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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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to shedding new light on mode of action, our most recent studies have indicated that the A. actinomycetemcomitans ISF is a product of the cdtB gene, one of three genes encoding the cytolethal distending toxin (Cdt). The Cdts are a newly described family of heat-labile protein cytotoxins produced by several Gram-negative bacterial species. These include diarrheal disease-causing enteropathogens such as some Escherichia coli isolates, Campylobacter jejuni, and Shigella dysenteriae (13, 14, 15, 16). 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 strains Y4 and 652 (17, 18). The Cdts cause cell cycle arrest and in some cell lines a progressive cellular distension and finally death; it should be noted that the gross cellular change associated with Cdt activity is clearly different from those caused by other known toxins that induce rapid morphological alterations culminating in cell death (17, 19). There is now clear evidence that the 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.
To date, limited information is available that addresses the functional relationship(s) between the three Cdt proteins. In this regard, we have shown that highly purified CdtB from A. actinomycetemcomitans is not only capable of causing cell cycle arrest in human T cells, but is also able to induce all of the biological properties previously associated with the Cdt family: cell cycle arrest and morphological alterations in cell lines such as HeLa cells. In contrast, Stevens et al. (20) have reported contradictory findings and propose that CdtC is the biologically active component of H. ducreyi Cdt. Indeed, the possibility exists that the highly purified preparation of CdtB could have been contaminated with small amounts of CdtC (or CdtA). Therefore, the focus of this investigation was to further analyze the toxic activity of CdtB. In this study, we show that the A. actinomycetemcomitans cdtA, cdtB, and cdtC genes are coexpressed with two small open reading frames that reside upstream of cdtA. Furthermore, recombinant expression of cdtB alone generates an active toxin that is capable of inducing G2 arrest in human lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human PBMC were prepared by buoyant density centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech, Piscataway, NJ), and purified T cells were obtained by E-rosette formation, as described previously (8). 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 human PBMC 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 found to contain >98% T cells when stained with anti-CD3 mAb (Becton Dickinson Immunocytometry Systems, San Jose, CA) and analyzed by flow cytometry.
To measure Cdt-induced cell cycle arrest, 1-ml cultures of T cells (1 x 106 cells/ml) were activated with PHA (1 µg/ml; Abbott Laboratories, Abbott Park, IL), following pretreatment for 45 min with appropriate bacterial cell extract; the cells were incubated in RPMI 1640, antibiotics, and 2% heat-inactivated human AB sera. Flow cytometric analysis was used to analyze cell cycle distribution 72 h later, as previously reported (10). Briefly, T 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. Samples were analyzed on a Becton Dickinson FACStarPLUS flow cytometer. Propidium iodide fluorescence was excited by an argon laser operating at 488 nm and fluorescence measured with a 630/22-nm bandpass filter using linear amplification. A minimum of 15,000 events was collected on each sample; cell cycle analysis was performed using Modfit (Verity Software House, Topsham, ME).
Sequencing of A. actinomycetemcomitans cdt gene cluster
We previously isolated the complete cdt locus from an
A. actinomycetemcomitans strain 652 library in 
EMBL3 by
screening with the cdtB gene as a probe (12).
One clone with a 23-kbp insert containing the entire cdt
operon was chosen for further study; restriction analysis of this clone
indicated that the cdt gene cluster could be localized to a
9-kb fragment following digestion with EcoRI. Further
digestion of the 9-kb fragment with SmaI produced a 4-kb
fragment that was isolated and cloned into pUC19 to generate pUCAacdt2.
The sequences of the orf1, orf2, cdtA, and cdtC
were determined from DNA fragments flanking the cdtB gene,
which were derived from this clone. The A.
actinomycetemcomitans strain 652 cdt gene sequence has
been submitted to the GenBank database (AF102554).
Construction of plasmids expressing cdt genes
Several of the cdt gene constructs used in this study
were derived from the pUCAacdt2 plasmid, which contains the 4-kb insert
described above (also see Table I
). This
DNA fragment contains cdtA, cdtB, cdtC, a small upstream
open reading frame (orf2), and an additional 2.5 kb of
sequence downstream of the cdtC gene. To assess the role of
individual genes in generating biologically active toxin, a series of
plasmids were constructed that lacked the region downstream of
cdtC and/or one or more of the open reading frames present
in pUCAacdt2. These plasmids were prepared by first digesting pUCAacdt2
with NheI, which cleaves within the cdtA gene,
and EcoRI, which cleaves in the pUC multiple cloning region,
as shown in Fig. 1. The resulting 3.2-kb
DNA fragment contains the plasmid vector, orf 2, and the
first 250 residues of the cdtA gene (Fig. 1). PCR products
were then generated from primer pairs P3/P4, P3/P5, and P3/P6 (see
Table I) to generate products that
possess an upstream NheI site and a downstream
EcoRI site. The resulting fragments were subsequently
ligated to the 3.2-kbp product from the restriction digestion above.
Ligation with the 1946-bp P3/P4 product yielded pUCAacdt3, which
contains orf2, cdtA, cdtB, and
cdtC, but lacks the 2.5-kb sequence downstream of the
cdt operon. Plasmid pUCAacdt4, which contains
orf2, cdtA, and cdtB, was produced by
ligation with the 1350-bp P3/P5 product, and pUCAacdt7, containing only
orf2 and cdtA, resulted from ligation with the
430-bp P3/P6 product.
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). The resulting plasmid was ligated with the 716-bp PCR product
generated from primer pair P1/P2, which introduced the complete
orf1 sequence along with 466 bp of additional upstream
sequence and regenerated an intact copy of orf2. Two additional plasmids, pUCAacdt5 and pUCAacdt6, in which cdtB and cdtA, respectively, were inactivated, were also prepared. To construct pUCAacdt5, a 273-bp segment near the end of the gene was excised from cdtB by digestion of pUCAacdt3 with MscI and PmlI. The resulting plasmid contains orf2, cdtA, the truncated cdtB gene, and cdtC. pUCAacdt6 was prepared by digesting pUCAacdt3 with NheI, then blunting with Klenow polymerase followed by religation. The resulting plasmid thus contains a frameshift mutation in the cdtA gene.
All ligation mixtures were transformed into E. coli DH5
.
For analysis of immunosuppressive activity, 10-ml cultures were grown
in LB media supplemented with 100 µg/ml ampicillin to an
OD610 of 0.4. Samples of the culture supernatant
and cell pellet were collected for analysis. The cell pellet was
sonicated following a wash in 50 mM Tris (pH 8). Immunosuppressive
activity was defined as growth arrest of PHA-activated cells and was
assessed as an accumulation of cells in the G2
phase of the cell cycle over that observed in control (PHA alone)
cultures (12). It should be noted that cells were exposed
to the same amount of each cell extract based upon total protein
content, and the plasmids were classified based upon their ability to
express immunoinhibitory activity (G2
accumulation). Relative differences in immunosuppressive potency are
difficult to determine since it was not possible to account for
variations in the levels of expression between cultures of E.
coli transfected with each of the plasmids.
Expression of individual cdt genes and isolation of recombinant proteins
Plasmids that contain only cdtA, cdtB, or
cdtC were constructed as follows. PUCAacdtB-his encodes CdtB
with a C-terminal histidine tag, but lacking the signal sequence. The
plasmid was prepared by ligating the 801-bp P7/P8 PCR product (see
Table I; Fig. 1) into pGEM-T. The resulting plasmid was digested with
PstI and BamHI to remove the insert, which was
subsequently ligated into pUC19 and used to transform E.
coli DH5.
A plasmid that directs the expression of the CdtC protein was
constructed in pGEX-6p-2 to generate a GST fusion protein.
pUCAacdtC-GST was prepared by ligation of the 511-bp P9/P10 PCR product
(see Table I) into pGEM-T. The insert was then isolated from the
resulting plasmid by digestion with EcoRI and
BamHI, ligated into pGEX-6p-2, and transformed into E.
coli DH5.
Cultures of transformed E. coli pUCAacdtB-his were grown in 500 ml LB broth and induced with 0.1 mM IPTG; bacterial cells were harvested, washed, and resuspended in 50 mM Tris (pH 8) containing 100 mM NaCl, 2 mM EDTA, 1 mM PMSF, and 0.4 mg/ml lysozyme. The cells were frozen overnight, thawed, and sonicated. The expressed protein was contained in inclusion bodies that were isolated, solubilized, and refolded, as described by Li et al. (21). Briefly, the inclusion bodies were isolated by centrifugation (10,000 x g) and washed in 50 mM Tris (pH 8) containing 2 M Urea. The inclusion bodies were solubilized in 50 mM Tris (pH 8) containing 8 M urea and 100 mM 2-ME; solubilization was allowed to proceed for 2 h at 37°C. Following centrifugation, the solubilized protein was refolded by transferring the sample (1/100 dilution) into 100 mM Tris (pH 8) containing 0.5 M L-arginine and 1 mM glutathione disulfide; the refolded protein was then transferred to 20 mM Tris, pH 8, containing 0.5 M NaCl by passage through a Sephadex G25 column. The recombinant protein was then isolated on a histidine-binding column (His-Bind Quick Columns; Novagen, Madison, WI) and eluted from the column with 60 mM imidazole.
E. coli pUCAacdtC-GST was also found to produce inclusion bodies upon induction of expression. The inclusion bodies were isolated as described above. The refolded protein was transferred to PBS by passage through a Sephadex G25 column. The fusion protein was then purified using a commercial kit (Amersham Pharmacia Biotech Bulk Purification Module), and the recombinant protein separated from GST by digestion with PreScission Protease (Amersham Pharmacia Biotech), according to the manufacturer’s recommendations.
RNA extraction and RT-PCR
Total RNA was isolated from 200-ml cultures of A. actinomycetemcomitans strain 652, JP-2, and Y4 (22). Briefly, the bacteria were harvested by centrifugation and resuspended in cold 200 mM Tris (pH 8) containing 20 mM EDTA, 20 mM sodium azide, and 20 mM aurintricarboxylic acid (Sigma, St. Louis, MO). The cells were centrifuged at 9000 x g for 5 min and resuspended in 50 mM Tris, pH 7, containing 8% sucrose, 5% Triton X-100, 5 mM EDTA, and 10 mM vanadyl ribonucleoside complex (Life Technologies, Grand Island, NY). RNA was then isolated by phenol-chloroform extraction. Following precipitation in 3 M sodium acetate (pH 6), the pellets were resuspended in diethyl pyrocarbonate (DEPC)-water and the RNA isolated by centrifugation in CsCl. The RNA pellet was suspended in TE buffer, treated with RNase-free DNase (Promega, Madison, WI), and precipitated with ethanol at -70°C.
RT-PCR was conducted using a commercial kit (Perkin-Elmer RNA PCR kit;
Foster City, CA), according to the manufacturer’s protocol. Briefly,
1.5 µg RNA was converted to single-strand cDNA with primers derived
from the cdt operon sequence determined above. A series of
oligonucleotide primers were synthesized (see Table II) to generate
products that spanned all of the intergenic regions between
orf1 and cdtC. PCR reactions were performed on
the cDNA template after the addition of Taq DNA polymerase,
deoxynucleoside triphosphates (1 mM), and each primer (0.15 µM) with
a Perkin-Elmer Thermocycler under the following conditions (1 min,
45 s at 95°C; 50 cycles, 15 s at 95°C, 15 s at
50°C, 1 min at 72°C; 7 min at 72°C). DNA products were visualized
by ethidium bromide staining after electrophoresis in 1% agarose.
Controls included samples either lacking the reverse transcriptase or
the RNA template.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, we isolated and sequenced the A.
actinomycetemcomitans cdtB gene (12). Subsequent
sequencing upstream and downstream from the A.
actinomycetemcomitans strain 652 cdtB gene identified
genes exhibiting homology to known cdtA and cdtC
genes. In addition, two short open reading frames, designated
orf1 and orf2, were identified upstream of the
cdtA gene. As shown in Fig. 2, the size of these genes are 263 bp (orf1), 258 bp
(orf2), 669 bp (cdtA), 852 bp (cdtB),
and 589 bp (cdtC). Each of the genes is preceded by a
ribosome binding site, and relatively short intergenic domains lie
between each gene. The entire nucleotide sequence of the A.
actinomycetemcomitans strain 652 cdt locus was
submitted to GenBank (accession no. AF102554).
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, the deduced
amino acid sequence of the five A. actinomycetemcomitans
strain 652 open reading frames is highly conserved in A.
actinomycetemcomitans strains, Y4, 29522, and HK1651. One major
difference was noted: the nucleotide sequence upstream of
orf1 in strain 652 diverges from the corresponding sequence
in strain Y4. In addition, the deduced peptide sequences of CdtA, CdtB,
and CdtC from A. actinomycetemcomitans strain 652 are highly
similar to the corresponding Cdt proteins of H. ducreyi
(87–91% identity), but share only partial homology with the Cdt
proteins of C. jejuni (36–48%) and E. coli
(29–49%).
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). The results are
shown in Fig. 2. For example, as shown in lanes 1–3,
primers that span regions of orf1, orf2, and
cdtA generated the predicted product of 327 bp encompassing
the intergenic region between orf1 and orf 2, the
predicted 264-bp product spanning the intergenic region between
orf2 and cdtA, and the 521-bp product of both of
the above intergenic sequences, respectively. Two sets of primers were
used to link cdtA and cdtB (see Table II). The
first set of primers spanned the downstream half of cdtA
through the first 35 residues of cdtB and generated a RT-PCR
product of 518 bp, as predicted (Fig. 2, lane 4). A second
set of primers generated a 772-bp product that spanned the region,
including the last 53 residues of cdtA and approximately the first half
of cdtB (Fig. 2, lanes 5 and 6).
Finally, two sets of primers were used to show linkage of
cdtB and cdtC. These primer sets generated the
expected products of 772 and 157 bp (Fig. 2, lanes 6 and
7). As shown in Fig. 2A–C, similar results were
obtained from all three of the A. actinomycetemcomitans
strains examined. Control reactions consisted of samples that lacked
the RNA template or were not treated with the reverse transcriptase.
None of the controls produced detectable products (not shown). These
observations suggest that the five open reading frames are coexpressed
in a manner consistent with that of an operon. Analysis of A. actinomycetemcomitans strain 652 gene product immunoinhibitory activity
We previously showed that A. actinomycetemcomitans
expresses an ISF that is capable of impairing human T and B cell
function, and that this activity was associated with the CdtB protein
(10, 12). However, we could not exclude the possibility
that the CdtB preparations also contained other Cdt proteins such as
CdtC. To determine the contribution of each cdt gene in
generating toxic activity, plasmids containing defined deletions of
cdtA, cdtB, and/or the cdtC were
constructed (see Fig. 3), and cell
extracts derived from recombinant E. coli cells transformed
with each of the plasmids were analyzed for activity, as described in
Materials and Methods. As shown in Fig. 4A, control cultures exposed
to medium alone do not proliferate and exhibit a single peak of
propidium iodide fluorescence representing cells in the
G0G1 phase. In contrast,
activation of cells with PHA alone elicits a cell cycle profile
consisting of cells not only in the
G0G1 phase (80.1%), but
also in the S (14%) and G2/M (5.9%) phases as
well (Fig. 5B). Plasmid
pUCAacdt1, which contains all five of the cdt open
reading frames, expresses immunosuppressive activity that is evident
from the accumulation of T cells in the G2/M
phase (14.7%) of the cell cycle (Fig. 5D); similar results
were found using extracts of A. actinomycetemcomitans
containing native CdtB (not shown) (12). Extracts from
cells transformed with pUC alone had no effect on PHA-induced cell
cycle progression (Fig. 4C).
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E, treatment of T cells with
extracts from E. coli transformed with pUCAacdt2, which
lacks orf1, results in the accumulation of
G2/M cells (18.3%) over that observed in the PHA
control cultures. A third plasmid, pUCAacdt3, which lacks
orf1 as well as the 2.5-kb sequence downstream of
cdtC (see Fig. 3), also induces a G2
arrest (10.7% G2/M cells) (Fig. 4F).
These results strongly suggest that the entire functional
cdt gene cluster is contained in a fragment comprising only
orf2, cdtA, cdtB, and cdtC,
but does not exclude a possible role for orf1 in the
expression of toxin activity in A.
actinomycetemcomitans.
To determine the contribution of the individual cdtA,
cdtB, and cdtC genes, three additional plasmid
constructs were tested. Plasmid pUCAacdt4 contains the complete
sequence for cdtA and cdtB, but lacks the
cdtC gene. As shown in Fig. 4G, T cells
pretreated with cell extracts from E. coli transformed with
pUCAacdt4 exhibit cell cycle arrest; 13.8% of the cells are in the
G2/M phase representing a 2-fold increase over
that observed in the PHA control cultures (B). The
requirement for cdtA was assessed with pUCAacdt6, which
contains the complete cdtB and cdtC genes, but in
which cdtA has been functionally deleted by a frameshift
mutation near its 5' end. As shown in Fig. 4H, T cells
treated with pUCAacdt6 extracts exhibit 17.4%
G2/M cells. Another plasmid, pUCAacdt7,
containing only the cdtA gene and orf2, failed to
produce toxin activity (I). Finally, the requirement for
cdtB was probed with pUCAacdt5, which contains intact
cdtA and cdtC genes, but is capable of expressing
a truncated cdtB gene that lacks the last 250 residues of
the sequence. Surprisingly, this construct was also capable of inducing
cell cycle arrest, as indicated by an accumulation of
G2/M phase cells (23.2%; Fig. 4J).
The results above suggest that Cdt-induced cell cycle arrest in human T
cells may be associated with both CdtB and CdtC. To further evaluate
these proteins, two isogenic plasmids were constructed that express
only cdtB or cdtC. The CdtC peptide was expressed
as a GST fusion protein, while the cdtB gene was modified so
that the peptide contained a his tag at the C terminus (see
Materials and Methods). E. coli transformed with
each of the plasmids contained inclusion bodies. However,
solubilization of the inclusion bodies from each strain was effected in
8 M urea, and the protein obtained was subsequently refolded and
purified over a nickel column (CdtB) or glutathione-Sepharose (CdtC).
The isolation and subsequent purification of CdtB and CdtC from
inclusion bodies are shown in Fig. 5A. The purified peptides
were then assayed for their ability to cause cell cycle arrest in human
T cells (see Fig. 5B–E). B and C,
Represent cell cycle profiles from control (medium alone) and
PHA-activated T cells, respectively. The latter contains cells in the
G0G1 (80.2%), S (12.9%),
and G2/M (6.9%) phases of the cell cycle. As
shown in D, recombinant CdtB was capable of altering cell
cycle progression; 14% of the cells were in the
G2 phase, representing a 2-fold increase over the
PHA control cultures. These results suggest that the CdtB protein is
capable of inducing cell cycle arrest of human lymphocytes in the
absence of CdtC. In contrast, cells treated with recombinant CdtC (Fig. 5E) exhibit a cell cycle profile similar to that observed
with cells incubated with PHA alone; 5.3% of the cells were in
G2/M. It should be noted that a wide range of
concentrations was employed in preliminary studies; in all instances,
extracts prepared from the isogenic construct containing the
cdtC gene failed to alter the cell cycle progression of
PHA-activated T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There are conflicting reports regarding whether a single gene or multiple genes of the E. coli or H. ducreyi cdt operons encode the structural Cdt (reviewed in Ref. 24). Our previous study suggested that cdtB encoded the biologically active immunosuppressive factor in A. actinomycetemcomitans. However, we could not completely rule out the possibility that small amounts of CdtC were present in the CdtB preparations used for these studies. In contrast, Stevens et al. (20) suggest that cdtC encodes the structural toxin of H. ducreyi, and other reports suggest that two or even all three cdt genes are required for toxic activity in E. coli (24). Our results clearly show that the CdtB protein of A. actinomycetemcomitans, when expressed in a cdtA-/cdtC- background, induces G2 arrest in lymphocytes. Thus, cdtB encodes a biologically active component of the A. actinomycetemcomitans Cdt that is capable of acting independently of the other gene products encoded by the cdt operon.
Little information is available for any of the Cdts regarding the nature of the holotoxin that is secreted by the bacterial cell. Our results show that the CdtC protein, when expressed in a cdtA-/cdtB- background, had little effect on human lymphocytes. However, we cannot rule out a role for CdtC in immunosuppression in vivo. Indeed, pUCAacdt5, which contains the intact cdtA and cdtC genes as well as a truncated cdtB gene, also induced G2 arrest in lymphocytes. One possible explanation for this result is that the truncated CdtB protein, which is presumably expressed from pUCAacdt5, retains immunosuppressive activity. Alternatively, cdtC may also encode a biologically active immunosuppressive factor that was unable to refold properly when isolated from inclusion bodies in E. coli. 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. This alternative 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 (17, 25).
The role of the Cdts in pathogenesis remains to be clarified. Our results suggest that these proteins may be involved in modulating the immune response of the host. Such immunosuppressive activity could lead to a state of hyporesponsiveness that may contribute to the disease process by favoring colonization by the organism itself or by that of other opportunistic organisms. Thus, we propose that infection by Cdt-producing microbial species would impair host defense mechanisms and that this disturbance would, in turn, adversely affect the development of normal immunologic defense mechanisms. In addition to our demonstration that A. actinomycetemcomitans CdtB is a potent immunosuppressive factor, Gelfanova et al. (26) have recently shown that the H. ducreyi Cdt is also capable of impairing lymphoid function. It will be interesting to determine whether the Cdts of other Gram-negative organisms such as E. coli, Shigella, or Campylobacter are also capable of impairing lymphoid function. Demonstrating conservation of function in these cdt operons, which have diverged in sequence from the A. actinomycetemcomitans and H. ducreyi genes, might highlight important regions of the Cdt proteins that are required for interactions with lymphocytes.
In conclusion, our results show that CdtB derived from A. actinomycetemcomitans is active against human lymphocytes in the absence of other Cdt proteins and when expressed in a cdtA-/cdtC- background. However, it is possible that the mature toxin is actually comprised of multiple subunits including CdtC. It is also important that any definition of biologic activity must be made in the context of the target cell. In this regard, our studies clearly define the A. actinomycetemcomitans Cdt as an immunoinhibitory protein(s).
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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.
3 Abbreviations used in this paper: ISF, immunosuppressive factor; Cdt, cytolethal distending toxin; orf, open reading frame.
Received for publication February 11, 2000. Accepted for publication June 12, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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K. Deng and E. J. Hansen A CdtA-CdtC Complex Can Block Killing of HeLa Cells by Haemophilus ducreyi Cytolethal Distending Toxin Infect. Immun., November 1, 2003; 71(11): 6633 - 6640. [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. B. Lee, D. C. Hassane, D. L. Cottle, and C. L. Pickett Interactions of Campylobacter jejuni Cytolethal Distending Toxin Subunits CdtA and CdtC with HeLa Cells Infect. Immun., September 1, 2003; 71(9): 4883 - 4890. [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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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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L. A. Svensson, P. Henning, and T. Lagergard The Cytolethal Distending Toxin of Haemophilus ducreyi Inhibits Endothelial Cell Proliferation Infect. Immun., May 1, 2002; 70(5): 2665 - 2669. [Abstract] [Full Text] [PDF]
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 B. Henderson Oral bacterial disease and the science of cellular conversation J R Soc Med, January 2, 2002; 95(2): 77 - 80. [Full Text] [PDF]
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