for the experiments described in this
study.
The Cdt preparations were analyzed by Western blot. Briefly, samples were separated by 12% SDS-PAGE and then transferred to nitrocellulose. The membrane was blocked with BLOTTO and then incubated with polyclonal rabbit sera to either CdtA, CdtB, or CdtC for 18 h at 4°C. Membranes were washed, incubated with goat anti-rabbit Ig sera conjugated to HRP (Southern Biotechnology Associates, Birmingham, AL); the blots were developed using chemiluminescence (New England Nuclear Life Sciences, Boston, MA).
Cell isolation, culture, and transfections
Human PBMC (HPBMC) were prepared as described previously (13). Briefly, HPBMC were isolated from 100 to 200 ml of heparinized venous blood obtained from healthy donors. The blood was diluted with an equal volume of HBSS, and the HPBMC were isolated by buoyant density centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech, Piscataway, NJ). HPBMC were washed twice with RPMI 1640, and viable cell counts were performed by assessing trypan blue dye exclusion. Lymphocytes (1 x 106 cells/ml) were pretreated for 45 min with rCdtABC or CdtB and then activated with either PHA (1 µg/ml; Abbott Laboratories, Abbott Park, IL) or anti-CD3 (20 ng/ml; PharMingen, San Diego, CA) and anti-CD28 (5 µg/ml; PharMingen) mAbs. The cells were incubated in RPMI 1640, antibiotics, and 2% heat-inactivated human AB sera for the time periods indicated.
The human B lymphoblastoid cell line, JY, was obtained from J. Strominger (Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA) and was grown in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FCS (Gemini Bio-Product, Calabasas, CA), 0.1 mm modified Eagle medium nonessential amino acids, modified Eagle medium vitamin solution, 2 mm L-glutamine, and 50 µg gentamicin per milliliter. Bcl-2 cDNA was a gift from S. Korsmeyer (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA). The bcl-2 gene was amplified by PCR and cloned into pcDNA3.1+ (Invitrogen, Carlsbad, CA). Plasmids were transfected into JY cells (JY/bcl-2) by electroporation (Gene ZAPPER 450/2500; IBI, Madison, WI; 290 V, 950 µf) along with pcDNA3.1 (JY/gen) and selected in the presence of Geneticin (1 mg/ml; Life Technologies). Clones were isolated by limiting dilution and maintained at 37°C under 5% CO2.
Expression of Bcl-2 in JY/gen and JY/bcl-2 cells was monitored by flow cytometry. Briefly, cells (1 x 106) were fixed in 4% paraformaldehyde for 20 min at room temperature. Following permeabilization in 0.1% Triton X-100, the cells were stained with anti Bcl-2 mAb conjugated to FITC (Serotec, Raleigh, NC). The relative amount of anti-Bcl-2 FITC fluorescence was detected, as previously described (12).
Flow cytometric analysis of cell cycle
To measure Cdt-induced cell cycle arrest, 1-ml cultures of HPBMC (1 x 106 cells/ml) were activated with PHA (1 µg/ml; Abbott Laboratories), following pretreatment for 45 min with either native CdtB or rCdtABC; the cells were incubated in RPMI 1640, antibiotics, and 2% heat-inactivated human AB sera. JY cells (2 x 105/ml) were incubated, as described earlier, in the absence or presence of CdtB or rCdtABC. Flow cytometry was used to analyze cell cycle distribution, as previously reported (12). Briefly, cells were washed and fixed for 60 min with cold 80% ethanol. After washing, the cells were stained with propidium iodide (10 µg/ml containing 1 mg/ml RNase) for 30 min, and samples were analyzed on a BD Biosciences (Mountain View, CA) FACStarPlus flow cytometer. Propidium iodide fluorescence was excited by an argon laser operating at 488 nm, and fluorescence was measured with a 630/22-nm bandpass filter using linear amplification. A minimum of 15,000 events was collected for each sample; cell cycle analysis was performed using Modfit (Verity Software House, Topsham, ME).
Analysis of apoptosis
We first assessed the induction of apoptosis in Cdt-treated HPBMC by measuring DNA fragmentation using the TUNEL assay (In Situ Cell Death Detection Kit; Boehringer Mannheim, Indianapolis, IN). HPBMC cultures were prepared as described above; at the end of the incubation period, cells were centrifuged, resuspended in 1 ml freshly prepared 4% formaldehyde, and vortexed gently. After 60 min at room temperature, the cells were washed with PBS and permeabilized in 0.1% Triton X-100 for 2 min at 4°C. The cells were then washed with PBS and incubated in a solution containing FITC-labeled nucleotide and TdT, according to the manufacturer’s specifications, and analyzed by flow cytometry.
Alterations in mitochondrial transmembrane potential
(
m) and reactive oxygen species (ROS)
generation were monitored simultaneously by flow cytometry using a
modification of the method described by Castedo et al.
(14). Briefly, T cells were exposed to CdtB or rCdtABC for
72 h; 
m and ROS were measured
using 40 nM 3,3'-dihexyloxacarbocyanine
(DiOC6(3)) and 2 µM
dihydroethidium (Molecular Probes, Eugene, OR), respectively.
Fluorescence was measured after staining the cells for 15 min at 37°C
with each probe. DiOC6(3) was excited with a
laser at 488 nm (250 mW), and emission was measured through a 530/30-nm
bandpass filter. Ethidium fluorescence was excited with a laser at 488
nm (250 mW), and emission was detected with a 575/26-nm bandpass
filter. Logarithmic amplification was used to detect the fluorescence
of the probes; at least 15,000 cells were analyzed per sample. Forward
light scatter (FSC) and side light scatter were acquired in linear
mode.
Mitochondria were also monitored for the expression of Apo2.7, a 38-kDa membrane protein that appears on cells undergoing apoptosis (15). HPBMC were incubated as described and then permeabilized with digitonin (10 µg) for 20 min at 4°C. The cells were then washed and stained with anti-Apo2.7 Ab conjugated to PE (Beckman Coulter, Miami, FL). PE fluorescence was monitored by flow cytometry, as previously described (12).
A hallmark of apoptosis is the activation of a cascade of proteolytic enzymes commonly referred to as caspases. We monitored caspase activation in HPBMC by flow cytometry using the following carboxy fluorescein-labeled fluoromethyl ketone (FMK) peptide inhibitors of caspases 8, 9, and 3: carboxyfluorescein (FAM)-leucylglutamylthreonylaspartic acid (LETD)-FMK (caspase 8), FAM-leucylglutamylhistidylaspartic acid (LEHD)-FMK (caspase 9), and FAM-aspartylglutamylvalylaspartic acid (DEVD)-FMK (caspase 3) (Intergen, Purchase, NY). These inhibitors irreversibly bind to the active caspase. For analysis of caspase activation in JY cells, we used the tripeptide inhibitor, FAM-VAD-FMK, which recognizes the active sites of several caspases, including 1–10 and 12 (Intergen). Following 72-h exposure to CdtB or rCdtABC, HPBMC or JY cells were stained with the caspase inhibitor, according to the manufacturer’s specifications. FITC fluorescence was detected as previously described (16).
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Results |
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A) was first used. Treatment
of HPBMC with 20–300 ng/ml rCdtABC not only induces cell cycle arrest
as we have previously shown (11), but also induces DNA
fragmentation in a dose-dependent manner (Fig. 1B); DNA
fragmentation was observed in 17–34% of the cells 72 h after
treatment with rCdtABC and activation by PHA (Fig. 1). DNA fragmentation was observed in
2.5% of control cells exposed to PHA alone. Minimal DNA fragmentation
(<2%) occurred in HPBMC exposed to extracts prepared from E.
coli transformed with a control plasmid. We have previously shown
that the A. actinomycetemcomitans CdtB peptide alone is able
to induce cell cycle arrest both in human lymphocytes as well as HeLa
cells. As shown in Fig. 1A, this highly purified protein
contains only the CdtB peptide. HPBMC pretreated with 2–20 ng/ml CdtB
and then activated with PHA exhibit DNA fragmentation in 10–32% of
the cells 72 h later (Fig. 1B).
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, minimal DNA fragmentation was observed with either
rCdtABC (10.9%) or CdtB (9.1%) at 48 h; this compares to 5% in
control cultures (PHA alone). There was a significant increase in the
percentage of apoptotic cells at 72 h with 47% of both rCdtABC-
and CdtB-treated cells exhibiting DNA fragmentation. This percentage
was sustained at 96 h as well. Minimal apoptosis was detected in
control cells; 2 and 4% of the cells exhibit DNA fragmentation at 72
and 96 h, respectively. It is important to note that the induction
of apoptosis by the Cdt requires that the cells are in an activated
state. As shown in Fig. 3, A
and B, treatment of lymphocytes in the absence of mitogenic
activation for 72 h does not lead to apoptosis; 3 and 5.2% of the
cells treated with medium or rCdtABC, respectively, exhibit DNA
fragmentation. Although the induction of apoptosis requires that the
cells are activated, the type of mitogenic activation is not critical.
In addition to PHA, we also demonstrate that rCdtABC induces apoptosis
in lymphocytes activated with anti-CD3 and anti-CD28 Abs (Fig. 3, C and D); 0.5 and 24.1% of the cells
exhibited DNA fragmentation in activated control cells and
rCdtABC-treated cells, respectively. Similar results were observed with
CdtB (data not shown).
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A) to 436 (Fig. 4C). Cells treated with extracts derived from
pUC19-transformed E. coli (Fig. 4B),
which do not express cdt genes, exhibit FSC similar to that
of the PHA control (mean channel fluorescence (MCF) = 596). A
decrease in FSC was also observed in lymphocytes treated with CdtB; the
MCF is 412 (Fig. 4D). We also observed a small, but
reproducible, increase in the side light scatter patterns of rCdtABC-
and CdtB-treated cells over that of the control cells (data not shown).
These changes are indicative of an overall shift in the optical
properties of the cell consistent with a reduction in cell size and an
increase in nuclear condensation, respectively, and represent
morphological alterations associated with apoptosis.
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m, and ROS generation. As shown in Fig. 5, increases in Apo2.7 expression were
observed in lymphocytes pretreated with rCdtABC or CdtB; 49.4 and
50.2% of the cells were Apo 2.7 positive, respectively. This compares
to 17.9% in control (PHA alone) cultures, and 19.8% in cells treated
with the pUC19 control extract. The ability of Cdt to induce a
mitochondrial permeability transition state was next assessed by
measuring the m and generation of ROS.
HPBMC were cultured in the presence of PHA and PHA, plus pUC19,
rCdtABC, or CdtB for 72 h, and then stained with the fluorescent
probes DiOC6(3) and hydroethidine to measure
m and ROS generation, respectively.
Multiparametric FACS analysis indicates that 73% of the control cells
are characterized as exhibiting bright DiOC6(3)
fluorescence and virtually no ethidium fluorescence
(DiOC6(3)brightEthdim);
the MCF is 1740 and 11, respectively (Fig. 6A, lower right
quadrant). This is consistent with high
m and no ROS production in the viable
cells. Similar results were observed with cells exposed to pUC19
extract (Fig. 6B). Exposure to rCdtABC results in a
reduction of DiOC6(3) fluorescence in 51.2% of
the cells (DiOC6(3)dim);
the MCF decreases to 269 (Fig. 6C, upper leftand lower left quadrants). Of the
DiOC6(3)dim cells, 25.5%
also exhibit an increase in ethidium fluorescence with a MCF of 287
(Fig. 6C, upper left quadrant). Likewise, HPBMC
exposed to CdtB also manifest a reduction of
DiOC6(3) fluorescence in 48.6% of the cells; the
MCF decreases to 299 (Fig. 6D, upper leftand lower left quadrants). Of the
DiOC6(3)dim cells, 23.6%
also exhibit an increase in ethidium fluorescence with an MCF of 280
(Fig. 6D, upper left quadrant). Thus, the
mitochondria of these cells not only exhibit a decline in
m, but also increased generation of
superoxide anion.
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, A, E, and
I, there is minimal caspase 8, 9, or 3 activity in control
cells; the percentage of cells treated with PHA alone that exhibited
caspase activity was below 22%. Similar results were observed for
cells treated with PHA and the pUC19 extract (Fig. 7, B,
F, and J). In contrast, both rCdtABC and CdtB
induce significant caspase activity. Activation of caspase 8 was
observed in 61% (Fig. 7C) and 65% (Fig. 7D) of
the rCdtABC- and CdtB-treated cells, respectively. Likewise, caspase 9
was activated in 53% (Fig. 7G) of the rCdtABC-treated cells
and 46% (Fig. 7H) of the cells exposed to CdtB. Finally,
>65% of the cells treated with either rCdtABC or CdtB (Fig. 7, K and L) contained active caspase 3.
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, the transfected cells clearly
express higher levels of the Bcl-2 protein than do control cells
transfected with the pcDNA3.1 plasmid lacking an insert
(JY/gen); the MCF for anti-Bcl-2 fluorescence is 49
(JY/bcl-2) and 9 (JY/gen). The two JY cell lines
were then exposed to tissue culture medium, pUC19 extract, rCdtABC, or
CdtB, and incubated for 72 h. Induction of apoptosis was monitored
by measuring total caspase activation with the FAM-VAD-FMK inhibitor
(Fig. 8). Both rCdtABC and CdtB induce apoptosis in 79 and 72% of the
JY/gen cells, respectively; minimal apoptotic cells were
detected in cultures incubated in medium (15%) or extracts derived
from pUC19 alone (16%). In contrast, JY/bcl-2 cells were
relatively resistant to the apoptotic effects of Cdt; treatment with
rCdtABC results in 35% apoptosis and 27% in CdtB-treated cells. Six
percent of the control JY/bcl-2 cells were apoptotic. In
contrast to apoptosis, we observed that both the JY/gen and
JY/bcl-2 cell lines are susceptible to CdtB-induced cell
cycle arrest (Fig. 9). Seventy-two hours
after treatment of JY/gen cells with CdtB, 29% of the cells
are found in the G2 phase; this compares with
11% in JY/gen cells incubated in medium alone.
JY/Bcl-2 cells also exhibit signs of
G2 arrest following exposure to CdtB; at 72
h, 9% of JY/bcl-2 cells incubated in medium alone are in
the G2 phase; in contrast, 67% of the
CdtB-treated cells are in the G2 phase of the
cell cycle. Similar effects were observed for rCdtABC as well (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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m and generation of ROS. Development of the mitochondrial permeability transition state is linked to the release of cytochrome c into the cytosol and, hence, the activation of the caspase cascade. Therefore, we monitored Cdt-treated cells for evidence of caspase activation. Our data clearly indicate that three members of this family of proteases are indeed activated in Cdt-treated lymphocytes: caspases 8, 9, and 3. We focused on these caspases because they are situated at critical points in apoptotic pathways. For instance, caspase 9 is positioned early in the caspase cascade, and its activation is consistent with perturbation of mitochondrial function and cytochrome c release. Likewise, caspase 3 is a component of the downstream protease pathway and not only amplifies signals that lead to caspase 9 activation, but also orchestrates complete destruction of the cell. Of particular interest is our observation that Cdt treatment also led to caspase 8 activation. Caspase 8 is a critical element of the upstream apoptotic cascade, and its activation is often associated with initiation of apoptosis via cell surface receptors such as Fas. Collectively, these results clearly demonstrate that exposure of lymphocytes to Cdt results in G2 arrest and subsequent cell death by activation of the caspase cascade. It is particularly relevant to note that recent studies by Gelfanova et al. (22) also suggest that H. ducreyi Cdt induces lymphocyte apoptosis.
An important issue raised by these investigations is whether the primary effect of Cdts is to induce cell cycle arrest as opposed to apoptosis. Indeed, one of the frequent sequelae of cells that exit the cell cycle at the G2 checkpoint is that they eventually undergo apoptosis and elimination. Based upon our observations, we conclude that cell cycle arrest is the principal effect of lymphocyte exposure to Cdt. First, for cells to become apoptotic as a consequence of Cdt treatment, they must be in an activated state. Indeed, we observed that inactive lymphocytes do not undergo apoptosis in response to Cdt. However, it should be noted that upon exposure to other apoptogens, such as mercurial compounds, resting lymphocytes are capable of an apoptotic response (23). Second, the induction of apoptosis is not dependent upon a specific mitogen, because treatment with Cdt in the presence of other stimuli such as with anti-CD3/CD28 mAbs also led to apoptosis. Third, the kinetics of Cdt-induced G2 arrest and apoptosis suggests that the block in cell cycle progression occurs first with maximal G2 accumulation detected at 72 h and maximal apoptosis between 72 and 96 h. It is interesting to note that the percentage of G2 cells declines after 72 h, while the percentage of apoptotic cells is maintained or slightly increased at 96 h. The basis for these observations is most likely related to the loss of DNA in the G2-arrested cells as they undergo apoptosis. Consequently, the decline in DNA content results in a shift of the arrested cells from the G2 peak of the cell cycle profiles to either the S or G1 phase. This is indeed a common feature of apoptosis and may eventually lead to the appearance of a sub-G1 population (24).
Experiments using bcl-2-transfected JY cells provide further support that induction of cell cycle arrest is the primary effect of Cdts. Overexpression of this antiapoptotic protein in JY cells (JY/bcl-2) inhibited apoptosis following exposure to CdtB relative to control cells (JY/gen). In contrast to apoptosis, JY/bcl-2 cells were still susceptible to Cdt-induced G2 arrest. In fact, treatment with either CdtB or rCdtABC led to a significant increase in the G2 population over that observed in similarly treated JY/gen cells. It is likely that this increase in G2 accumulation results from the Bcl-2 block in apoptosis. Because JY/bcl-2 cells are no longer capable of an apoptotic response, the arrested cells do not lose DNA, and as a consequence, are able to retain their tetraploid DNA phenotype for an extended period of time. Similar results were also noted with unregulated expression of the Bcl-2 homologue, Bcl-xL (data not shown). Thus, our observations clearly demonstrate the A. actinomycetemcomitans Cdt induces an irreversible G2 arrest; furthermore, these data are consistent with activation of the G2 checkpoint, and subsequently of the apoptotic death pathway as well.
It is interesting to note that before our studies, the primary effect of the family of Cdts was the induction of distinct morphologic changes in target cell lines. These Cdt-specific changes include cell elongation and distension. However, in our previous investigations, we did not detect such alterations in human lymphocytes. Furthermore, we now report that rather than an increase in cell size, treatment of human lymphocytes with Cdt leads to a decrease in cell size and increased cellular condensation. Such changes are consistent with the induction of apoptosis. Thus, the descriptive name applied to this family of toxins is clearly misleading and may not accurately reflect their effect on host target cells. Based upon our studies as well as that of other investigators, it is more likely that the Cdts represent a class of immunoregulatory toxins (7, 22).
Our studies address another important issue pertaining to the biology of the Cdt family of toxins. Little information is available for any of the Cdts regarding the nature of the holotoxin that is secreted by the bacterial cell. We have previously demonstrated that CdtB alone is capable of inducing lymphocyte cell cycle arrest as well as eliciting the morphological changes typically associated with Cdts in cell lines (7). Moreover, we have shown that the CdtB protein, when expressed in a cdtA-/cdtC- background in E. coli, is able to induce lymphocyte G2 arrest (10). In this study, we also demonstrate that the CdtB peptide is at least partially responsible for inducing apoptosis. These results are in contrast to those of Stevens et al. (25), who suggest that cdtC encodes the structural toxin of H. ducreyi. Although our own observations clearly indicate that the CdtB peptide derived from A. actinomycetemcomitans is a biologically active toxin unit, we cannot eliminate the possibility that CdtC is also active. In fact, we have constructed two plasmids that contain the cdtA and cdtC gene, but lack the complete cdtB gene; extracts derived from E. coli transformed with these plasmids are capable of inducing G2 arrest in human lymphocytes. Thus, it is possible that the cytolethal distending holotoxin of A. actinomycetemcomitans may be a heterodimer of CdtB and CdtC, with one or both of the individual proteins being capable of inducing G2 arrest and apoptosis. This possibility is also consistent with the work of several investigators demonstrating that Abs against the H. ducreyi CdtC polypeptide neutralize the Cdt activity of that organism (26, 27).
In summary, it is becoming increasingly clear that the host immune system is a target of many pathogenic organisms. Avoidance or modulation of the immune response by invading pathogens may be a critical event in determining the outcome of numerous infectious processes. Moreover, subversion of the immune response not only affects the course of initial infection by facilitating spread, multiplication, and persistence, but may also lead to enhanced susceptibility to infection by secondary pathogens as well. The ability of lymphocytes to undergo a proliferative response to antigenic challenge is essential for all immune responses. Thus, the mode of action of the Cdts is well suited to disruption of immunologic defense mechanisms. We propose that such immunologic perturbations could contribute to the pathogenesis of diseases associated not only with A. actinomycetemcomitans, but with other Cdt-producing organisms as well.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bruce J. Shenker, Department of Pathology, University of Pennsylvania, 4010 Locust Street, Philadelphia, PA 19104-6002. E-mail address: shenker{at}path.dental.upenn.edu
3 Abbreviations used in this paper: Cdt, cytolethal distending toxin; DiOC6(3), 3,3'- dihexyloxacarbocyanine; FMK, fluoromethyl ketone; FSC, forward light scatter; HPBMC, human PBMC; ISF, immunosuppressive factor; MCF, mean channel fluorescence; ROS, reactive oxygen species; FAM, carboxyfluorescein; LETD, leucylglutamylthreonylaspartic acid; LEHD, leucylglutamylhistidylaspartic acid; DEVD, aspartylglutamylvalylaspartic acid; m, mitochondrial transmembrane potential.
Received for publication October 6, 2000. Accepted for publication April 26, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 integrin on the surface of human target cells. J. Biol. Chem. 272:30463.This article has been cited by other articles:
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Z. Ge, Y. Feng, M. T. Whary, P. R. Nambiar, S. Xu, V. Ng, N. S. Taylor, and J. G. Fox Cytolethal Distending Toxin Is Essential for Helicobacter hepaticus Colonization in Outbred Swiss Webster Mice Infect. Immun., June 1, 2005; 73(6): 3559 - 3567. [Abstract] [Full Text] [PDF]
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S. Kato, N. Sugimura, K. Nakashima, T. Nishihara, and Y. Kowashi Actinobacillus actinomycetemcomitans induces apoptosis in human monocytic THP-1 cells J. Med. Microbiol., March 1, 2005; 54(3): 293 - 298. [Abstract] [Full Text] [PDF]
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M. Ohara, T. Hayashi, Y. Kusunoki, M. Miyauchi, T. Takata, and M. Sugai Caspase-2 and Caspase-7 Are Involved in Cytolethal Distending Toxin-Induced Apoptosis in Jurkat and MOLT-4 T-Cell Lines Infect. Immun., February 1, 2004; 72(2): 871 - 879. [Abstract] [Full Text] [PDF]
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B. J. Shenker, D. Besack, T. McKay, L. Pankoski, A. Zekavat, and D. R. Demuth Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin (Cdt): Evidence That the Holotoxin Is Composed of Three Subunits: CdtA, CdtB, and CdtC J. Immunol., January 1, 2004; 172(1): 410 - 417. [Abstract] [Full Text] [PDF]
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D. C. Hassane, R. B. Lee, and C. L. Pickett Campylobacter jejuni Cytolethal Distending Toxin Promotes DNA Repair Responses in Normal Human Cells Infect. Immun., January 1, 2003; 71(1): 541 - 545. [Abstract] [Full Text] [PDF]
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B. HENDERSON, M. WILSON, L. SHARP, and J. M. WARD Actinobacillus actinomycetemcomitans J. Med. Microbiol., December 1, 2002; 51(12): 1013 - 1020. [Full Text] [PDF]
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; 20–300
ng/ml), or CdtB (
, 2–20 ng/ml), and then activated with PHA. After
incubation for 72 h, the cells were analyzed for DNA fragmentation
using the TUNEL assay. The percentage of control cells (PHA alone)
exhibiting DNA fragmentation was 3.5%. Data are plotted as percentage
of apoptosis (TUNEL-positive cells) vs Cdt concentration (µg/ml).
Results represent the mean ± SEM of three experiments, each
performed in triplicate.
) cells were incubated under the conditions
indicated for 72 h. The percentage of apoptotic cells was
determined by measuring caspase activation using the fluorescent
caspase inhibitor FAM-VAD-FMK. Results represent the mean ± SD of
three experiments, each performed in triplicate. Inset,
Relative amount of Bcl-2 protein in JY/gen (broken line)
and JY/bcl-2 (solid line) cells; Bcl-2 content was
measured by immunofluorescence and analyzed by flow cytometry.