Cytolethal Distending Toxin Sequence and Activity in the Enterohepatic Pathogen Helicobacter hepaticus

Abstract

Little is known about the molecular pathogenesis of hepatitis and enterocolitis caused by enterohepatic Helicobacter species. Sonicates of the murine pathogen Helicobacter hepaticus were found to cause progressive cell distension, accumulation of filamentous actin, and G(2)/M cell cycle arrest in HeLa cell monolayers. The genes encoding this cytotoxic activity were cloned from H. hepaticus. Three open reading frames with closest homology to cdtA, cdtB, and cdtC from Campylobacter jejuni were identified. Sonicates of a laboratory strain of Escherichia coli carrying the cloned cdtABC gene cluster from H. hepaticus reproduced the cytotoxic activities seen with sonicates of H. hepaticus. Cytolethal distending toxin activity is a potential virulence determinant of H. hepaticus that may play a role in the pathogenesis of Helicobacter-associated hepatitis and enterocolitis.

Full text

INFECTION AND IMMUNITY,
0019-9567/00/$04.00⫹0Jan. 2000, p. 184–191 Vol. 68, No. 1
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Cytolethal Distending Toxin Sequence and Activity in the
Enterohepatic Pathogen Helicobacter hepaticus
VINCENT B. YOUNG,
1,2
KIMBERLY A. KNOX,
3
AND DAVID B. SCHAUER
1,3
*
Division of Bioengineering and Environmental Health
1
and Division of Comparative Medicine,
3
Massachusetts Institute
of Technology, Cambridge, Massachusetts 02139, and Infectious Diseases Unit, Department of Medicine,
Massachusetts General Hospital, Boston, Massachusetts 02114
2
Received 27 July 1999/Returned for modification 10 September 1999/Accepted 14 October 1999
Little is known about the molecular pathogenesis of hepatitis and enterocolitis caused by enterohepatic
Helicobacter species. Sonicates of the murine pathogen Helicobacter hepaticus were found to cause progressive
cell distension, accumulation of filamentous actin, and G
2
/M cell cycle arrest in HeLa cell monolayers. The
genes encoding this cytotoxic activity were cloned from H. hepaticus. Three open reading frames with closest
homology to cdtA,cdtB, and cdtC from Campylobacter jejuni were identified. Sonicates of a laboratory strain of
Escherichia coli carrying the cloned cdtABC gene cluster from H. hepaticus reproduced the cytotoxic activities
seen with sonicates of H. hepaticus. Cytolethal distending toxin activity is a potential virulence determinant of
H. hepaticus that may play a role in the pathogenesis of Helicobacter-associated hepatitis and enterocolitis.
Enterohepatic Helicobacter species (EHS) are emerging as
important pathogens in the genus Helicobacter (15). In contrast
to Helicobacter pylori and other gastric Helicobacter species, the
EHS colonize the lower gastrointestinal tract, including the
ileum, cecum, colon, and biliary tree. However, in a manner
similar to the gastric Helicobacter species, the EHS cause per-
sistent infections associated with chronic inflammation and
epithelial cell hyperproliferation that can lead to neoplastic
disease (15, 19, 36).
H. hepaticus is an EHS that causes chronic active hepatitis
and typhlocolitis in immunocompetent mice (18, 37). In male
mice of susceptible strains, H. hepaticus infection leads to
chronic active hepatitis and liver cancer (16, 18, 20, 36). In
addition, H. hepaticus infection is sufficient to induce inflam-
matory bowel disease in certain immunodeficient mice (9, 23,
34, 35).
Little is known about the molecular pathogenesis of the
EHS. Urease, which has been demonstrated to be a virulence
factor in the gastric Helicobacter species H. pylori and H. mus-
telae, is not present in all of the EHS. H. hepaticus strains do
possess urease activity (16), but the role of urease in coloni-
zation or disease is not clear. Genes homologous to vacA,
encoding the vacuolating cytotoxin (Vac) (13), and cag, the
cytotoxin-associated genes that are part of a pathogenicity is-
land in H. pylori (1, 10), have not been definitively identified in
any EHS.
A cytolethal distending toxin (CDT) has been described in a
number of mucosal pathogens, including Campylobacter jejuni
(21) and other Campylobacter species (27), certain Escherichia
coli strains (7, 22), Shigella dysenteriae (26), Haemophilus du-
creyi (12), and Actinobacillus actinomycetemcomitans (31).
CDT causes progressive cell enlargement and eventual death.
The mechanism of CDT activity is reported to involve G
2
/M
cell cycle arrest in target cells, possibly by preventing activation
of cdc2 (11, 38). Additionally, the appearance of abnormal
accumulations of polymerized actin has been reported in Chi-
nese hamster ovary cells treated with the CDT from E. coli
9142-88 (5).
Campylobacter species are closely related to members of the
genus Helicobacter. Members of both genera are microaerobic,
motile, spiral- to curved-shaped, gram-negative bacteria that
colonize the mucus of the gastrointestinal tract. C. jejuni and
other Campylobacter species are an important cause of acute
gastroenteritis (3). EHS have also been recognized to cause
gastroenteritis, and similarities between these two groups of
organisms have resulted in misidentification of some EHS as
Campylobacter species in clinical and epidemiologic studies (6,
8, 28, 29). It has been suggested that CDT plays a role in the
pathogenesis of C. jejuni-induced gastroenteritis (38). Given
the similarities between campylobacters and helicobacters,
particularly the EHS, we examined H. hepaticus for nucleotide
sequence homology to the cdtABC gene cluster and for CDT
activity.
MATERIALS AND METHODS
Bacteria and cell lines. H. hepaticus ATCC 51449 was obtained from the
American Type Culture Collection (ATCC), Manassas, Va., and was cultured on
tryptic soy agar plates supplemented with 5% sheep blood. A microaerobic
environment was maintained in vented GasPak jars which were evacuated to ⫺20
mm Hg and then equilibrated with a gas mixture consisting of 80% N
2
, 10% H
2
,
and 10% CO
2
. An incubation temperature of 37°C was used for growth. Long-
term storage of bacteria was at ⫺70°C in tryptic soy broth with 30% glycerol.
E. coli XL-1 Blue and SOLR were obtained from Stratagene (La Jolla, Calif.)
and maintained on Luria-Bertani broth agar plates with the appropriate antibi-
otic selection.
HeLa cells (CCL-2) were obtained from the ATCC and cultured in Dulbecco’s
modified Eagle’s medium supplemented with glutamine and 10% fetal calf se-
rum.
PCR and DNA sequence determination. Genomic DNA from plate-grown
bacteria was isolated by using a Qiagen QIAamp kit for small-scale preparations
or a Qiagen genomic G-100 kit for large-scale purification (Qiagen Inc., Santa
Clarita, Calif.). Kits were used in accordance with the recommendations of the
manufacturer.
The degenerate primers VAT2 and WMI1, originally used to identify the cdtB
gene in C. jejuni (27), were synthesized (IDT, Coraville, Iowa) and used for PCR.
PCR was performed by using Pharmacia Ready-To-Go PCR beads (Amersham
Pharmacia Biotech Inc., Piscataway, N.J.). Reactions were set up with 1 ␮l
(approximately 100 ng) of template DNA, 20 pmol of each primer, and enough
water for a total volume of 25 ␮l. This yielded a reaction containing 1.5 U of Taq
polymerase, 10 mM Tris-HCl (pH 9.0 at room temperature), 50 mM KCl, 1.5
mM MgCl
2
, 200 ␮M each nucleotide, and stabilizers, including bovine serum
albumin (BSA). The reaction mixtures were overlaid with 50 ␮l of mineral oil
and subjected to amplification in a DNA thermal cycler (Perkin-Elmer model
* Corresponding author. Mailing address: MIT, Rm. 56-787, Cam-
bridge, MA 02139. Phone: (617) 253-8113. Fax: (617) 258-0225. E-
mail: schauer@mit.edu.
184

FIG. 1. Nucleotide sequence of the cdtABC gene cluster from H. hepaticus. The three presumed ribosomal binding sites (underlined) for each open reading frame
are shown along with the deduced amino acid sequences. Also indicated (bold) are the sites at which the degenerate PCR primers VAT2 and WMI1 bind. Forward
primer VAT2 and the predicted site of reverse primer WMI1 are expected to yield a 505-bp amplicon. The actual binding site of the reverse primer WMI1* along with
forward primer VAT2 produced an observed product of 768 bp.
185

480; PE Biosystems, Foster City, Calif.). The cycling conditions were as follows:
initial denaturation at 94°C for 4 min, followed by 30 cycles of denaturation at
94°C for 1 min, annealing at 42°C for 1.5 min, and extension at 72°C for 1.5 min.
A final extension at 72°C for 4 min was performed. Ten microliters of each
reaction mixture was analyzed by electrophoresis in a 1.0% agarose gel and
visualized after staining with ethidium bromide.
Bands of interest were excised from the gel and purified by using a gel band
purification kit (Amersham Pharmacia Biotech) in accordance with the recom-
mendations of the manufacturer. Purified fragments were ligated into the Pro-
mega pGEM T-easy vector (Promega, Madison, Wis.).
DNA sequencing on an ABI model 377 PRISM automated DNA sequencer
was performed by the core laboratory at the Massachusetts General Hospital
(Boston). DNA sequence analysis was performed on a Macintosh G3 computer
using the MacVector 6.5 software package (Oxford Molecular, Campbell, Calif.).
Preparation of bacterial sonicates. H. hepaticus cultures grown for 48 h on
three 100-mm-diameter plates were harvested into 1 ml of phosphate-buffered
saline (PBS). The bacteria were disrupted by six 30-s pulses on ice with a
VirSonic 50 sonicator (Virtis, Gardiner, N.Y.). Debris was removed by centrif-
ugation at 16,000 ⫻gin an Eppendorf model 5415 centrifuge (Eppendorf
Scientific, Westbury, N.Y.), followed by filtration through a 0.2-␮m-pore-size
filter. Aliquots of the preparations were stored at ⫺70°C.
Cultures of E. coli harboring the cloned cdtABC gene cluster from H. hepaticus
were grown for 18 h in 5 ml of Luria-Bertani broth supplemented with ampicillin
at 100 ␮g/ml. Bacteria were harvested by centrifugation and then suspended in
1 ml of PBS. Preparation of sonicates was then performed as described above.
Tissue culture assay for CDT activity. HeLa cells were seeded onto 13-mm-
diameter circular glass coverslips in 24-well tissue culture plates at a density of
2⫻10
3
per well. Twenty microliters of bacterial sonicate was added to each well,
and the plates were incubated in 5% CO
2
at 37°C. At appropriate time points,
coverslips were washed with PBS and then stained with Diff-Quik modified
Wright stain (Baxter Healthcare, Miami, Fla.) and mounted for visualization by
light microscopy.
Immunofluorescence microscopy. Coverslips were washed with PBS and then
fixed with a solution of 3.7% formaldehyde in PBS for 10 min at room temper-
ature. After washing with PBS, cells were permeabilized with a solution of 0.1%
Triton X-100 in PBS for 10 min at room temperature. The coverslips were
washed again with PBS and then stored at 4°C in PBS with 0.5% BSA until they
were stained.
Polymerized actin was stained with phalloidin labeled with Texas red (Molec-
ular Probes, Eugene, Oreg.), and the nuclei were stained with Hoechst 33342 as
described previously (39). Photographs were taken on a Nikon Labophot micro-
scope (Nikon, USA, Melville, N.Y.) with T-Max 100 film (Kodak, Rochester,
N.Y.).
Flow cytometry. Twenty-five-square-centimeter tissue culture flasks were
seeded with 3 ⫻10
5
HeLa cells. One hundred microliters of bacterial sonicate
was added to each flask, and then the flasks were incubated in 5% CO
2
at 37°C.
After 24, 48, and 72 h, cells were removed by trypsinization and transferred to a
1.5-ml microcentrifuge tube. Cells were pelleted by centrifugation at 735 ⫻gfor
3 min and resuspended in 3% polyethylene glycol 8000–2.5 ␮g of propidium
iodide per ml–9 U of RNase per ml–0.1% Triton X-100–0.001% BSA in 4 mM
sodium citrate. The cells were incubated for 20 min at 37°C and then mixed with
an equal volume of 3% polyethylene glycol 8000–2.5 ␮g of propidium iodide per
ml–0.1% Triton X-100–0.001% BSA in 0.4 M NaCl. Cells were incubated at 4°C
for at least 1 h before performance of DNA content analysis on a FACScan flow
cytometer (Becton Dickinson, Franklin Lakes, N.J.) using the Cell Quant soft-
ware for data acquisition. Data analysis was performed by using the ModFit
program on 10
4
cells for each experiment.
Genomic library construction and screening. Two genomic H. hepaticus li-
braries were constructed by using the insertion vector ␭ZAPII (Stratagene).
Briefly, genomic DNA from H. hepaticus ATCC 51449 was partially digested with
FIG. 2. Comparison of the predicted amino acid sequences of CdtA, CdtB, and CdtC from H. hepaticus ATCC 51449 (Hh) and C. jejuni 81-176 (Cj). Colons indicate
identical amino acids, and conserved amino acids are indicated by periods. Dashes indicate gaps introduced by the MacVector ClustalW alignment program.
186 YOUNG ET AL. INFECT.IMMUN.

the restriction enzyme Tsp509I. DNA with a length of ⱖ5 kb was ligated into the
EcoRI site of the vector. The libraries were screened by DNA hybridization using
the PCR amplicon generated by amplification of H. hepaticus genomic DNA with
the primers VAT2 and WMI1 (27). An [␣-
32
P]dCTP-labeled probe was gener-
ated by using a random primer DNA labeling kit (Ready-To-Go DNA labeling
beads; Amersham Pharmacia Biotech). The radioactive probe was used to screen
10
5
recombinant bacteriophage. Probe-positive plaques were identified and sub-
cloned into the plasmid vector pBluescript SK(⫺) using the in vivo excision and
recircularization features of the ␭ZAPII vector. E. coli bacteria carrying these
recombinant plasmids were further characterized by restriction mapping and by
screening for CDT activity.
Nucleotide sequence accession number. The nucleotide sequence of the H.
hepaticus cdtABC gene cluster has been entered in the GenBank database under
accession no. AF163667.
RESULTS
H. hepaticus possesses cdtABC nucleotide sequence homol-
ogy. The degenerate primers VAT2 and WMI1 (27) amplify a
494-bp fragment of the cdtB gene from C. jejuni. Amplification
of H. hepaticus genomic DNA with these primers produced a
larger than expected amplicon of approximately 750 bp (data
not shown). The complete nucleotide sequence of this ampli-
con was determined, and the deduced amino acid sequence
exhibited significant homology to the published CdtB sequence
of C. jejuni. The predicted H. hepaticus peptide fragment ex-
hibited 57% identity and 72% similarity to the C. jejuni gene
product. The larger than expected size of the amplicon was a
consequence of the WMI1 primer annealing to a site 264 bp
downstream of the anticipated target site (Fig. 1).
By using this PCR product, lambda clones containing ho-
mology to cdtB were isolated and corresponding plasmid
clones were generated. DNA sequence analysis of these clones
revealed that H. hepaticus has three closely linked open read-
ing frames corresponding to the entire cdtABC gene cluster
(Fig. 1). The deduced amino acid sequences had the closest
homology to the proteins encoded by the cdtABC gene cluster
from C. jejuni (Fig. 2). The amino acid homologies were as
follows: CdtA, 30% identity and 44% similarity; CdtB, 56%
identity and 72% similarity; CdtC, 40% identity and 55% sim-
ilarity.
Sonicates of H. hepaticus cause morphologic changes in
HeLa cell monolayers. Because the presence of cdtABC nucle-
otide sequence homology was demonstrated in DNA from H.
hepaticus, we sought to determine whether sonicates of this
organism exhibited CDT activity. HeLa cells treated with soni-
cates of H. hepaticus showed marked cellular distension (Fig.
3B). The distended cells also exhibited nuclear enlargement,
and approximately 15% of the affected cells were found to be
multinucleated. Occasionally, nuclear irregularities and frag-
mentation were also seen in HeLa cells treated with sonicates
of H. hepaticus.
The cytoplasmic distension induced by sonicates of H. he-
paticus was readily apparent, but the extent of cellular margins
was underestimated by using light microscopy following the
application of this modified Wright stain. To further examine
the cellular structure of treated cells and to detect associated
cytoskeletal changes, monolayers were stained with fluores-
cently labeled phalloidin to visualize filamentous actin (F-ac-
tin). Phalloidin staining revealed the full extent of the enlarge-
ment of cells in monolayers treated with sonicates of H.
hepaticus (Fig. 4). There also appeared to be an increase in the
amount of F-actin present in cells in affected monolayers. Nu-
clear staining with the fluorescent DNA stain Hoechst 33342
confirmed the nuclear enlargement, multinucleation, and nu-
clear fragmentation visualized by light microscopy.
Sonicates of H. hepaticus cause G
2
/M cell cycle arrest in
cultured cells. To examine whether the morphologic changes
observed in cells treated with sonicates of H. hepaticus were
also associated with cell cycle arrest, the DNA content of
treated cells was determined by flow cytometry. In untreated
cell monolayers, the fraction of cells with a DNA content of 4N
was consistently 8 to 10% (Fig. 5A). In monolayers treated
with sonicates of H. hepaticus, an increase in the fraction of
cells with a DNA content of 4N was seen 24 h after sonicate
addition (Fig. 5D). The fraction of cells with a DNA content of
4N increased at 48 h and reached a maximum by 72 after
sonicate addition (Fig. 5G and J). In addition, by 72 h, there
was a significant fraction of cells with a DNA content of 8N
among cells treated with H. hepaticus sonicates. Examination
of the size of these cells, as judged by fluorescent width, indi-
cates that these are probably multinucleated cells, as opposed
to cellular aggregates. This is consistent with the observation
that multinucleated cells were present in treated monolayers
(Fig. 3).
Cytopathic activity of E. coli carrying the cdtABC gene clus-
ter from H. hepaticus. Sonicates of the 15 E. coli clones har-
boring the recombinant plasmids generated from the genomic
library screen were examined for a cytopathic effect on HeLa
cell monolayers. Sonicates of 3 of the 15 clones produced a
cytopathic effect on cultured HeLa cell monolayers which was
indistinguishable from that produced by sonicates of H. hepati-
cus. Representative clones were selected for further character-
ization. HeLa cell monolayers treated with sonicates of E. coli
carrying the H. hepaticus cdt locus were examined for cytoskel-
etal and nuclear rearrangements over time by fluorescence
FIG. 3. Cytopathic effect of H. hepaticus CDT on HeLa cells. Compared to
untreated cells (A), cells treated with sonicates from H. hepaticus (B) exhibited
marked cytoplasmic distension along with nuclear enlargement, multinucleation,
and nuclear fragmentation. Magnification, ⫻250.
VOL. 68, 2000 HELICOBACTER HEPATICUS CDT 187

microscopy. At 24 h after sonicate addition, nuclear fragmen-
tation was observed (Fig. 5F) but the majority of cells still had
a normal size and actin ultrastructure. By 48 h, more nuclear
abnormalities were observed (Fig. 5I) and cell distension be-
came apparent, along with an increase in the amount of F-actin
(Fig. 5H). At 72 h, the cells in monolayers treated with soni-
cates of an E. coli CDT clone (Fig. 5K and L) were indistin-
guishable from those treated with H. hepaticus sonicates (Fig.
3).Cell cycle analysis demonstrated that the cytopathic effect
produced by the E. coli clones was also accompanied by G
2
/M
cell cycle arrest (Fig. 6). Mapping of the inserts from a number
of clones demonstrated that induction of a cytopathic effect
and cell cycle arrest required the presence of the entire
cdtABC gene cluster (Fig. 6).
DISCUSSION
In this study, we identified and characterized CDT activity in
the EHS H. hepaticus. The term CDT was coined to describe
an activity in culture supernatants of certain E. coli strains, as
well as C. jejuni, that causes progressive distension and even-
tually death of cultured mammalian cells (21, 22). We demon-
strate here that sonicates of H. hepaticus induce G
2
/M cell
cycle arrest in cultured HeLa cells and cause progressive cel-
lular enlargement of HeLa cells, accompanied by the appear-
ance of abnormal accumulations of F-actin. Coupled with the
presence of DNA sequences homologous to the cdtABC gene
cluster from C. jejuni and the cytopathic activity of E. coli
strains carrying the cloned H. hepaticus genes, these results
indicate that H. hepaticus possesses a toxin that is a novel
member of the CDT family.
The predicted CDT gene products from H. hepaticus had the
closest homology to the CDT from C. jejuni. As reported
previously (27, 31), the greatest homology is seen in the CdtB
amino acid sequence. This may indicate a conserved function
for the CdtB subunit; however, our results are in agreement
with others indicating that all three gene products are neces-
sary for cytotoxic activity in laboratory strains of E. coli.
The exact role of CDT in pathogenesis has not been clearly
determined. It has been proposed that CDT plays a role in the
pathogenesis of diarrheal illness. CDT activity in E. coli was
originally described in clinical isolates associated with gastro-
enteritis (22). A study of children with acute diarrhea showed
a trend toward an increased rate of isolation of CDT-produc-
ing E. coli among children with diarrhea compared to controls,
but this did not reach statistical significance (2). It should be
noted that several different virotypes of E. coli are associated
FIG. 4. Cytoplasmic and nuclear distension of HeLa cells treated with sonicates of H. hepaticus. Photomicrographs of untreated cells (A and B) and cells treated
with sonicates of H. hepaticus (C and D) for 72 h are shown. Fixed and permeabilized cells were double labeled for epifluorescence microscopy with Texas red-labeled
phalloidin (A and C) and Hoechst 33342 (B and D). Treated cells exhibited increased cell size and prominent stress fiber-like structures (C) as well, as increased nuclear
size (D). Magnification, ⫻500.
188 YOUNG ET AL. INFECT.IMMUN.

with diarrheal illness, each with distinct virulence determinants
(24). The presence of CDT in a particular E. coli strain may
represent only one of several virulence factors required for
gastrointestinal pathogenesis. Conversely, CDT has been dem-
onstrated in all C. jejuni isolates, as well as in other members
of the genus Campylobacter (14, 27). Demonstration of a role
for CDT in gastrointestinal pathogenesis mediated by C. jejuni
has not been reported. The well-characterized colonization
models for C. jejuni may not be optimal for establishing the
contribution of CDT to disease outcome, since they do not
reproduce the clinical syndrome of gastroenteritis associated
with C. jejuni infection. Partially purified preparations of the
FIG. 5. Time dependence of DNA content and cytopathic effect of HeLa cells treated with bacterial sonicates with CDT activity. Compared to control cells (A),
cells treated with sonicates from H. hepaticus (D, G, and J) showed a progressive increase in the fraction of cells with a DNA content of 4 N, with a maximal effect
being reached 72 h after sonicate addition (J). Compared to control cells (B and C), cells treated with sonicates from an E. coli strain harboring the cloned H. hepaticus
cdt locus showed a progressive increase in size with accumulations of polymerized actin (E, H, and K) and progressive nuclear abnormalities (F, I, and L). These changes
in morphology mirrored the progressive cell cycle block observed by flow cytometry. Magnification, ⫻400. PI, propidium iodide.
VOL. 68, 2000 HELICOBACTER HEPATICUS CDT 189

CDT from S. dysenteriae expressed in a laboratory strain of E.
coli have been shown to induce watery diarrhea in suckling
mice (25). However, the role of CDT in intact S. dysenteriae or
in diarrheagenic E. coli has not been demonstrated. An iso-
genic H. ducreyi cdtC mutant has been shown to maintain
virulence in the temperature-dependent rabbit model of ex-
perimental chancroid (30).
CDT is a candidate virulence factor in the EHS H. hepaticus
that may play a role in the pathogenesis of gastrointestinal
disease caused by these organisms. In mice, infection with H.
hepaticus is associated with a proliferative typhlitis and prolif-
erative hepatitis. In vitro, CDT appears to induce cell cycle
arrest, which suggests that the true targets of CDT activity are
not enterocytes or hepatocytes. It is possible that CDT causes
arrest of a cell type that inhibits epithelial cell proliferation.
Alternatively, CDT in H. hepaticus may have a role in modu-
lation of the immune response that allows persistence of the
organism. However, it should be noted that CDT activity and
nucleotide sequence homology do not appear to be present in
all EHS that cause gastroenteritis. We have identified CDT
activity and a homologue of the cdtB locus in the EHS H.
pullorum but have failed to demonstrate either CDT activity or
DNA homology to cdtB in the EHS H. cinaedi or H. fennelliae
(data not shown). It also remains to be determined if CDT is
a candidate virulence determinant for any of the gastric Heli-
cobacter species, but CDT homology is not present in the
genomic sequence of H. pylori (4, 33).
Another cytotoxic activity called granulating cytotoxin has
been described previously in H. hepaticus (32). This activity is
distinct from the Vac characterized in H. pylori. This cytotoxic
activity can be demonstrated on the CCL9.1 mouse liver cell
line and is characterized by the appearance of cytoplasmic
granules in intoxicated cells. The role of this toxin in patho-
genesis is also unknown. Other cell lines, including HeLa cells,
do not display cytopathic effects when treated with granulating
cytotoxin. The lack of effect on HeLa cells, which were used to
demonstrate CDT activity, indicates that the previously de-
scribed granulating cytotoxin is distinct from the CDT activity
described here.
We are currently characterizing CDT genes and activities in
other Helicobacter species. We have also developed methods
for targeted gene disruption in EHS (unpublished results). By
using these techniques and our well-characterized small-ani-
mal models of EHS infection and disease (9, 17, 37), we expect
to define the role of CDT in the pathogenesis of Helicobacter-
associated experimental inflammatory bowel disease and he-
patic disease.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grants AI01398 to
V.B.Y. and DK52413 to D.B.S. from the National Institutes of Health.
We thank James G. Fox and Stephen B. Calderwood for critical
review of the manuscript and Glenn Paradis for help with flow cytom-
etry.
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FIG. 6. Cytolethal distending toxin activity exhibited by sonicates of E. coli clones carrying plasmids containing various sections of the H. hepaticus cdtABC gene
cluster. The ability of sonicates of each clone to cause a typical CDT cytopathic effect and cell cycle arrest in cultured HeLa cell monolayers is indicated. Cell cycle
arrest is indicated by the percentage of cells in G
2
/M relative to the percentage seen in monolayers treated with H. hepaticus sonicates. Only sonicates of a clone
(pVBY9) that carries a plasmid with the entire cdtABC gene cluster were able to cause a cytopathic effect or cell cycle arrest. ND, not done.
190 YOUNG ET AL. INFECT.IMMUN.

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Editor: P. E. Orndorff
VOL. 68, 2000 HELICOBACTER HEPATICUS CDT 191