INFECTION AND IMMUNITY, Aug. 2006, p. 4496–4504
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 8
Modulation of Host Immune Responses by the Cytolethal
Distending Toxin of Helicobacter hepaticus
Jason S. Pratt,1,2Kacey L. Sachen,1Heather D. Wood,1
Kathryn A. Eaton,4,5and Vincent B. Young1,2,3*
Department of Microbiology and Molecular Genetics,1National Food Safety and Toxicology Center,2and Infectious Diseases Unit,
Department of Internal Medicine,3Michigan State University, East Lansing, Michigan 48824, and Unit for
Laboratory Animal Medicine4and Department of Microbiology and Immunology,5University of
Michigan School of Medicine, Ann Arbor, Michigan 48109
Received 28 March 2006/Returned for modification 3 May 2006/Accepted 11 May 2006
Persistent murine infection with Helicobacter hepaticus leads to chronic gastrointestinal inflammation and
neoplasia in susceptible strains. To determine the role of the virulence factor cytolethal distending toxin (CDT)
in the pathogenesis of this organism, interleukin-10-deficient (IL-10?/?) mice were experimentally infected
with wild-type H. hepaticus and a CDT-deficient isogenic mutant. Both wild-type H. hepaticus and the CDT-
deficient mutant successfully colonized IL-10?/?mice, and they reached similar tissue levels by 6 weeks after
infection. Only animals infected with wild-type type H. hepaticus developed significant typhlocolitis. However,
by 4 months after infection, the CDT-deficient mutant was no longer detectable in IL-10?/?mice, whereas
wild-type H. hepaticus persisted for the 8-month duration of the experiment. Animals infected with wild-type H.
hepaticus exhibited severe typhlocolitis at 8 months after infection, while animals originally challenged with the
CDT-deficient mutant had minimal cecal inflammation at this time point. In follow-up experiments, animals
that cleared infection with the CDT-deficient mutant were protected from rechallenge with either mutant or
wild-type H. hepaticus. Animals infected with wild-type H. hepaticus developed serum immunoglobulin G1
(IgG1) and IgG2c responses against H. hepaticus, while animals challenged with the CDT-deficient mutant
developed significantly lower IgG2c responses and failed to mount IgG1 responses against H. hepaticus. These
results suggest that CDT plays a key immunomodulatory role that allows persistence of H. hepaticus and that
in IL-10?/?mice this alteration of the host immune response results in the development of colitis.
Helicobacter species are responsible for chronic human and
veterinary infections (44). In humans, H. pylori infection can
last for decades, associated with a subclinical gastritis. Long-
term infection with H. pylori can lead to the development of
neoplastic disease, including gastric cancer and mucosa-asso-
ciated lymphoid tissue lymphomas (37).
In addition to H. pylori and other gastric Helicobacter spe-
cies, the enterohepatic Helicobacter species (EHS) have
emerged as veterinary and human pathogens also associated
with long-term infection and the development of neoplastic
disease (13, 44). The EHS H. hepaticus was originally discov-
ered as the causative agent for the development of chronic
hepatitis and hepatocellular cancer in A/JCr mice (15, 46). It
was subsequently determined that H. hepaticus infection in
mice with altered immune function was also associated with
the development of a condition that mimicked human inflam-
matory bowel disease (IBD) (3, 5, 22). Long-term infection
with H. hepaticus in animals that develop IBD can lead to the
development of colon cancer (9, 10, 28).
H. hepaticus and a number of other EHS have been shown to
produce a cytotoxin that is a member of the cytolethal distend-
ing toxin (CDT) family (4, 52, 54). CDT is a tripartite bacterial
toxin that is encountered in a number of pathogenic gram-
negative organisms, including Campylobacter jejuni and other
Campylobacter species, certain Escherichia coli strains, Shigella
dysenteriae, Haemophilus ducreyi, and Actinobacillus actinomy-
cetemcomitans (reviewed in references 24, 35, and 36).
The active subunit of CDT, CdtB, has structural and func-
tional homology to mammalian DNase I (8, 23, 32). It has been
proposed that this DNase activity is responsible for the cell
cycle arrest that is a key feature of the CDT-mediated cyto-
pathic effect in vitro (7, 17, 20, 33).
The role of CDT in the in vivo pathogenesis of organisms
that elaborate this toxin has been investigated. Fox and col-
leagues demonstrated that wild-type C. jejuni, but not an iso-
genic mutant lacking CDT, triggered gastroenteritis in NF-?B-
deficient mice (16). The same group recently reported that
CDT expression by H. hepaticus is required for long-term col-
onization of outbred Swiss Webster mice (18). We recently
reported that an isogenic H. hepaticus mutant that lacked CDT
production was able to colonize C57BL/6 interleukin-10-defi-
cient (IL-10?/?) mice, but colonization with the CDT-deficient
strain was associated with a significant reduction in IBD activ-
ity 6 weeks after infection compared to that in animals infected
with wild-type H. hepaticus (53).
These results suggest that CDT expression may represent a
bacterial adaptation that influences the interaction between
the bacterium and the host immune system. Therefore, to
determine more precisely the role of CDT in the modulation of
the host response to H. hepaticus, multiple infection studies
were carried out with C57BL/6 IL-10?/?mice challenged with
wild-type H. hepaticus and a CDT-deficient isogenic mutant.
* Corresponding author. Mailing address: Room B42, Food Safety
and Toxicology Building, Michigan State University, East Lansing, MI
48824. Phone: (517) 432-3100. Fax: (517) 432-2310. E-mail: youngvi
MATERIALS AND METHODS
Bacterial strains and cell lines. The wild-type H. hepaticus strain 3B1 (the type
strain, ATCC 51488) was obtained from the American Type Culture Collection
(Manassas, VA). The isogenic mutant 3B1::Tn20 was generated by transposon
shuttle mutagenesis with allelic exchange into H. hepaticus (53). 3B1::Tn20 has a
transposon inserted near the start of cdtA and no longer produces cytolethal
distending toxin (53).
Wild-type H. hepaticus and the CDT-deficient isogenic mutant strain were
grown at 37°C for 3 to 4 days in a microaerobic environment, which was main-
tained in vented GasPak jars without a catalyst after evacuation to ?20 mm Hg
and equilibration with a gas mixture consisting of 80% N2, 10% CO2, and 10%
H2. H. hepaticus was grown on tryptic soy agar (TSA) supplemented with 5%
sheep blood and with 20 ?g/ml chloramphenicol (all from Sigma, St. Louis, MO)
for the chloramphenicol-resistant transposon mutant.
Animals. All animal protocols were reviewed and approved by the Michigan
State University All University Committee on Animal Use and Care. Breeding
pairs of Helicobacter-free C57BL/6 IL-10?/?mice were obtained from the Jack-
son Laboratories (Bar Harbor, ME) and housed with autoclaved food, bedding,
and water, with cage changes performed in a laminar flow hood. For infection
studies, 4- to 6-week-old C57BL/6 IL-10?/?mice were transferred to the Uni-
versity Research Containment Facility at Michigan State University and housed
under the same conditions as the breeding pairs. Animals were housed in groups
of up to five animals per microisolator cage (Table 1).
Murine infection with H. hepaticus. H. hepaticus was harvested after 48 h of
growth on agar plates and resuspended in a small volume of tryptic soy broth.
The optical density (OD) at 600 nm of the inoculum was measured and 10-fold
serial dilutions of the inoculum plated to quantify the CFU used for infection.
Mice were inoculated with a single dose of a suspension of bacteria with an OD
of 1.0 at 600 nm (?1 ? 108CFU) in a volume of 0.2 to 0.3 ml. Bacteria were
introduced directly into the stomach with a 24-gauge ball-tipped gavage needle.
Control mice were inoculated with sterile tryptic soy broth.
Detection of H. hepaticus in mouse feces and tissues. Fecal pellets from the
animals in one cage were collected and pooled to monitor colonization status.
Culture for H. hepaticus was accomplished by homogenizing feces in 0.5 ?l of
phosphate-buffered saline and plating 50 ?l on TSA supplemented with 5%
sheep blood, 20 ?g/ml cefoperazone, 10 ?g/ml vancomycin, and 2 ?g/ml ampho-
tericin B. DNA was isolated from fecal pellets as described previously (47).
Briefly, fecal pellets were homogenized, and following a low-speed centrifu-
gation to remove large particulate matter, DNA was isolated using a com-
mercial kit (QIAGEN tissue kit; QIAGEN, Valencia, CA). DNA was isolated
from gastrointestinal tissue collected at the time of necropsy by using the
QIAGEN tissue kit.
Direct, single-stage PCR amplification to detect H. hepaticus was performed
using the primers 5? GCA TTT GAA ACT GTT ACT CTG 3? (B38) and 5? CTG
TTT TCA AGC TCC CC 3? (B39), which produce a 417-bp amplicon (42). PCR
was performed using 5 ml of template at approximately 100 ng/?l of DNA
extracted from fecal or tissue samples. Each 25-?l PCR mixture contained 20
pmol of each primer, 200 ?M of each deoxynucleoside triphosphate, and 1.5 U
of Taq DNA polymerase in a final concentration of 10 mM Tris-HCl, 50 mM
KCl, and 1.5 mM MgCl2(Ready To Go PCR beads; Amersham Pharmacia
Biotech, Piscataway, NJ). Cycling conditions included 30 cycles of 30 seconds at
94°C, 45 seconds at 54°C, and 45 seconds at 72°C. PCR products were visualized
by agarose gel electrophoresis.
To provide a measure of relative levels of fecal shedding of H. hepaticus, a
nested PCR system was developed. The first-stage PCR employs primers 5? GCT
ATG ACG GGT ATC C 3? (C97) and 5? ACT TCA CCC CAG TCG CTG 3?
(C05), which amplify the small-subunit rRNA genes from all known Helicobacter
species (14), yielding an approximately 1,200-bp amplicon. PCR was performed
using Ready To Go PCR beads. Cycling conditions included 30 cycles of 30
seconds at 94°C, 60 seconds at 58°C, and 90 seconds at 72°C. The nested stage
was performed using primers B38 and B39 (described above) with 1 ?l of the
reaction mixture from the primary PCR as the template. PCR products were
visualized by agarose gel electrophoresis. In our experience, this nested PCR has
sufficient sensitivity to detect 25 to 50 genome equivalents of H. hepaticus in a
volume of 1 ?l (approximately 100 ng) of DNA purified from mouse feces.
Mouse necropsy and histologic procedures. Mice were euthanatized by CO2
asphyxiation. Collection of the ileocecocolic junction and preparation of hema-
toxylin-and-eosin-stained sections for histopathologic examination were per-
formed as described previously (53). Histologic sections were examined using the
following scoring system for inflammation: 0, normal; 1, small multifocal lamina
proprial and/or transepithelial leukocyte accumulations; 2, coalescing mucosal
inflammation with or without early submucosal extension; 3, coalescing mucosal
inflammation with prominent multifocal submucosal extension with or without
follicle formation; and 4, severe diffuse inflammation of mucosa, submucosa, and
T-RFLP analysis. Terminal restriction fragment length polymorphism (T-
RFLP) analysis was performed as detailed previously (21). Briefly, PCR ampli-
fication employing primers targeting bacterial 16S rRNA genes (8F and 1492R
(41) was performed on each DNA sample. The 8F primer was linked to the
fluorescent dye 6-carboxyfluorescein (Integrated DNA Technologies, Coralville,
IA), and the 1492R primer was unlabeled. The PCR products were purified using
GFX purification columns (Amersham Pharmacia Biotech). Two hundred nano-
grams of purified PCR amplicon was cut individually with the restriction enzymes
HhaI and MspI (New England Biolabs, Beverly, MA) for 1 to 2 h at 37°C (27).
The DNA fragments were separated on an ABI 3100 Genetic Analyzer auto-
mated sequence analyzer (Applied Biosystems Instruments, Foster City, CA) in
GeneScan mode. The 5? terminal restriction fragments (TRFs) were detected by
excitation of the 6-carboxyfluorescein molecule attached to the forward primer.
The sizes and abundances of the fragments were calculated using GeneScan 3.7.
T-RFLP profiles were analyzed as follows. To standardize each profile for the
quantity of labeled DNA present in each sample, the sum of TRF peak heights
in each profile being compared was calculated. The sum of peak heights generally
varied less than twofold over all of the profiles. Each sum of TRF peak heights
was normalized to the lowest sum of peak heights of the comparison samples.
This yielded a correction factor that was applied to each peak in a given profile.
The resultant peak heights were filtered to eliminate peaks with a height below
the noise threshold (set at a relative fluorescence value of 50). The fraction of the
total signal provided by the H. hepaticus TRF was determined by dividing the
peak height of the H. hepaticus TRF by the sum of the total peak height in a given
Preparation of H. hepaticus antigens. H. hepaticus was cultured on TSA blood
plates and suspended in phosphate-buffered saline at an OD at 600 nm of 1.5 to
3.0. The suspension was then frozen at ?70°C, thawed, and pelleted at 5,000 rpm
for 10 min. The pellet was resuspended in B-PER I reagent (Pierce Chemical
Co., Rockford, IL) and then centrifuged at 13,000 rpm for 5 min. The protein
concentration of the supernatant was measured by the Lowry technique (Bio-
Rad protein assay; Bio-Rad Laboratories, Hercules, CA). Aliquots of the super-
natant were stored at ?70°C.
Evaluation of serum antibody responses to H. hepaticus. At necropsy, blood
was collected via cardiac puncture from CO2-asphyxiated mice. For a subset of
mice, blood was collected every 3 weeks during the course infection by saphenous
vein puncture. Blood was centrifuged at 10,000 rpm for 10 min, and the serum
was preserved with 15 mM sodium azide. An enzyme-linked immunosorbent
assay (ELISA) was used to detect H. hepaticus-specific serum immunoglobulin G
(IgG) and IgA.
Nunc Polysorb 96-well plates (Nalge Nunc International, Rochester, NY) were
coated with 100 ml of a 10-mg/ml concentration of H. hepaticus protein in
carbonate buffer (pH 9.6) overnight at 4°C. Plates were blocked with 1% bovine
serum albumin in phosphate-buffered saline. Serum was diluted 1/100, and bio-
tinylated secondary antibodies included goat anti-mouse IgG1 (Sigma, St. Louis,
MO) diluted 1/5,000 and goat anti-mouse IgG2c (Southern Biotech, Birming-
ham, AL) diluted 1/1,000. Incubation with extravidin-peroxidase (Sigma) at a
dilution of 1/1,000 was followed by incubation with 2,2?-azine-di(3-ethylbenzthia-
zoline sulfonate) (ABTS) horseradish peroxidase substrate (Pierce) for color
development. Optical density at 405 nm was recorded by an ELISA plate reader
(VERSAmax; Molecular Devices, Sunnyvale, CA).
Statistical analysis. Statistical analysis was performed using the JMP statistical
package (SAS, Cary, NC). Categorical inflammation scores were compared by
the nonparametric Wilcoxon rank sum test with statistical significance set to a P
value of ?0.05. ELISA data were analyzed using a one-way analysis of variance
(ANOVA), and the Tukey-Kramer honestly significant difference test was used
TABLE 1. Design of infection studies
No. of animals in study (sacrifice, days postchallenge)
4, 8, 14, 42)
Wild type (3B1)
VOL. 74, 2006 H. HEPATICUS CDT AND IMMUNITY 4497
to identify groups with significantly different means with an alpha level set to
An isogenic CDT-deficient H. hepaticus mutant demon-
strates delayed colonization kinetics and triggers minimal
typhlocolitis. We have previously shown that infection of IL-
10?/?mice with a CDT-deficient H. hepaticus mutant is asso-
ciated with decreased IBD activity 6 weeks after challenge
compared to that in animals infected with wild-type H. hepati-
cus (53). Both the CDT-deficient mutant and wild-type H.
hepaticus were detected by culture and PCR in the feces and
tissue of all animals at the end of this 6-week infection study.
In order to follow the early colonization kinetics and the
development of typhlocolitis in H. hepaticus-infected C57BL/6
IL-10?/?mice, a time course infection study was performed.
The H. hepaticus mutant 3B1::Tn20 is deficient in CDT pro-
duction due to a transposon insertion in the cdtA gene of the
type strain of H. hepaticus (53). Groups of three mice were
challenged via oral gavage with either wild-type H. hepaticus or
the isogenic CDT-deficient mutant (Table 1). Animals were
sacrificed at 2, 4, 8, 14, and 42 days after experimental chal-
lenge and the cecal tissue harvested to assess the development
of inflammation and to determine relative levels of coloniza-
tion of the mucosa by H. hepaticus.
Animals infected with the CDT-deficient mutant 3B1::Tn20
did not exhibit significant cecal inflammation at any time after
challenge (Fig. 1). Animals infected with wild-type H. hepati-
cus, however, developed histologically significant typhlocolitis
as early as 8 days after challenge (Fig. 1). Uninfected control
animals did not develop any significant inflammation (data not
Although all animals challenged with either wild-type H.
hepaticus or the CDT-deficient mutant had the organism de-
tectable by culture or H. hepaticus-specific PCR of feces, we
wished to compare the colonizations of the cecal mucosa by
each isogenic strain. We have previously reported that the 16S
rRNA gene-encoding sequence-based technique of T-RFLP
analysis can be used to temporally follow the colonization of
the cecal mucosa of wild-type mice by wild-type H. hepaticus
(21). We used T-RFLP analysis to compare the colonization
kinetics of wild-type H. hepaticus and the CDT-deficient mu-
tant in the IL-10?/?mice. Wild-type H. hepaticus rapidly col-
onized the cecal mucosae of IL-10?/?mice, comprising ap-
proximately 50% of mucosa-associated microbiota by 14 days
postchallenge (Fig. 2). These kinetics are similar to those for
the colonization of the cecal mucosae of wild-type mice as
reported previously (21). The CDT-deficient mutant appeared
to have somewhat delayed kinetics, not being detectable as a
major TRF at 8 days after challenge and not becoming the
dominant TRF until 42 days after challenge. However, at 42
days, the CDT-deficient mutant was the dominant component
of the mucosa-associated microbiota in two of the three mice
and was easily detectable in the remaining animal (Fig. 2).
Thus, the CDT-deficient mutant can still become a significant
member of the cecal-associated microbiota, but it does so with
delayed kinetics compared to wild type and does not trigger
An isogenic CDT-deficient H. hepaticus mutant exhibits a
long-term colonization defect. To determine the role of CDT
in the development of long-term sequelae of H. hepaticus in-
fection, additional infection studies were initiated using the
isogenic CDT mutant. Four- to 6-week-old C57BL/6 IL-10?/?
mice were infected with wild-type H. hepaticus strain 3B1 and
the CDT-deficient isogenic mutant 3B1::Tn20. An equal num-
FIG. 1. Categorical inflammation scores following the temporal de-
velopment of typhlocolitis in IL-10?/?mice infected with wild-type H.
hepaticus or a CDT-deficient isogenic mutant. Animals infected with
wild-type H. hepaticus developed severe inflammation within 8 days
after experimental challenge, whereas animals infected with the CDT-
deficient mutant did not develop significant colitis.
FIG. 2. Monitoring of the colonization of IL-10?/?mice with H.
hepaticus. T-RFLP analysis was used to determine the fraction of the
mucosa-associated microbiota represented by H. hepaticus in animals
infected with the wild type or the CDT-deficient mutant. The CDT-
deficient mutant colonizes with slightly delayed kinetics compared to
the wild type but by 42 days after infection reaches a similar level
among the cecal microbiota.
4498 PRATT ET AL.INFECT. IMMUN.
ber of control mice were sham infected with sterile culture
broth. There were 10 animals in each experimental group, for
a total of 30 animals in this study (Table 1). Animals in all
experimental groups were monitored for H. hepaticus coloni-
zation by H. hepaticus-specific PCR amplification of DNA iso-
lated from fresh fecal pellets (42) and, at certain time points,
by selective culture for H. hepaticus from feces. Since mice are
coprophagic, we used the “cage” as the unit of infection when
assessing colonization. Thus, there were two cages in each
experimental group, and at all times, the results were concor-
dant for each of the two cages for each group.
C57BL/6 IL-10?/?mice were initially colonized by wild-type
H. hepaticus 3B1 and the isogenic CDT-deficient mutant
(Table 2). Animals challenged with wild-type H. hepaticus
remained colonized for the entire duration of each experiment.
Conversely, although the CDT-deficient mutant 3B1::Tn20 was
detectable at 61 days postinfection (p.i.) in IL-10?/?animals,
this mutant was not found by PCR or culture at 115 days p.i.
These mice remained negative for H. hepaticus for the remain-
der of the experiment. At all times when colonization was
assessed by both culture and PCR for H. hepaticus, there was
concordance between the two methods (Table 2).
Mice that clear infection with a CDT-deficient H. hepaticus
mutant exhibit minimal typhlocolitis. Histologic examination
of the ileocecocolic junctions of the IL-10?/?mice challenged
with wild-type 3B1 and the isogenic mutant was performed at
251 days postinfection. Of the 30 animals in this experiment,
one had to be sacrificed prior to this time. One animal infected
with wild-type 3B1 developed rectal prolapse at 130 days p.i.
Nine of the 10 control animals had no or minimal evidence
of inflammation at the ileocecocolic junction at the time of
necropsy (Fig. 3A). One of the control animals had significant
inflammation and hyperplasia (Fig. 4). Feces and tissue from
this animal were examined carefully for H. hepaticus infection
by culture and PCR, and H. hepaticus was not detected in this
Animals with infected with wild-type H. hepaticus exhibited
moderate to severe chronic typhlocolitis (Fig. 3B). These ani-
mals developed marked mucosal hyperplasia with infiltration
of the lamina propria with mononuclear inflammatory cells.
This disease was significantly different from findings for unin-
fected controls (Fig. 4). IL-10?/?mice that were initially in-
fected with the CDT-deficient mutant 3B1::Tn20 but eventu-
ally cleared the infection at between 61 and 115 days p.i. had
minimal signs of inflammation. Two animals had no visible
inflammatory infiltrates, while the majority of the remaining
animals only had small, scattered aggregates of chronic inflam-
matory cells with minimal hyperplasia (Fig. 3C). The scores of
the animals infected with the CDT-deficient mutant 3B1::Tn20
FIG. 3. Histolopathologic lesions in the cecae of IL-10?/?mice 8
months after challenge with isogenic strains of H. hepaticus. IL-10?/?
mice were sham infected (A) or infected with wild-type H. hepaticus
strain 3B1 (B) or the CDT-negative mutant 3B1::Tn20 (C). Mice
challenged with wild-type H. hepaticus developed moderate to severe
inflammation and hyperplasia. Mice challenged with the CDT-defi-
cient mutant, which was cleared by 4 months after infection, exhibited
minimal disease at 8 months.
TABLE 2. Chronological monitoring of colonization of IL-10?/?
mice with H. hepaticus by H. hepaticus-specific PCR and culture
PCR result (culture result) at day p.i.:
9 32 45 61 115251
VOL. 74, 2006 H. HEPATICUS CDT AND IMMUNITY4499
were not significantly different from the scores of uninfected
controls (Fig. 4) at 8 months postinfection.
Monitoring of progressive clearance of a CDT-deficient H.
hepaticus mutant. Given the above results, we wished to follow
the clearance of the CDT-deficient H. hepaticus mutant from
the gastrointestinal tract. To achieve this, an additional, lon-
gitudinal study was performed (Table 1). Groups of C57BL/6
IL-10?/?mice were infected with wild-type H. hepaticus and
the isogenic CDT-deficient mutant 3B1::Tn20. Animals were
housed in groups of three to five animals per cage. A total of
40 animals in 11 cages were infected with 3B1::Tn20. Ten
animals, divided into four cages, remained as uninfected con-
trols, and an additional 10 animals (also in four cages) were
infected with wild-type H. hepaticus strain 3B1. Once again, we
used the “cage” as the unit of infection, and the colonization
status was assessed using pooled fecal samples taken approxi-
mately every 3 weeks from each cage of animals. To provide a
measure of the relative levels of colonization, a nested PCR
assay was used to monitor colonization. An initial PCR ampli-
fication was performed with a pair of primers that amplify the
small-subunit rRNA genes of all known Helicobacter species
(14), followed by amplification with a nested pair of primers
specific for H. hepaticus (42), which were used to monitor
colonization in the initial experiment described above. In our
hands, this nested PCR assay has the ability to detect between
25 and 50 genome equivalents of H. hepaticus DNA in 1 ?l
(approximately 100 ng) of fecal DNA (data not shown).
As before, C57BL/6 IL-10?/?mice were initially colonized
by wild-type H. hepaticus 3B1 and 3B1::Tn20. Wild-type H.
hepaticus colonized the mice for the entire duration of each
experiment (Fig. 5). Conversely, although the CDT-deficient
mutant 3B1::Tn20 was detectable by the primary PCR in all
cages of infected mice sampled at 75 days p.i. in IL-10?/?
animals, an increasing fraction of the cages were negative by
the primary PCR assay. All cages of 3B1::Tn20-infected ani-
mals remained positive by the more sensitive nested PCR assay
until 117 days postinfection, but then cages became negative by
this nested assay with kinetics similar to those seen with the
primary PCR assay (Fig. 5).
H. hepaticus-specific humoral and cellular immune re-
sponses in IL-10?/?mice infected with wild-type or CDT-
deficient bacteria. It has been demonstrated that mice infected
with H. hepaticus develop a significant H. hepaticus-specific
antibody response (47). To measure antibody responses
against H. hepaticus in infected animals, an ELISA was devel-
oped based on a previously published assay (48). Antigens
were extracted from plate-grown H. hepaticus via the use of a
nonanionic detergent mixture (B-PER; Pierce Chemical Co.,
Rockford, IL) and used to coat 96-well microtiter plates. The
sera from IL-10?/?animals in the longitudinal follow-up study
were used in this ELISA to monitor the development of an
anti-H. hepaticus humoral immune response in animals in-
fected with wild-type H. hepaticus and the CDT-deficient mu-
tant 3B1::Tn20. Thirty animals in the experiment (10 controls,
10 challenged with wild-type H. hepaticus, and 10 challenged
with the CDT-deficient mutant) had serum samples taken ev-
ery 3 weeks, allowing us to follow the development of H.
hepaticus-specific humoral immune responses.
None of the sera from uninfected C57BL/6 IL-10?/?ani-
mals contained antibody reactive to H. hepaticus antigens in
ELISA (Fig. 6). All animals infected with wild-type H. hepati-
FIG. 4. Categorical inflammation scores for uninfected IL-10?/?
mice and mice infected with wild-type H. hepaticus strain 3B1 and the
CDT-deficient isogenic mutant strains 8 months after challenge. Com-
parisons between groups were performed using the Wilcoxon rank sum
FIG. 5. Monitoring of the loss of colonization with a CDT-deficient
H. hepaticus mutant by using a nested PCR assay. H. hepaticus could be
detected in the feces of animals challenged with wild-type (WT) H.
hepaticus for the entire 225-day duration of the experiment. There was
progressive loss of colonization with the CDT-deficient mutant starting
75 days after infection as judged by the primary stage of the PCR assay
and starting at day 125 as judged by the nested assay.
4500PRATT ET AL.INFECT. IMMUN.
cus or the CDT-deficient mutant developed IgG2c H. hepati-
cus-specific humoral immune responses by 34 days after infec-
tion (Fig. 6, top panel). However, the magnitude of the
responses was significantly greater (P ? 0.05 by ANOVA and
the Tukey-Kramer honestly significant difference test at 34
days and all subsequent time points) in the animals that were
challenged with wild-type H. hepaticus. This differential re-
sponse was even more dramatic when H. hepaticus-specific
IgG1 responses were measured. Animals challenged with wild-
type H. hepaticus developed robust anti-H. hepaticus IgG1 re-
sponses by 34 days after infection, but the responses in animals
challenged with the CDT-deficient mutant were not signifi-
cantly different from those in uninfected controls at any time
following infection (Fig. 6, bottom panel).
At the end of the experiment (225 days after initial infec-
tion), analysis of sera from all mice revealed that these differ-
ences in response were sustained. Animals challenged with
wild-type H. hepaticus had average IgG2c responses of 2.12 ?
0.36 (mean ? standard deviation) and average IgG1 responses
of 0.91 ? 0.71. This is in contrast to the responses of animals
challenged with the CDT-deficient mutant, which had average
IgG2c responses of 0.88 ? 0.58 and IgG1 responses of 0.12 ?
Mice that clear infection with a CDT-deficient H. hepaticus
mutant are protected from rechallenge. Once we determined
that mice could clear infection with the CDT-deficient H. he-
paticus transposon mutant, we wished to determine if clear-
ance was associated with the development of protective immu-
nity. Three cages of IL-10?/?mice that were infected with the
CDT-deficient mutant 3B1::Tn20 and had cleared infection (as
judged by the nested PCR assay) were rechallenged with H.
hepaticus. Two cages of mice (a total of six animals, three in
each cage) were rechallenged with 3B1::Tn20, and one cage of
mice (four animals) was rechallenged with wild-type H. hepati-
cus 3B1. Colonization status was monitored by the nested PCR
assay described above.
One cage of mice rechallenged with 3B1::Tn20 did not have
H. hepaticus detectable by either the primary or nested PCR
assay at 7 and 21 days postinfection. The other cage of three
mice rechallenged with 3B1::Tn20 was positive by both primary
and nested PCR on day 7 but only by nested PCR on day 21.
The cage of animals rechallenged with wild-type H. hepaticus
was positive only by the nested PCR assay on both days 7 and
Since the PCR assays were performed on a pooled fecal
sample from each cage, at 35 days after infection, an individual
fecal pellet was taken from each animal for PCR analysis of
colonization. Of the four mice rechallenged with wild-type H.
hepaticus, three had cleared the organism by 35 days after
rechallenge. One of the animals was still shedding relatively
large numbers of H. hepaticus organisms, since a fecal pellet
from this animal was positive by both the primary and nested
PCR assays. H. hepaticus was cultured from the feces of this
animal and was confirmed to be the wild-type H. hepaticus used
for rechallenge and not the CDT-deficient mutant originally
used to challenge this animal. Of the animals rechallenged with
3B1::Tn20, two of the six animals were negative for H. hepati-
cus by PCR. Overall, 5 of 10 animals rechallenged with H.
hepaticus were not infected with the organism at 35 days after
challenge (P ? 0.0015 by Fisher’s exact test, compared to the
animals challenged initially with wild-type H. hepaticus in this
Chronicity is a key feature of Helicobacter infections. In
some cases, this is of minimal significance to the host. How-
ever, chronic Helicobacter infection and associated long-term
inflammation can lead to the development of neoplastic dis-
ease. It is estimated that at least half of the world’s population
is chronically infected with H. pylori. Although only a minority
of chronically infected individuals go on to develop gastric
adenocarcinoma, the sheer magnitude of chronic infections
makes this the second leading cause of cancer-related death
Bacteria that are able to cause persistent infections have
FIG. 6. Development of H. hepaticus-specific humoral immune re-
sponses in IL-10?/?animals infected with wild-type and CDT-deficient
H. hepaticus. (Top) Animals infected with the wild type and the CDT-
deficient mutant developed significant IgG2c responses by 34 days
after challenge, but the magnitude of the response was greater in
animals infected with wild-type H. hepaticus. (Bottom) Significant
IgG1 responses were seen only in animals infected with the wild type.
Animals infected with the CDT-deficient mutant did not develop IgG1
responses greater than those in negative controls. Each data point
represents the mean OD values for 10 animals, with standard devia-
tions depicted by error bars. ?, P ? 0.05 compared to control mice; §,
P ? 0.05 compared to CDT-deficient mice. All mean comparisons
were performed by ANOVA and the Tukey-Kramer test.
VOL. 74, 2006 H. HEPATICUS CDT AND IMMUNITY 4501
developed varied strategies to evade immune surveillance (29,
39, 50). H. pylori produces the virulence factor vacuolating
cytotoxin A (VacA), which has been theorized to allow H.
pylori to evade immune surveillance and chronically colonize
the gastric mucosa (30). VacA has been shown to inhibit T-cell
activation and thus may interfere with the generation of a
protective immune response by the host (2, 19).
H. hepaticus does not possess a homologue of vacA, the
structural gene for VacA. Cytolethal distending toxin, which
has not been demonstrated to be present in H. pylori, may serve
in a role parallel to that of VacA as an immunoregulatory toxin
in H. hepaticus. CDT has been shown to induce apoptosis in
primary human peripheral blood mononuclear cells and cul-
tured T-cell lines (34, 40, 43). In addition to a direct effect on
T cells, CDT may be able to interfere with immune responses
by interfering with antigen-presenting cells. It was recently
shown that primary human macrophages and dendritic cells
treated with CDT were deficient in cytokine production and in
the stimulation of T-cell proliferation (49).
In the current study, we provide evidence that CDT is nec-
essary for long-term colonization of the murine gut by H.
hepaticus. A CDT-deficient isogenic mutant of H. hepaticus can
maintain colonization of IL-10?/?mice for at least 2 months
but is subsequently cleared by 4 months after experimental
infection. This implies that IL-10?/?mice infected with the
CDT-deficient mutant are able to mount an immune response
that results in clearance of the organism.
Mice infected with either wild-type H. hepaticus or the CDT-
deficient mutant develop significant H. hepaticus-specific hu-
moral immune responses as measured by ELISA. It has been
previously reported that mice experimentally challenged with
wild-type H. hepaticus develop robust anti-H. hepaticus anti-
body titers, despite being unable to clear the infection (48). We
demonstrate here that IL-10?/?mice infected with wild-type
H. hepaticus develop greater humoral responses than animals
infected with a CDT-deficient mutant. This diminished hu-
moral immune response cannot be explained by lower levels of
tissue colonization by the CDT-deficient mutant. T-RFLP
analysis revealed that the CDT-deficient mutant has slightly
delayed colonization kinetics of the mucosa but is able to reach
levels comparable to those reached by wild-type H. hepaticus.
Thus, the decreased levels of H. hepaticus-specific antibody
seen in animals challenged with the CDT-deficient mutant
suggest that CDT expression modulates the host response to
Additional support for the role of CDT in modulating host
immune responses comes from the observation that IL-10?/?
mice infected with the CDT-deficient mutant develop signifi-
cantly less colonic inflammation than IL-10?/?animals in-
fected with wild-type bacteria. In the present study, we dem-
onstrate that over the first 6 weeks following experimental
infection and at 8 months after infection, minimal inflamma-
tion is seen in mice challenged with a CDT-deficient mutant.
The degree of inflammation in animals challenged with the
CDT-deficient mutant was not different from that seen in un-
infected, control animals. This is in marked contrast to the case
for IL-10?/?animals infected with wild-type H. hepaticus,
where significant colitis was seen as early as 8 days after infec-
tion. It is important to note that one of the 20 control animals
in the two long-term studies presented here did develop sig-
nificant typhlocolitis (Fig. 2). This animal was carefully exam-
ined for evidence of H. hepaticus infection, and this was not
found by culture and PCR of fecal specimens and tissue spec-
imens. We have noted the generation of “spontaneous” colitis
in ?5% of the non-H. hepaticus-infected mice in our breeding
colony during the 3 years that it has been in existence, partic-
ularly among older animals. This emphasizes that although H.
hepaticus is sufficient to trigger significant colitis in virtually all
experimentally infected IL-10?/?animals, it is not a necessary
factor. Other, presumably noninfectious triggers, such as me-
chanical mucosal injury from materials in the feed, can initiate
an uncontrolled inflammatory response in these colitis-prone
animals, albeit at a much lower rate than H. hepaticus infec-
Our results are in general agreement with those of Ge and
colleagues, who reported that an independent CDT-deficient
H. hepaticus mutant was unable to maintain persistent coloni-
zation of outbred Swiss Webster mice (18). In their study, mice
infected with either wild-type H. hepaticus or the CDT-defi-
cient mutant developed significant H. hepaticus-specific ELISA
reactivity compared to uninfected controls. They also noted
that mice infected with the CDT-deficient mutant had de-
creased H. hepaticus-specific ELISA reactivity compared to
animals infected with the wild type. As Ge and colleagues
assessed ELISA reactivity only at the termination of their
experiment, the lower levels of H. hepaticus-specific antibody
in animals challenged with their CDT-deficient mutant may
have reflected the time that passed between clearance of the
organism and the collection of serum at the end of the exper-
iment. However, our data regarding the longitudinal develop-
ment of anti-H. hepaticus antibody reactivity in animals sug-
gests that this is a reflection of the differential immune
response to wild-type H. hepaticus and a CDT-deficient mu-
As a final demonstration of the differences in the immune
responses to wild-type H. hepaticus and a CDT-deficient mu-
tant, we show that animals that clear infection with the CDT-
deficient mutant are provided with a degree of protection
against reinfection. Initial infection with the CDT-deficient
mutant 3B1::Tn20 is associated, in a subset of individuals, with
the development of a protective immune response against re-
challenge with either 3B1::Tn20 or wild-type H. hepaticus. Al-
though the number of animals rechallenged with H. hepaticus
following clearance of the CDT-deficient mutant was small, the
development of protective immunity in half of the rechal-
lenged animals was statistically significant. Animals infected
with wild-type H. hepaticus (either experimentally or via verti-
cal transmission) remain colonized with the organism for their
entire life span. “Spontaneous” loss of colonization does not
occur, and antibiotic treatment directed at H. hepaticus is char-
acterized by inconsistent efficacy in eliminating carriage (6).
It is formally possible that 3B1::Tn20 has a defect (perhaps
unrelated to lack of CDT) that is responsible for the long-term
colonization defect. However, the fact that animals that have
lost colonization with 3B1::Tn20 rapidly clear rechallenge with
wild-type H. hepaticus provides much stronger evidence that
this is due to the generation of protective immunity. Therefore,
although mice infected with either wild-type H. hepaticus or
the CDT-deficient mutant develop H. hepaticus-specific hu-
moral immune responses, there is a significant qualitative (as
4502PRATT ET AL.INFECT. IMMUN.
well as quantitative) difference in the immune response, since
protective immunity does not develop in animals infected with
wild-type organisms. It remains to be determined if the actual
H. hepaticus antigens/epitopes that are targeted by the H. he-
paticus-specific immune responses differ and if this explains the
development of protective immunity in animals infected with
the CDT-deficient mutant.
The results from the current study suggest that CDT pro-
duction by H. hepaticus represents a bacterial adaptation that
allows long-term persistence within the mammalian host. CDT
expression serves to modify the development of host immunity
such that the resulting H. hepaticus-specific immune responses
fail to clear the organism. In a host with an altered immune
system, such as an IL-10?/?mouse, this modulation of the H.
hepaticus-specific immune response results in the development
of dysregulated immunity and colitis. Although the develop-
ment of severe typhlocolitis in IL-10?/?mice following H.
hepaticus infection appears to be dependent on CDT produc-
tion by the bacterium, the resulting disease could be consid-
ered to be a reflection of the underlying host defect rather than
enhanced “virulence” of CDT-producing H. hepaticus.
This line of reasoning can help explain the results obtained
by other investigators who have examined the role of CDT in
the pathogenesis of bacterial infections. CDT does not appear
to play a significant role in the acute pathogenesis of H. ducreyi.
Isogenic H. ducreyi CDT mutants were found to be fully viru-
lent in human volunteers and in the temperature-dependent
rabbit model of chancroid (26, 51). However, these models
examine only the acute state of infection, up to 14 days in the
human volunteers and 7 days in the rabbit. However, chancroid
ulceration due to H. ducreyi infection is a chronic condition,
persisting for months in the absence of effective antimicrobial
therapy (25). The authors of these studies speculated that CDT
may have a role in the chronic phase of chancroid but not in
the acute phase (45).
CDT expression is also found in most strains of C. jejuni (11,
12, 38). Isogenic CDT-deficient C. jejuni strains are unable to
maintain long-term gastrointestinal colonization of wild-type
mice, again suggesting a role for CDT in the escape of immune
surveillance (16). These CDT-deficient C. jejuni strains also
caused less severe chronic gastrointestinal disease (gastritis
and duodenitis) in NF-?B-deficient mice (16). However, the
role for CDT in the acute pathogenesis of Campylobacter-
associated human disease is not clear. CDT-negative C. jejuni
strains have been found to be associated with human enteric
disease, causing some investigators to question the role of
CDT in the pathogenic process (1). These same authors dem-
onstrated that an isogenic CDT-deficient C. jejuni strain was
able to colonize newborn chicks for up to 5 days, but they did
not examine the ability of this mutant to persist long term.
Interestingly, they also reported that sera from patients with
campylobacteriosis developed neutralizing anti-CDT antibod-
ies, but similar neutralizing antibodies were not found in chick-
ens experimentally infected with CDT-producing C. jejuni (1).
The results of experiments with H. hepaticus and C. jejuni
suggest that it is not entirely accurate to consider CDT a strict
virulence factor for the Epsilonproteobacteria, at least not in the
sense of being central to the development of acute bacterially
mediated pathology. It might be more appropriate to consider
CDT an evolutionary adaptation that plays a key role in the
establishment of symbiotic relationships between these bacte-
ria and natural eukaryotic hosts. In the case of H. hepaticus,
which is widespread in wild and laboratory rodents and thus
can be considered to be part of the indigenous murine gastro-
intestinal microbiota (44), CDT is a bacterial adaptation that
allows the bacterium to occupy this environmental niche as a
commensal. Intriguing evidence for this hypothesis has been
derived from studies that suggest that CDT may play a key role
in a symbiotic system involving insects, viruses, and bacteria
In summary, we confirm that cytolethal distending toxin has
an essential role in maintaining long-term intestinal coloniza-
tion by H. hepaticus via the modulation of host immune re-
sponses. It will be interesting to determine if there are similar
roles for CDT in other bacteria that elaborate this toxin.
We thank Jennifer Cortez for expert technical assistance.
This work was supported by a USDA National Needs Fellowship to
J.S.P., a Michigan State University Intramural Research Grant Pro-
gram (IRGP) New Investigator Award to V.B.Y., and a Crohn’s and
Colitis Foundation of America Senior Investigator Award to V.B.Y.
Additional support was provided by the Michigan State University
Center for Microbial Pathogenesis.
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Editor: J. T. Barbieri
4504PRATT ET AL.INFECT. IMMUN.