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Departments of
*
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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95%
identity with Hemophilus ducreyi CdtB protein and 
60%
homology with Escherichia coli or Campylobacter
jejuni CdtB. Pretreatment of PHA-activated lymphocytes with
5–25 ng ISF results in G2 arrest of CD4+ and
CD8+ T cells. Similarly, treatment of HeLa cells results in
G2 arrest and cell elongation and distension. However,
lymphocytes are at least 5 times more sensitive to ISF than HeLa cells
and do not undergo the elongation and distension that characterizes
interactions of Cdts with cell lines. ISF-treated lymphocytes express
normal cyclin A and B1 levels, but contain reduced levels of cell
cycle-dependent kinase-1 (Cdk1). Additionally, the majority of Cdk1 is
in the hyperphosphorylated, inactive, form. In contrast, PHA-induced
G2 cells contain elevated levels of the hypophosphorylated,
active Cdk1. Failure of ISF-treated cells to dephosphorylate Cdk1 is
not associated with decreased availability of Cdc25. These studies
suggest that the CdtB protein alone is capable of inducing
G2 arrest in lymphocytes and cell cycle arrest, elongation,
and distension of HeLa cells. Our studies also suggest that lymphocytes
may be primary targets for A. actinomycetemcomitans CdtB
(ISF) and possibly for other Cdt family members as well. Thus, Cdts may
function to impair host immunity and contribute to the pathogenesis of
disease associated with Cdt-producing organisms. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Actinobacillus actinomycetemcomitans, a nonmotile, Gram-negative coccobacillus, is associated with several human diseases. These include endocarditis, meningitis, osteomyelitis, s.c. abscesses, and periodontal disease (3, 4, 5, 6, 7, 8). Although the pathogenic mechanism(s) by which A. actinomycetemcomitans acts to cause disease is not known, it does produce several potential virulence factors capable of facilitating colonization, destroying host tissue, inhibiting tissue repair, and interfering with host defenses (reviewed in 7). With respect to the latter, several studies suggest that impaired host defense mechanisms may contribute to infectious diseases associated with A. actinomycetemcomitans (reviewed in 7 and 9). In this regard we have previously shown that A. actinomycetemcomitans produces a heat-labile immunosuppressive factor (ISF)4 that is capable of inhibiting both human T and B cell function (10, 11, 12, 13). While the exact mechanism by which this immunoregulatory agent acts is not yet known, our previous studies provide strong support for the ability of ISF to induce aberrations in the normal progression of the lymphocyte cell cycle. This is characterized by both an accumulation of cells in the G2 and M phase of the cell cycle as well as the generation of a subset of T cells with an abnormal phenotype. The latter cells are not only CD3 positive, but are CD4+ and CD8+ as well; furthermore, these cells are predominantly in the S and G2 phases of the cell cycle.
The cytolethal distending toxins (Cdts) are a newly described family of heat-labile protein cytotoxins produced by several different bacterial species. These include diarrheal disease-causing enteropathogens such as some Escherichia coli isolates, Campylobacter jejuni and Shigella dysenteriae (14, 15, 16, 17). More recently, related toxins have been identified in Haemophilus ducreyi, a human pathogen responsible for the formation of chancroid ulcers and buboes, and A. actinomycetemcomitans strain Y4 (18, 19). The Cdts cause progressive cellular distension and finally death in some cell lines; it should be noted that the gross cellular changes associated with Cdt activity are clearly different from those caused by other known toxins that induce rapid morphological alterations culminating in cell death (18, 20, 21). There is now clear evidence that Cdt is encoded by three genes, designated cdtA, cdtB, and cdtC, which are arranged in an apparent operon. These three genes specify polypeptides with predicted or apparent molecular masses of approximately 25–35 kDa. We now report that the A. actinomycetemcomitans ISF 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 (18). Subsequently, the entire gene encoding ISF was isolated and was shown to be 95% identical with the CdtB protein of H. ducreyi. Moreover, we have determined that the purified ISF is capable of inducing a G2 arrest in the cell cycle of both lymphocytes and human cell lines. However, lymphocytes are 5-fold more sensitive to the toxin than are HeLa cells and do not exhibit the morphologic effects that are commonly observed with cell lines. The G2 arrest appears to be associated with a failure to dephosphorylate cell cycle-dependent kinase-1 (Cdk1). These studies suggest that the primary host target of A. actinomycetemcomitans ISF and possibly all Cdt toxins may be lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ISF was prepared from A. actinomycetemcomitans strain 652 using a modification of the procedure 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 8.0, containing 10 mM NaCl, 5 mM EDTA, 0.1 mM PMSF, lysozyme (10 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 ISF was then purified to homogeneity as described in Results.
Protein and gene sequencing A. actinomycetemcomitans ISF (CdtB)
The purified ISF was immobilized onto PVDF membrane (Bio-Rad, Hercules, CA) and subjected to N-terminal amino acid analysis using Edmund degradation. Sequencing was performed on an ABI Procise sequencer (Applied Biosystems, Foster City, CA) using the manufacturer’s software by the Wistar Protein and Molecular Biology Core facility (Wistar Institute; Philadelphia, PA). The derived N-terminal peptide sequence was compared with known protein sequences using the NCBI Blast program.
To clone the gene encoding ISF, PCRs were conducted using A.
actinomycetemcomitans strain 652 genomic DNA as the template and
degenerate oligonucleotide primers based upon the ISF N-terminal amino
acid sequence determined above and the deduced amino acid sequence of
CdtC of H. ducrei (residues 23–28) (18). The resulting
fragment was cloned in pGEM-T (Promega) and transformed into E.
coli DH5
. Sequencing was conducted on an ABI 377 by the DNA
Sequencing Core Facility at the University of Pennsylvania. To complete
the sequencing of the ISF gene (cdtB), additional primers
were synthesized based upon the A. actinomycetemcomitans
sequence already determined. Subsequently, the complete cdt
locus was isolated from an A. actinomycetemcomitans library
in 
EMBL3 by screening with the cdtB gene as a probe. One
clone with a 15-kbp insert containing the entire cdt operon was chosen
for further study. The sequences of the A. actinomycetemcomitans
cdtA and cdtC genes were determined from DNA fragments
flanking the cdtB gene that were derived from this clone.
The A. actinomycetemcomitans strain 652 cdtB gene
sequence has been submitted to the GenBank database; the accession
number is AF102554.
Cell isolation and culture
Human PBMC (HPBMC) were prepared as described previously (10). Briefly, HPBMC were isolated from 100–200 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 (Pharmacia, Piscataway, NJ). The HPBMC were washed twice with RPMI 1640, and counts of viable cell were performed by assessing trypan blue dye exclusion.
For selected experiments purified populations of T cells were obtained by E rosette formation as described previously (12). Briefly, sheep erythrocytes were washed and treated with 0.14 M 2-aminoethylisothiouronium bromide at pH 9 for 15 min. After four washes, the erythrocytes were incubated with HPBMC as a cell pellet for 60 min. The cells were then gently resuspended; nonrosetted cells were separated from rosetted cells on Ficoll-Hypaque as described above. The rosetted cells found in the pellet were lysed to remove erythrocytes and contained >98% T cells when stained with anti-CD3 mAb (Becton Dickinson Immunocytometry Systems, San Jose, CA) and analyzed by flow cytometry. HPBMC or purified T cells were incubated in 1-ml cultures in 24-well plates containing 1.5 x 106 cells/ml, PHA (1 µg/ml; Murex, Dartfield, U.K.), and ISF (5–25 ng/ml) in RPMI 1640, antibiotics, and 2% heat-inactivated human AB serum. The cells were incubated for 72 h (or as indicated), harvested, and analyzed by flow cytometry or extracts prepared for Western blot analysis as described below.
Column fractions were monitored for ISF activity by measuring their relative ability to inhibit [3H]thymidine incorporation of HPBMC as previously described (10). Briefly, 2 x 105 cells were placed into each well of flat-bottom microculture plates. Each culture received 0.1 ml of medium or 0.1 ml of varying amounts of individual column fractions diluted in medium. The cells were incubated for 60 min at 37°C, at which time the cells received an optimal mitogenic dose of Con A (1 µg; Calbiochem, La Jolla, CA). The cells were incubated for 96 h, labeled with [3H]thymidine, and harvested as previously reported (12). One unit of ISF activity was calculated as that volume of material required to reduce [3H]TdR incorporation to 50% maximum (Con A alone).
Assessment of A. actinomycetemcomitans ISF on HeLa cells
HeLa cells were obtained from the American Type Tissue Culture Collection (Manassas, VA) and maintained in RPMI 1640 containing antibiotics and 10% FBS. For cell cycle analysis, 1-ml cultures were established containing 2.5 x 104 cells in 24-well plates. Cultures received varying amounts of ISF or medium (control) and incubated for 72–96 h, at which time the cells were trypsinized and subjected to cell cycle analysis by flow cytometry (see below). Any nonadherent cells found in the culture were retained and included in the cell cycle analysis. For morphological evaluation, 1 x 103 cells were added to microscope chamber slides (Lab-Tek, Naperville, IL) in 0.4 ml of medium in the presence or the absence of ISF. Following 96-h incubation, the cells were stained with hematoxylin and eosin and analyzed.
Flow cytometric analysis of cell cycle
Cell cycle analysis was performed on lymphocytes and HeLa cells as previously reported (12). Briefly, T cells (1.5 x 106) were first washed (PBS containing 0.1% NaN3) and then stained with anti-CD4- or anti-CD8-conjugated FITC (Becton Dickinson Immunocytometry Systems) 72 h after exposure to medium, PHA, or PHA and ISF. The cells were washed and fixed for 60 min with cold 0.25% paraformaldehyde. After washing, the cells were permeabilized with 0.2% Tween 20 in PBS for 15 min at 37°C. DNA was then stained by incubating cells with propidium iodide (10 µg/ml containing 1 mg/ml RNase) for 30 min. Samples were analyzed on a Becton Dickinson FACStarPlus flow cytometer. FITC and propidium iodide fluorescence were excited by an argon laser operating at 488 nm, and fluorescence was measured with a 530/30-nm bandpass filter (FITC) or a 630/22-nm bandpass filter (propidium iodide). A minimum of 30,000 events were collected on each sample. Immunofluorescence data were collected on a log scale, and propidium iodide emissions were collected using linear amplification.
For the analysis of total RNA, protein, and cyclins, the cells were harvested and washed in PBS and then resuspended in cold 80% ethanol. To measure cell cycle along with total RNA and protein, the cells were washed in PBS and stained with Hoechst 33342 (2 µg/ml; Molecular Probes, Eugene, OR) and pyronine Y (4 µg/ml; Sigma, St. Louis, MO) or with FITC (0.1 µg/ml; Molecular Probes), respectively (22, 23). The cells were analyzed by flow cytometry; Hoechst fluorescence was excited with one laser operating in UV emission detected through a 424/44-nm bandpass filter. Both pyronine Y and FITC were excited with a second laser operating at 488 nm; emission was detected with a 575/26-nm bandpass filter (pyronine Y) or a 535/30-nm bandpass filter (FITC). The cyclins were analyzed after fixation for 2 h at -20°C when the cells were washed, resuspended in 0.25% Triton X-100/PBS, and incubated for 5 min at 2–5°C. The cells were washed and then stained with FITC-conjugated Abs to cyclin A or B1 (PharMingen, San Diego, CA) for 30 min; control cells were stained with appropriate isotype controls. After washing, the cells were resuspended in propidium iodide solution (10 µg/ml) and analyzed as described above.
Western blot analysis for the analysis of Cdc2 and Cdc25
T cells were incubated with PHA in the presence or the absence of ISF for 72 h. The cells were harvested, and replicate wells were pooled and solubilized in 50 mM Tris containing 0.5% Nonidet P-40, 150 mM NaCl, 1 mM sodium vanadate, 50 mM sodium fluoride, 1 mM benzamidine, and 25 µg/ml aprotinin, leupeptin, and trypsin inhibitor (Sigma). Cell debris was removed by centrifugation (10,000 x g). Proteins of interest were detected by Western blot analysis using mAbs to the Cdc-2 or using actin (PharMingen) or goat polyclonal antisera to Cdc25B and Cdc25C (PharMingen) as previously described (24). Briefly, total solubilized cellular protein (10 µg) was separated by 12% SDS-PAGE and then transferred to nitrocellulose. The membrane was blocked with BLOTTO and then incubated with one of the primary Abs listed above for 18 h at 4°C. Membranes were washed, incubated with rabbit anti-mouse Ig (or anti-goat) serum conjugated to horseradish peroxidase (Fisher Scientific, Pittsburgh, PA); the blots were developed using chemiluminescence (New England Nuclear, Boston, MA). The relative quantity of each band was determined by scanning densitometry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). Fractions were collected
and monitored for both A280 and ISF activities; for these
purposes we defined 1 LD50 unit of ISF as that amount
(volume) of material required to reduce Con A-induced
[3H]TdR incorporation to 50% of the maximum. As shown in
Fig. 1A, ISF activity was eluted with 300–400 mM NaCl; the
active fractions were pooled, dialyzed, and applied to a
chromatofocusing column equilibrated at pH 7 (Mono P, Pharmacia). After
extensive washing, the column was eluted with a linear pH 7.0–6.0
gradient (Polybuffer, Pharmacia); >90% of the ISF activity was
obtained in a single fraction corresponding to pH 6.6 (Fig. 1B). The ISF activity was concentrated and further
fractionated by gel filtration chromatography (Superdex 75, Pharmacia).
Column fractions were monitored for ISF activity as described above
(Fig. 1C); ISF activity eluted from the column in a volume
that corresponds to a m.w. of 30–40 kDa. These fractions were further
assessed for purity by SDS-PAGE analysis; as shown in Fig. 1D, the ISF obtained by this purification scheme yields a
single band corresponding to a Mr of
approximately 35 kDa.
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, the first 15 amino acids of the
N-terminus of the protein were identified. Comparison of this sequence
to known protein sequences revealed 95% identify with the deduced
amino acid sequence of the CdtB protein of H. ducreyi.
Therefore, we used a series of degenerative primers based upon both the
A. actinomycetemcomitans and H. ducreyi protein
sequences to determine the entire gene sequence of the A.
actinomycetemcomitans ISF (cdtB) gene (Fig. 2). The
gene consists of 848 bp with a RBS site just upstream to the start
codon (residue 34). Analysis of the deduced amino acid sequence
revealed that the A. actinomycetemcomitans cdtB encodes for
a protein consisting of 283 amino acids with a molecular mass of 32
kDa. Moreover, the gene encodes an additional 22 amino acids that
comprise a signal sequence. Comparison of the complete A.
actinomycetemcomitans cdtB gene sequence with other cdt
genes indicates 94% identify with the cdtB of H.
ducreyi and only 66 and 59% identities with the cdtB
genes of C. jejuni and E. coli, respectively.
Likewise, as shown in Table I, analysis
of the deduced amino acid sequence of the CdtB proteins indicates that
the A. actinomycetemcomitans protein is almost identical
(94%) with the H. ducreyi CdtB protein; identities with
C. jejuni or E. coli are 60 and 57%,
respectively. Further sequencing upstream and downstream from the
A. actinomycetemcomitans cdtB gene identified genes
corresponding to cdtA and cdtC, respectively (not
shown), suggesting that the structure of the A.
actinomycetemcomitans cdtB locus is similar to those of other
cdt gene clusters. Subsequent to these analyses Sugai et al.
(19) reported the cloning of a cdt toxin gene cluster from
A. actinomycetemcomitans strain Y4. Comparison of the
cdtB genes from the two A. actinomycetemcomitans
strains was identical with the exception of a single nucleotide; this
translated to a single amino acid difference in the deduced sequence of
the polypeptides.
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A and F). By contrast, the addition of PHA to T
lymphocyte cultures resulted in cell activation characterized by the
appearance of cells in both the S (14–20%) and G2M
(4–5%) phases of the cell cycle (Fig. 3, B and
G). Treatment of lymphocytes with 5–25 ng of ISF resulted
in a 4- to 6-fold increase in the population of G2M cells
(Fig. 3, C–E and H–J). In the presence of 5 ng
of ISF, the percentage of G2M cells increased to 34 and
17% for CD4+ and CD8+ T cells, respectively.
Increasing the ISF concentration to 25 ng did not further increase
the numbers of CD4+ cells in the G2/M phase of
the cell cycle, whereas the percentage of CD8+ T cells in
this phase of the cell cycle increased to 25%. No effect was observed
when the ISF concentration was reduced to <1 ng/ml (data not shown).
Furthermore, incubation of the ISF-treated T cells for 96 h failed
to significantly increase the percentage of G2/M cells over
that observed at 72 h.
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, treatment of HeLa cells with 25 ng of
ISF for 96 h resulted in a 2-fold increase in the percentage of
G2 cells over that observed in untreated cultures. In
contrast to human T cells, no effect of ISF was noted at either 10 or 5
ng of ISF. Similarly, treatment of HeLa cells with 25 ng of ISF
resulted in the typical morphologic changes previously described for
other toxins of this family (Fig. 5,
A and B). These cells were severalfold larger
than the untreated cells and exhibited the cytoplasmic elongation and
distension previously noted (25). These morphological alterations were
not a feature of human T cells arrested in the G2 phase of
the cell cycle as a result of exposure to ISF (Fig. 5, C and
D).
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, A and D)
contained amounts of RNA and protein below those detectable with the
fluorochromes employed. Both the G1 and G2M
populations of the PHA-activated cells contained elevated levels of
RNA; the mean channel fluorescence (MCF) values for these cells were
283 and 608, respectively. Treatment with ISF resulted in a small, but
reproducible, decrease in the content of RNA in both G1
(MCF = 240) and G2M (MCF = 587) cells. Similarly,
PHA-activated cells contained high levels of protein in both the
G1 (MCF = 272) and G2M (MCF = 616)
populations; the total protein content was also slightly reduced in
ISF-treated cells. The MCF values in these cells were 192
(G1 cells) and 560 (G2/M cells).
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in
relationship to the DNA content of the cells. Cyclin A was found
essentially only in G2/M cells; 66% of the
G2/M cells in the PHA-activated cultures stained positive
for cyclin A with a MCF of 438 (Fig. 7B). This compares to
70% cyclin A-positive cells with a MCF of 473 in the G2/M
population induced under the influence of ISF (Fig. 7C).
Likewise, cyclin B was expressed in 88% of the G2/M cells
of both the PHA-treated cells (control) and ISF-treated cells (Fig. 7, E and F); the MCF values were 421 and 472,
respectively. It should be noted that cyclins A and B were not detected
in the resting populations (no PHA; data not shown), nor were they
detected in any of the G1 cells from PHA activated cultures
with or without ISF.
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A, lane
1) do not express detectable levels of either form of Cdk1. By
comparison, PHA-activated T cells contain significantly elevated levels
of Cdk1 (Fig. 8A, lane 2); the majority of Cdk1
in these cells is present as the active, dephosphorylated kinase.
Activation of T cells in the presence of 5–25 ng of ISF results in
reduced expression of Cdk1 (Fig. 8A, lanes 3–5);
moreover, the majority of Cdk1 in these cells is in the inactive,
hyperphosphorylated form. Finally, we evaluated cells for the presence
of Cdc25, the phosphatase responsible for dephosphorylating Cdk1. As
shown in Fig. 8B, active T cells contain minimally
detectable levels of Cdc25; these levels increase in PHA-activated T
cells. Surprisingly, cells also treated with ISF contain up to more
than twice the amount of Cdc25 as that observed in cells activated by
PHA alone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The family of Cdts represent a novel toxic activity whose members have been identified in several Gram-negative bacterial species. These include some strains of E. coli, S. dysenteriae, Campylobacter sp., and more recently H. ducreyi and A. actinomycetemcomitans (14, 15, 17, 18, 19, 25). Regardless of the species, the genes encoding Cdt form a three-gene operon, cdtA, cdtB, and cdtC, encoding three proteins with similar molecular masses (20–35 kDa). The Cdt were originally defined based upon their effects on mammalian cell lines, HeLa, Hep-2, Vero, and Chinese hamster ovary cells. Exposure to Cdt causes these cells to arrest in the G2 phase of the cell cycle; these cells also exhibit morphologic alterations, including elongation and distension (20, 25). To date there is no documentation or evidence pointing to a possible host target cell for these putative virulence factors. Furthermore, several questions remain regarding the relationship among the three Cdt gene products: CdtA, CdtB, and CdtC. For instance, are the three proteins required to act together as a holotoxin for full expression of biologic activity or is each of the Cdt toxins fully capable of functioning individually to induce cell cycle arrest? Alternatively, only one of the peptides may be the active toxin. The other two components may be required for proper expression and/or secretion of the active toxin by the bacteria.
The studies described in this paper address several issues regarding the possible role that Cdt toxins might play during microbial infection. For instance, it is clear from our studies that at least one of the Cdt polypeptides, CdtB, is capable of expressing all of the biologic properties previously associated with this family of toxins. Thus, cell cycle arrest, cell elongation, and cell distention are induced in HeLa cells treated with 25 ng of highly purified A. actinomycetemcomitans ISF (CdtB), which is free of CdtA and CdtC. This was confirmed by the presence of a single protein band on SDS-PAGE and the generation of a single unambiguous N-terminal sequence by Edmund degradation of the purified ISF sample. However, our results do not exclude the possibility that CdtA and CdtC are biologically active as well or are required to activate CdtB. Indeed, Sugai et al. (19) report that none of the cdt genes expressed individually in E. coli possessed biologic activity, but expression of all three genes generated active toxin. In light of our results showing that purified CdtB is active, this would suggest that post-translational modification of CdtB may occur. Also, it is noteworthy that we previously reported that partially pure preparations of A. actinomycetemcomitans extracts were capable of inhibiting fibroblast proliferation (26). This activity was separate from the ISF and perhaps represents activity associated with either CdtA or CdtC.
Our studies also define the lymphocyte as a target for the family of Cdt toxins. Human T cells are at least 5-fold more sensitive to the action (i.e., cell cycle arrest) of CdtB then are HeLa cells. The basis for the tropism of CdtB for human T cells is not clear and is currently under investigation. In addition, the morphologic changes, for example cell elongation and distension, often seen in Cdt-treated cell lines do not occur with lymphocytes treated with A. actinomycetemcomitans ISF (CdtB) and arrested in the G2 phase of the cell cycle. Thus, it is possible that the terminology for this family of potential virulence factors may be misleading, especially with regard to their effects on possible host target cells that may be relevant to the pathogenesis of disease-caused Cdt-producing bacteria. In this regard, it may be more appropriate to define Cdt toxins in relation to their effects on lymphocytes (i.e., as immunosuppressive factors) than in the context of cell lines. Thus, impairment of lymphocytes to progress normally through the cell cycle would certainly be expected to lead to impaired immunity and benefit the microbe, leading to increased susceptibility of the host to infection and resulting disease. Indeed, several studies provide support for the idea that A. actinomycetemcomitans infection is associated with immune abnormalities (reviewed in 7 and 9). It has also been suggested that the failure of the host to generate a rapid and robust immune response may be the determining factor as to whether A. actinomycetemcomitans produces a generalized and wide-spread infection as opposed to localized disease.
There is, in fact, growing evidence that perturbation of the ability of lymphocytes to properly transit through the cell cycle represents a strategy used by several human pathogens to interfere with host immunity. For instance, the activity of A. actinomycetemcomitans ISF (CdtB), in particular, and the family of Cdts, in general, closely resemble the activity of the Vpr protein of human immunodeficiency virus type (27, 28). In this instance, a G2 cell cycle arrest may benefit the virus by leading to increased rates of HIV-1 replication. We have also shown that other pathogens, such as Fusobacterium nucleatum, use a similar strategy to inhibit lymphocyte function by interfering with the cell cycle; this immunosuppressive factor inhibits mitogen-activated T cells by causing an arrest in the mid-G1 phase of the cell cycle (24). The G1 arrest is associated with the failure of these cells to express proliferating cell nuclear Ag. It is also noteworthy that this class of microbial-derived cell cycle inhibitors may eventually prove to be useful as immunosuppressive drugs, as other naturally occurring agents, such as cyclosporine A, have revolutionized organ transplantation through their ability to prevent graft rejection.
Cell cycle regulation is generally accepted to be controlled at discrete points, called checkpoints, by complexes of cyclins and a family of related protein kinases, the Cdks (reviewed in 29 and 30). Specific to the G2 phase of the cell cycle is the activation of Cdk1, which is also necessary for cells to progress into the M phase. The activation of Cdk1 is dependent upon the availability of both cyclin A and cyclin B1 as well as phosphatases (Cdc25) that dephosphorylate Cdk1 (31). Our observations indicate that although cells treated with A. actinomycetemcomitans contain less total RNA and protein than do control cells (activated by PHA alone), the cyclin A and cyclin B1 contents of G2 phase cells are comparable to those observed in the control population of G2 cells. As for Cdk1, the ISF-treated T cells not only express less kinase relative to PHA controls, but the majority of Cdk1 in these cells is in the inactive, hyperphosphorylated form. This is in contrast to control cells (PHA alone), which contain higher total levels of Cdk1 and approximately two-thirds of which is present in the active, hypophosphorylated form. These results are in agreement with those of Comayras et al. (32), who observed that E. coli Cdt also prevented Cdk1 dephosphorylation in HeLa cells. Likewise, the HIV-1 Vpr protein, which also induces a G2 arrest in lymphocytes, blocks Cdk1 activation by preventing its dephosphorylation (33). Furthermore, it is noteworthy that other agents unrelated to the Cdts, such as 12-O-tetradecanoyl phorbol 13-acetate, induce G2 arrest by specifically repressing Cdk1 expression (34). Thus, Cdk1 activation is critical for transit of cells through G2 and into the M phase of the cell cycle, and it appears that the failure to express sufficient quantities of Cdk1 as well as to dephosphorylate and active the kinase may be responsible for the ISF-induced cell cycle arrest.
To further define the mechanism for the failure to activate Cdk1 in A. actinomycetemcomitans ISF-treated lymphocytes, we explored the possibility that the defect was related to the availability of Cdc25, the phosphatase responsible for dephosphorylating the Cdk1. However, our studies indicate that Cdc25 is not only present in these cells, but the ISF-treated cells contain higher levels of Cdc25 than do the control PHA-activated cells. Studies are currently being conducted to further define the molecular lesions in these G2 arrested T cells. In this regard, one possibility is that the ISF-treated cells contain damaged DNA, which is known to result in cell cycle arrest at either the G1 or G2 checkpoint (29, 30). Consistent with this possibility is our preliminary observation that ISF-treated lymphocytes arrested in G2 eventually undergo apoptosis. However, it is not clear at this time how A. actinomycetemcomitans ISF (CdtB) might directly or indirectly induce DNA damage, which, in turn, results in an arrest of the cell cycle at the G2 checkpoint.
In summary, the avoidance or modulation of the immune response by invading pathogens may be a critical event in determining the outcome of numerous infectious processes. Our results suggest that the Cdts must now be considered members of the class of immunoregulatory agents capable of impairing immunologic responsiveness by interfering with lymphocyte activation. Such immunosuppressive factors could lead to a state of hyporesponsiveness that favors colonization by the initiating organism or by other opportunistic organisms. Although the immunologic mechanism involved in the pathogenesis of A. actinomycetemcomitans infections has not been clearly defined, there is growing evidence that suppressed host defense mechanisms may contribute to the disease process. This disturbance would, in turn, adversely affect the development of normal 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, by impairing host protection.
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Acknowledgments |
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Footnotes |
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2 The A. actinomycetemcomitans strain 652 cdtB gene sequence has been submitted to the GenBank database; the accession number is AF102554.
3 Address correspondence and reprint requests to Dr. Bruce J. Shenker, Department of Pathology, University of Pennsylvania School of Dental Medicine, 4010 Locust Street, Philadelphia, PA 19104-6002. E-mail address:
4 Abbreviations used in this paper: ISF, immunosuppressive factor; Cdt, cytolethal distending toxin; Cdk1, cell cycle-dependent kinase-1; HPBMC, human PBMC; MCF, mean channel fluorescence.
Received for publication November 2, 1998. Accepted for publication January 14, 1999.
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 M. Handfield, H.V. Baker, and R.J. Lamont Beyond Good and Evil in the Oral Cavity: Insights into Host-Microbe Relationships Derived from Transcriptional Profiling of Gingival Cells Journal of Dental Research, March 1, 2008; 87(3): 203 - 223. [Abstract] [Full Text] [PDF]
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 L. M. Schaeffer, M. L. Schmidt, and D. R. Demuth Induction of Aggregatibacter actinomycetemcomitans leukotoxin expression by IS1301 and orfA Microbiology, February 1, 2008; 154(2): 528 - 538. [Abstract] [Full Text] [PDF]
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 B. J. Shenker, M. Dlakic, L. P. Walker, D. Besack, E. Jaffe, E. LaBelle, and K. Boesze-Battaglia A Novel Mode of Action for a Microbial-Derived Immunotoxin: The Cytolethal Distending Toxin Subunit B Exhibits Phosphatidylinositol 3,4,5-Triphosphate Phosphatase Activity J. Immunol., April 15, 2007; 178(8): 5099 - 5108. [Abstract] [Full Text] [PDF]
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T. Kitagawa, H. Hoshida, and R. Akada Genome-Wide Analysis of Cellular Response to Bacterial Genotoxin CdtB in Yeast Infect. Immun., March 1, 2007; 75(3): 1393 - 1402. [Abstract] [Full Text] [PDF]
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L. Cao, A. Volgina, J. Korostoff, and J. M. DiRienzo Role of Intrachain Disulfides in the Activities of the CdtA and CdtC Subunits of the Cytolethal Distending Toxin of Actinobacillus actinomycetemcomitans Infect. Immun., September 1, 2006; 74(9): 4990 - 5002. [Abstract] [Full Text] [PDF]
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D. S. Pouniotis, O. Proudfoot, V. Bogdanoska, K. Scalzo, S. Kovacevic, R. L. Coppel, and M. Plebanski Selectively Impaired CD8+ but Not CD4+ T Cell Cycle Arrest during Priming as a Consequence of Dendritic Cell Interaction with Plasmodium-Infected Red Cells J. Immunol., September 15, 2005; 175(6): 3525 - 3533. [Abstract] [Full Text] [PDF]
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 P. Kang, J. Korostoff, A. Volgina, W. Grzesik, and J. M DiRienzo Differential effect of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans on co-cultures of human oral cells J. Med. Microbiol., August 1, 2005; 54(8): 785 - 794. [Abstract] [Full Text] [PDF]
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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. Akifusa, W. Heywood, S. P. Nair, G. Stenbeck, and B. Henderson Mechanism of internalization of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans Microbiology, May 1, 2005; 151(5): 1395 - 1402. [Abstract] [Full Text] [PDF]
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W. Heywood, B. Henderson, and S. P Nair Cytolethal distending toxin: creating a gap in the cell cycle J. Med. Microbiol., March 1, 2005; 54(3): 207 - 216. [Abstract] [Full Text] [PDF]
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B. J. Shenker, D. Besack, T. McKay, L. Pankoski, A. Zekavat, and D. R. Demuth Induction of Cell Cycle Arrest in Lymphocytes by Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Requires Three Subunits for Maximum Activity J. Immunol., February 15, 2005; 174(4): 2228 - 2234. [Abstract] [Full Text] [PDF]
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 R. P. Dassanayake, Y. Zhou, S. Hinkley, C. J. Stryker, G. Plauche, J. T. Borda, K. Sestak, and G. E. Duhamel Characterization of Cytolethal Distending Toxin of Campylobacter Species Isolated from Captive Macaque Monkeys J. Clin. Microbiol., February 1, 2005; 43(2): 641 - 649. [Abstract] [Full Text] [PDF]
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G. N. Belibasakis, A. Johansson, Y. Wang, C. Chen, S. Kalfas, and U. H. Lerner The Cytolethal Distending Toxin Induces Receptor Activator of NF-{kappa}B Ligand Expression in Human Gingival Fibroblasts and Periodontal Ligament Cells Infect. Immun., January 1, 2005; 73(1): 342 - 351. [Abstract] [Full Text] [PDF]
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 M. Ohara, E. Oswald, and M. Sugai Cytolethal Distending Toxin: A Bacterial Bullet Targeted to Nucleus J. Biochem., October 1, 2004; 136(4): 409 - 413. [Abstract] [Full Text] [PDF]
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K. Saiki, T. Gomi, and K. Konishi Deletion and Purification Studies to Elucidate the Structure of the Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin J. Biochem., September 1, 2004; 136(3): 335 - 342. [Abstract] [Full Text] [PDF]
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 E. Haghjoo and J. E. Galan Salmonella typhi encodes a functional cytolethal distending toxin that is delivered into host cells by a bacterial-internalization pathway PNAS, March 30, 2004; 101(13): 4614 - 4619. [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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C. Mitchell, L. Gao, and D. R. Demuth Positive and Negative cis-Acting Regulatory Sequences Control Expression of Leukotoxin in Actinobacillus actinomycetemcomitans 652 Infect. Immun., October 1, 2003; 71(10): 5640 - 5649. [Abstract] [Full Text] [PDF]
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R. Yamano, M. Ohara, S. Nishikubo, T. Fujiwara, T. Kawamoto, Y. Ueno, H. Komatsuzawa, K. Okuda, H. Kurihara, H. Suginaka, et al. Prevalence of Cytolethal Distending Toxin Production in Periodontopathogenic Bacteria J. Clin. Microbiol., April 1, 2003; 41(4): 1391 - 1398. [Abstract] [Full Text] [PDF]
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K. P. Fong, L. Gao, and D. R. Demuth luxS and arcB Control Aerobic Growth of Actinobacillus actinomycetemcomitans under Iron Limitation Infect. Immun., January 1, 2003; 71(1): 298 - 308. [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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T. Sato, T. Koseki, K. Yamato, K. Saiki, K. Konishi, M. Yoshikawa, I. Ishikawa, and T. Nishihara p53-Independent Expression of p21CIP1/WAF1 in Plasmacytic Cells during G2 Cell Cycle Arrest Induced by Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Infect. Immun., February 1, 2002; 70(2): 528 - 534. [Abstract] [Full Text] [PDF]
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K. P. Fong, W. O. Chung, R. J. Lamont, and D. R. Demuth Intra- and Interspecies Regulation of Gene Expression by Actinobacillus actinomycetemcomitans LuxS Infect. Immun., December 1, 2001; 69(12): 7625 - 7634. [Abstract] [Full Text] [PDF]
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D. A. Lewis, M. K. Stevens, J. L. Latimer, C. K. Ward, K. Deng, R. Blick, S. R. Lumbley, C. A. Ison, and E. J. Hansen Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC Mutants in In Vitro and In Vivo Systems Infect. Immun., September 1, 2001; 69(9): 5626 - 5634. [Abstract] [Full Text] [PDF]
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D. C. Hassane, R. B. Lee, M. D. Mendenhall, and C. L. Pickett Cytolethal Distending Toxin Demonstrates Genotoxic Activity in a Yeast Model Infect. Immun., September 1, 2001; 69(9): 5752 - 5759. [Abstract] [Full Text] [PDF]
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S. Akifusa, S. Poole, J. Lewthwaite, B. Henderson, and S. P. Nair Recombinant Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Proteins Are Required To Interact To Inhibit Human Cell Cycle Progression and To Stimulate Human Leukocyte Cytokine Synthesis Infect. Immun., September 1, 2001; 69(9): 5925 - 5930. [Abstract] [Full Text] [PDF]
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M. Lara-Tejero and J. E. Galan CdtA, CdtB, and CdtC Form a Tripartite Complex That Is Required for Cytolethal Distending Toxin Activity Infect. Immun., July 1, 2001; 69(7): 4358 - 4365. [Abstract] [Full Text] [PDF]
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B. J. Shenker, R. H. Hoffmaster, A. Zekavat, N. Yamaguchi, E. T. Lally, and D. R. Demuth Induction of Apoptosis in Human T Cells by Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Is a Consequence of G2 Arrest of the Cell Cycle J. Immunol., July 1, 2001; 167(1): 435 - 441. [Abstract] [Full Text] [PDF]
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C. Elwell, K. Chao, K. Patel, and L. Dreyfus Escherichia coli CdtB Mediates Cytolethal Distending Toxin Cell Cycle Arrest Infect. Immun., May 1, 2001; 69(5): 3418 - 3422. [Abstract] [Full Text] [PDF]
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A. Mooney, M. Clyne, T. Curran, D. Doherty, B. Kilmartin, and B. Bourke Campylobacter upsaliensis exerts a cytolethal distending toxin effect on HeLa cells and T lymphocytes Microbiology, March 1, 2001; 147(3): 735 - 743. [Abstract] [Full Text]
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 J.M. Guthmiller, E.T. Lolly, and J. Korostoff Beyond the Specific Plaque Hypothesis: Are Highly Leukotoxic Strains of Actinobacillus Actinomycetemcomitans a Paradigm for Periodontal Pathogenesis? Critical Reviews in Oral Biology & Medicine, January 1, 2001; 12(2): 116 - 124. [Abstract] [Full Text] [PDF]
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H.H. Zadeh, A. Nalbant, and K. Park Large-scale Early in vitro Response to Actinobacillus actinomycetemcomitans Suggests Superantigenic Activation of T-cells Journal of Dental Research, January 1, 2001; 80(1): 356 - 362. [Abstract] [PDF]
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T. E. Hickey, A. L. McVeigh, D. A. Scott, R. E. Michielutti, A. Bixby, S. A. Carroll, A. L. Bourgeois, and P. Guerry Campylobacter jejuni Cytolethal Distending Toxin Mediates Release of Interleukin-8 from Intestinal Epithelial Cells Infect. Immun., December 1, 2000; 68(12): 6535 - 6541. [Abstract] [Full Text] [PDF]
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X. Cortes-Bratti, E. Chaves-Olarte, T. Lagergard, and M. Thelestam Cellular Internalization of Cytolethal Distending Toxin from Haemophilus ducreyi Infect. Immun., December 1, 2000; 68(12): 6903 - 6911. [Abstract] [Full Text] [PDF]
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 S. C. Kachlany, P. J. Planet, M. K. Bhattacharjee, E. Kollia, R. DeSalle, D. H. Fine, and D. H. Figurski Nonspecific Adherence by Actinobacillus actinomycetemcomitans Requires Genes Widespread in Bacteria and Archaea J. Bacteriol., November 1, 2000; 182(21): 6169 - 6176. [Abstract] [Full Text]
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 M. Lara-Tejero and J. E. Galán A Bacterial Toxin That Controls Cell Cycle Progression as a Deoxyribonuclease I-Like Protein Science, October 13, 2000; 290(5490): 354 - 357. [Abstract] [Full Text]
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B. J. Shenker, R. H. Hoffmaster, T. L. McKay, and D. R. Demuth 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 J. Immunol., September 1, 2000; 165(5): 2612 - 2618. [Abstract] [Full Text] [PDF]
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D. PURDY, C.M. BUSWELL, A.E. HODGSON, K. McALPINE, I. HENDERSON, and S.A. LEACH Characterisation of cytolethal distending toxin (CDT) mutants of Campylobacter jejuni J. Med. Microbiol., May 1, 2000; 49(5): 473 - 479. [Abstract] [Full Text] [PDF]
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V. Gelfanova, E. J. Hansen, and S. M. Spinola Cytolethal Distending Toxin of Haemophilus ducreyi Induces Apoptotic Death of Jurkat T Cells Infect. Immun., December 1, 1999; 67(12): 6394 - 6402. [Abstract] [Full Text] [PDF]
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X. Cortes-Bratti, C. Karlsson, T. Lagergard, M. Thelestam, and T. Frisan The Haemophilus ducreyi Cytolethal Distending Toxin Induces Cell Cycle Arrest and Apoptosis via the DNA Damage Checkpoint Pathways J. Biol. Chem., February 9, 2001; 276(7): 5296 - 5302. [Abstract] [Full Text] [PDF]
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