INFECTION AND IMMUNITY, June 2005, p. 3559–3567
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 6
Cytolethal Distending Toxin Is Essential for Helicobacter hepaticus
Colonization in Outbred Swiss Webster Mice
Zhongming Ge,* Yan Feng, Mark T. Whary, Prashant R. Nambiar, Shilu Xu, Vivian Ng,
Nancy S. Taylor, and James G. Fox
Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02131
Received 10 December 2004/Returned for modification 14 January 2005/Accepted 8 February 2005
Helicobacter hepaticus, which induces chronic hepatitis and typhlocolitis in susceptible mouse strains, pro-
duces a cytolethal distending toxin (CDT) consisting of CdtA, CdtB, and CdtC. A cdtB-deficient H. hepaticus
isogenic mutant (HhcdtBm7) was generated and characterized for colonization parameters in four intestinal
regions (jejunum, ileum, cecum, and colon) of outbred Swiss Webster (SW) mice. Inactivation of the cdtB gene
abolished the ability of HhcdtBm7 to colonize female mice at both 8 and 16 weeks postinfection (wpi), whereas
HhcdtBm7 colonized all of four intestinal regions of three of five males at 8 wpi and then was eliminated by
16 wpi. Wild-type (WT) H. hepaticus was detected in the corresponding intestinal regions of both male and
female mice at 8 and 16 wpi; however, colonization levels of WT H. hepaticus in the cecum and colon of male
mice were approximately 1,000-fold higher than in females (P < 0.0079) at 16 wpi. Infection with WT H.
hepaticus, but not HhcdtBm7, at 8 wpi was associated with significantly increased mRNA level of ileal and cecal
gamma interferon (IFN-?) in females (P < 0.016 and 0.031 between WT H. hepaticus-infected and sham-dosed
females, respectively). In contrast, the mRNA levels of IFN-? were significantly higher in the colon (P < 0.0079)
and trended to be higher in the cecum (P < 0.15) in the HhcdtBm7-colonized male mice versus the sham-dosed
controls at 8 wpi. In addition, mRNA levels of ileal IFN-? were significantly higher in the control females than
males at 8 wpi (P < 0.016). There were significantly higher Th1-associated immunoglobulin G2a (IgG2a),
Th2-associated IgG1 and mucosal IgA (P < 0.002, 0.002, 0.002, respectively) responses in the mice infected with
WT H. hepaticus when compared to HhcdtBm7 at 16 wpi. Colonic interleukin-10 (IL-10) expressions at 16 wpi
were significantly lower in both female and male mice colonized by WT H. hepaticus or in males transiently
colonized through 8 wpi by HhcdtBm7 versus control mice (P < 0.0159). These lines of evidence indicate that
(i) H. hepaticus CDT plays a crucial role in the persistent colonization of H. hepaticus in SW mice; (ii) SW
female mice are more resistant to H. hepaticus colonization than male mice; (iii) there was persistent coloni-
zation of WT H. hepaticus in cecum, colon, and jejunum but only transient colonization of H. hepaticus in the
ileum of female mice; (iv) H. hepaticus colonization was associated with down-regulation of colonic IL-10
Multiple pathogenic gram-negative bacteria produce cytole-
thal distending toxins (CDTs) (reviewed in references 31 and
40). These CDTs are generally tripartite holotoxins, of which
subunit B contains an active domain with DNaseI-like activity,
whereas subunits A and C appear to function as accessory
proteins for the delivery of subunit B into target cells (25).
However, a recent study has reported that the Salmonella en-
terica serovar Typhi CdtB alone has CDT activity, given that
there are no homologs of cdtA and cdtC identified within its
genome (18). Upon entry into the cytosol, CdtB is translocated
into the nucleus, where it causes limited damage to host cell
DNA and thereby triggers the DNA damage repair mechanism
(26). CDT causes cell cycle arrest and subsequent cellular
distension and eventual cell death in cultured mammalian
cells. Current in vivo pathogenesis data on the role of bacterial
CDT have been inconsistent. The CDT activity in Haemophilus
ducreyi and Campylobacter jejuni was reported to be dispens-
able for colonization and has minimal contribution to pathol-
ogy in rabbits (37) and scid mice (32), respectively. However,
we recently demonstrated that CDT-deficient C. jejuni persis-
tently colonized NF-?B-deficient mice, while its colonization
persisted through 2 months postinfection (p.i.) but was elimi-
nated by 4 months p.i. from wild-type (WT) mice (14). In
addition, inactivation of cdtB in H. hepaticus significantly at-
tenuated the severity of typhlocolitis in interleukin-10?/?(IL-
10?/?) mice, although this mutation appeared to have no effect
on the colonization of the mutant (44). Thus, the type of
bacterial pathogen and the genetic background of experimen-
tal hosts influence colonization and clinical manifestations of
infection with CDT-deficient bacterial mutants.
Helicobacter hepaticus was originally isolated from the livers,
ceca, and colons of aged A/JCr mice that were controls for a
long-term chemical carcinogenesis study (12, 41). It has been
documented that the mouse cecum and colon are the primary
sites for H. hepaticus colonization (13). In contrast, the colo-
nization status of H. hepaticus in the ileum and small intestine
of mice has not been characterized. C. jejuni, which shares 953
protein orthologs with H. hepaticus (50.8% of the predicted
open reading frames) (38), colonizes proximal and distal small
intestine, cecum, and large intestine in chicks and mice (3, 10,
28). A recent study reported that C. jejuni colonization in the
jejunum of humans was associated with immunoproliferative
disease of the small intestine (27). Thus, characterization of H.
* Corresponding author. Mailing address: Division of Comparative
Medicine, Massachusetts Institute of Technology, 16-873, 77 Massa-
chusetts Avenue, Cambridge, MA 02131. Phone: (617) 253-5518. Fax:
(617) 258-5708. E-mail: firstname.lastname@example.org.
hepaticus colonization in the small intestine of mice will in-
crease our understanding of H. hepaticus pathogenesis.
Natural and experimental infection studies have established
that H. hepaticus in susceptible strains of mice causes chronic
active hepatitis, typhlocolitis, and hepatocellular carcinoma
(15, 19, 20, 41, 42). H. hepaticus-induced liver lesions are more
severe in infected male A/JCr mice (13, 33); however, the
gender factors contributing to this difference are not under-
stood. In addition, infection by H. hepaticus in immune dys-
regulated mice induces intestinal pathology that mimics some
features of inflammatory bowel disease (IBD) in humans (4, 6,
7, 24). Severity of H. hepaticus-induced typhlocolitis is associ-
ated with elevated production of proinflammatory cytokines
(e.g., IL-12, tumor necrosis factor ? [TNF-?], gamma inter-
feron [IFN-?]) (24, 29, 42).
Humans are infected by multiple enterohepatic helico-
bacters that have pathogenic potential (reviewed in reference
11). Human enterohepatic helicobacters H. canis, H. cinaedi,
and H. pullorum also contain CDT (5, 39, 43). The immune
responses of outbred mice to pathogenic infection should
mimic human immune responses and thus are useful models
for understanding host-pathogen interactions and identifying
potential bacterial virulence factors, particularly those influ-
encing colonization. In this study, we investigated the role of H.
hepaticus CDT in intestinal colonization using outbred Swiss
Webster (SW) mice. In addition, the colonization status of H.
hepaticus in the small intestine was determined; selected cyto-
kine profiles were characterized and compared between WT
and CDT-deficient H. hepaticus in both female and male SW
MATERIALS AND METHODS
Bacterial strains, growth media, and conditions. Escherichia coli strain Top10
was used as a recipient for cloning, mutagenesis, and plasmid propagation and
was cultured in Luria-Bertani (LB) broth or agar supplemented with antibiotics
ampicillin (50 ?g/ml) and chloramphenicol (25 ?g/ml) when appropriate. Wild-
type (WT) H. hepaticus strain 3B1 (12) was cultured on blood agar (Remel,
Lexignton, Kans.) for 2 to 3 days under microaerobic conditions (10% H2, 10%
CO2, 80% N2). Chloramphenicol-resistant H. hepaticus mutants were selected on
tryptic soy agar supplemented with 5% sheep blood and 25 ?g/ml of chloram-
phenicol (all from Sigma, St. Louis, MO).
Cloning of selected cytokines and GAPDH cDNA. The primers used for clon-
ing IL-10, IL-6, TNF-?, and IFN-? cDNA are listed in Table 1, whereas the
primers for the GAPDH gene were purchased from Applied Biosystems (Foster
City, CA). cDNA was synthesized from the RNA template (2 ?g) with a reverse
primer for each of IL-10, IL-6, TNF-?, IFN-?, and GAPDH genes and Super-
script RNaseH?reverse transcriptase according to the supplier’s protocol for
first-strand cDNA synthesis (Invitrogen, Carlsbad, CA). Amplicons were cloned
into a TOPO vector following the supplier’s instruction (Invitrogen), and plasmid
DNA was prepared using Aquick Mini kit (QIAGEN Inc., Valencia, CA). The
identity of recombinant plasmids was confirmed by DNA sequencing in the ABI
310 sequencer (Applied Biosystems). Concentrations of the plasmid DNA were
determined using a spectrophotometer GeneQuan Pro (Amersham Biosciences,
Real-time quantification of H. hepaticus, HH1450, and selected cytokines.
Chromosomal DNA from cultured bacteria was prepared using a High Pure
PCR Template kit according to the manufacturer’s protocol (Roche Applied
Science, Indianapolis, IN). Total DNA and RNA from jejunum, ileum, cecum,
and colon were isolated using Trizol reagents following the supplier’s procedure
(Invitrogen). The numbers of H. hepaticus in each of the intestinal segments were
determined by real-time quantitative PCR (Q-PCR) in the Prism Sequence
Detection System 7700 (Applied Biosystems) as described elsewhere (17).
For cytokine mRNA quantification, 5 ?g of total RNA from samples were
converted into cDNA using a High Capacity cDNA Archive kit following the
supplier’s recommendation (Applied Biosystems). The cDNA levels for IL-6,
IL-10, TNF-?, and IFN-? mRNA were measured by Q-PCR using commercial
primers and probes for each of the aforementioned murine cytokines. Briefly, a
25-?l mixture contained 5 ?l of cDNA (in duplicate), 1.25 ?l of a commercial
20? primer-probe solution (Applied Biosystems), 12.5 ?l of 2? master mix
(Applied Biosystems), and 6.25 ?l of double-distilled H2O. Concentrations of 10,
100, 1000, 104, 105, and 106copies of recombinant plasmids containing the
respective cytokine cDNA inserts were used to generate standard curves. The
copy number of each cytokine mRNA among the samples was normalized as
copies of each cytokine transcript per 106copies of GAPDH transcripts.
Transcript levels of H. hepaticus HH1450, which is located immediately down-
stream of the H. hepaticus cdt operon (38), were measured as described above for
murine cytokine quantification with some modifications. Q-PCR was carried out
using 2? SyBr Green I PCR master mix (Applied Biosystems) and 500 nM of
each of primers RTF2 and RTR2 (Table 1). In addition, the plasmid pVBY9
containing HH1450 was used to generate a standard curve of 10 to 107copies
Determination of the transcriptional terminator of the cdt operon by reverse
TABLE 1. The origin and sequences of the respective primersa
PrimerSequences (5? to 3?)Orientationb
aAll the primers were designed in this study. The numbers in parentheses denote the start nucleotide of each of the primers in the published nucleotide sequences.
bThe orientation of the primers to the coding strand of each of the genes.
cThe accession numbers of the respective genes in the sequence databases of the National Center for Biotechnology Information.
3560 GE ET AL.INFECT. IMMUN.
transcription-PCR (RT-PCR). Total RNA from H. hepaticus was isolated using
RNAeasy minikit, including a step of DNase treatment according to the suppli-
er’s instructions (QIAGEN Inc.). cDNA was synthesized from the RNA template
(4 ?g) with the respective reverse primer (either RTR1, TM1, or TM2) and
Superscript RNaseH-reverse transcriptase according to the supplier’s protocol
for first-strand cDNA synthesis (Invitrogen). Subsequently the reaction mixture
was incubated at 70°C for 10 min, 5 ?l of which was then subjected to conven-
PCR. The origins of the individual PCR primers used in this study are pre-
sented in Table 1. A 50-?l volume of PCR contained the following: 10 to 50 ng
of DNA template, 1 ? commercial buffer (Roche Applied Science), 100 ?g/ml
bovine serum albumin (BSA), 500 nM each of forward and reverse primers, and
2.5 units of High Fidelity DNA polymerase (Roche Applied Science). A ther-
mocycling program of 35 cycles in a Thermocycler Genius (Technie Incorpo-
rated, Princeton, NJ) was denaturation at 94°C for 1 min, followed by annealing
at 50°C to 60°C (based on the respective primers) for 30 s and extension at 72°C
for 1 min.
Primer extension. The primer PEP was labeled at 37°C for 15 min in the
mixture (5 ?l) containing 1 ?l of 5? forward buffer (Invitrogen), 20 ng of PEP,
10 ?Ci of [?-33P]ATP (NEN Life Science Products, Boston, MA), 2 units of T4
polynucleotide kinase. The labeled primer was mixed with 5 ?l of total H.
hepaticus RNA (5 ?g) and incubated at 70°C for 10 min; in the control reaction,
1 ?l of DNase-free RNase (Roche Applied Science) was added. Primer extension
was performed at 37°C for 1 h in a 20-?l volume containing 10 ?l of the
primer-RNA mixture, 4 ?l of 5? first-strand buffer (Invitrogen), 2 ?l of 0.1 mM
dithiothreitol, 2 ?l of 1 mM deoxynucleoside triphosphate solution (dATP,
dCTP, dGTP, and dTTP), and 200 units of Superscript RNaseH?reverse tran-
scriptase. Five microliters of sequencing loading dye was added to the reaction
mixture. Two microliters of the resulting samples along with the PEP-primed
sequencing reaction for plasmid pVBY9 were analyzed on a 5% polyacrylamide
Minitransposon mutagenesis. Minitransposon containing chloramphenicol
acetyltranferase gene (cat) was constructed using a kit according to the supplier’s
procedure (Epicenter Technologies, Madison, WI). Briefly, the cat cassette was
excised with HincII as previously described (16) and ligated into a HincII site of
construction vector pMOD-2. The cat-containing recombinants resistant to am-
picillin (Apr) and chloramphenicol (Cmr) were selected and then were subjected
to further characterization by DNA sequencing. The resultant Cmr-containing
pMOD (namely pMODCm-4) was used to generate the Cmr-minitransposon
(referred to as TnCm4) by PCR with primers PF and PR (Epicenter Technol-
ogies). The cdtB in plasmid pVBY9 was inactivated using TnCm4 and the in vitro
system according to the supplier’s recommendation (Epicenter Technologies).
Transformants with Aprand Cmrwere selected, and the cdtB-inactivated plas-
mids were screened by PCR with primers ZMG38 and ZMG41 (Table 1),
followed by DNA sequencing. The resultant plasmid pVBYMut7 was used for
generating isogenic H. hepaticus mutants.
Construction of isogenic mutants. WT H. hepaticus cells from frozen stock
were cultured on blood agar for 2 to 3 days and then washed twice with ice-cold
buffer containing 15% (vol/vol) glycerol and 7% (wt/vol) sucrose followed by
resuspension in the same buffer. A 50-?l aliquot of cells was mixed with 2 ?g of
plasmid pVBYMut7 DNA, followed by electroporation (2.5 kV, 12.5 kV/cm) in
an Escherichia coli pulser (Bio-Rad, Hercules, CA). Cells were incubated at 37°C
for 2 days on blood agar and scraped in Brucella broth containing 25% glycerol,
followed by application onto blood agar plates containing 25 ?g/ml of chloram-
phenicol. Isogenic mutants of H. hepaticus isolated after 5 to 10 days’ growth
under microaerobic conditions were characterized by PCR and DNA sequenc-
ing. Isogenic H. hepaticus mutant A7 (HhcdtBm7), which represented the mu-
tation in the cdtB gene, was selected for further characterization in vivo. The
CDT activity for WT H. hepaticus and HhcdtBm7 was measured in vitro on HeLa
cells as previously described (5).
Experimental design for in vivo infection. SW mice free of known murine
viruses, pathogenic bacteria including Helicobacter spp., and parasites were ob-
tained from Taconic Farms (Germantown, NY). The mice were maintained in an
Association for Accreditation and Assessment of Laboratory Animal Care In-
ternational-accredited facility in static microisolater cages. In a pilot study, fe-
male Helicobacter spp.-free SW mice (five per group, 4 to 6 weeks old) were
infected with WT H. hepaticus or its CDT-deficient mutant HhcdtBm7 for 6
weeks. Subsequently, 30 male and 30 female SW mice (4 to 6 weeks old) were
divided into six groups of 10 mice (either male or female) and were dosed with
WT H. hepaticus or HhcdtBm7 or sham-dosed with Brucella broth as a control,
respectively. For oral gavage, bacteria were cultured on blood agar, suspended in
Brucella broth, and adjusted to 108organisms/ml as estimated by spectropho-
tometry at an optical density of 660 nm (OD660). Mice received 0.2 ml of fresh
inocula by gastric gavage every other day for three doses.
Five male and five female mice from each group were necropsied at 8 and 16
wpi, respectively. Immediately after euthanasia, contents in the intestine were
removed by rinsing with sterile saline. Standardized 1-cm segments of jejunum,
ileum, cecum, and colon were collected for culture and RNA/DNA isolation.
Tissues for RNA/DNA isolation were frozen in liquid nitrogen immediately after
sampling and stored at ?70°C prior to use. Representative tissue sections were
fixed in 10% buffered formalin for histology.
Histopathology evaluation. The entire length of the small intestine from the
pylorus of the stomach to the ileoceco-colic (ICC) junction was measured and
evaluated under a dissection microscope for gross abnormalities. The small
intestine of each mouse was divided into three equal segments, Swiss-rolled and
embedded in paraffin, sectioned at 5-um thickness, and stained by hematoxylin
and eosin (HE) stain for histologic evaluation by a veterinary pathologist (PRN)
blinded to experimental groups. The ICC junction and colon were similarly
prepared and examined.
Serology and mucosal immunoglobulin A (IgA) responses to H. hepaticus
antigens. Sera was collected from mice at 8 and 16 wpi with WT H. hepaticus or
HhcdtBm7 and evaluated by enzyme-linked immunosorbent assay (ELISA) for
Th1-associated IgG2a and Th2-associated IgG1 antibody responses. Outer mem-
brane antigens of the type strain of H. hepaticus (ATCC 51449) were prepared
and used in the ELISA using standard methods as previously described (42).
Feces were collected at 16 wpi and extracted in protease inhibitor (Sigma) for
evaluation of IgA responses to H. hepaticus antigens as previously described (42).
Statistical analyses. Data on the levels of H. hepaticus and cytokine mRNA in
the tissues were analyzed using a Mann-Whitney nonparametric t test. Serology
and mucosal IgA results were compared using regression, analysis of variance,
and the Student’s t test. Values of P ? 0.05 are considered significant.
Determination of the cdt operon in H. hepaticus. To investi-
gate transcriptional regulation of the cdt genes and create
isogenic H. hepaticus mutants, the transcriptional start and
termination sites of H. hepaticus cdt operon were characterized
by primer extension and RT-PCR, respectively (Fig. 1). The 5?
end of a cDNA product generated by primer extension corre-
sponded to A145upstream of the start codon of cdtA; the
smaller labeled products on the gel probably represent the
partially degraded cdt transcripts. In addition, an oligonucleo-
tide sequence similar to the ?10 promoter box conserved
among bacterial promoters preceded this transcriptional start
site (Fig. 1B). These results indicate that nucleotide A145is the
transcriptional start site of the H. hepaticus cdt operon. For
determination of the transcriptional termination site, cDNA of
total RNA from H. hepaticus was synthesized using primers
TM1 (complementary to nucleotides 17 to 38 downstream of
the cdtC) and TM2 (complementary to nucleotides 91 to 108
downstream of the cdtC). The respective cDNA templates
were then amplified using the primer pair TM1/cdtCF (pro-
ducing a 500-bp fragment) and TM2/cdtCF (producing a
570-bp fragment). Amplicons of the correct sizes were pro-
duced from the TM1-primed cDNA but not from the TM2-
primed cDNA (Fig. 1C), indicating that the transcription of the
cdt operon was terminated in the region covering nucleotides
38 to 99 downstream of the cdtC stop codon. Successful am-
plification using RT-PCR with primers RTF1 and RTR1 (com-
plementary to the sequences cdtA and cdtC, respectively) in-
dicates cotranscription of cdtA, cdtB, and cdtC (data not
shown). The origins of these PCR products were confirmed by
DNA sequencing. These results confirm that the cdt operon in
H. hepaticus contains three structural genes cdtA, cdtB, and
cdtC previously described by Young et al. (45).
VOL. 73, 2005ROLE OF CDT IN H. HEPATICUS COLONIZATION3561
Colonization of WT H. hepaticus and HhcdtBm7 in outbred
SW mice. To investigate the role of H. hepaticus CDT in col-
onization, the transposon-mediated mutation in the cdtB gene
was created and the H. hepaticus mutant was administered to
outbred SW mice of both genders. HhcdtBm7 contained the
TnCm4-disrupted mutation within cdtB, which was confirmed
by PCR, genomic DNA sequencing, and lack of CDT in vitro
activity (data not shown). To investigate whether the TnCm4
insertion within the cdtB gave rise to polar effect on the down-
stream gene of the cdt operon, transcript copies of the HH1450
that locates immediately downstream of the H. hepaticus cdt
operon were quantified by RT-Q-PCR with primers RTF2 and
RTR2 (Fig. 1). The levels of the HH1450 transcripts per ng of
total RNA in three independent RNA preparations were 1,243
? 91 copies for HhcdtBm7 and 1,386 ? 176 copies for WT H.
hepaticus, indicating that there was no significant change in the
HH1450 transcription between WT H. hepaticus and Hhc-
dtBm7. Hence, this cdtB-disrupted mutation had no polar ef-
fect on its downstream gene.
In the pilot study, five female SW mice dosed with Hhc-
dtBm7 were negative by PCR for H. hepaticus in fecal DNA at
2 or 4 wpi and cecal DNA at 6 wpi, whereas the mice dosed
with WT H. hepaticus were PCR positive (data not shown). In
the longitudinal study, WT H. hepaticus at 8 wpi was variably
FIG. 1. Characterization of the transcriptional start and termination sites of the H. hepaticus cdt operon. (A) The gene organization of the cdt
operon: the primers for primer extension (PEP) and for RT-PCR as well as the insertional site of TnCm4 within the cdtB are denoted. (B) Primer
extension demonstrating the transcriptional start site (A145upstream of the start codon of the cdtA) is indicated by an arrow. Lanes C, T, A, G:
dideoxy-terminated sequence using plasmid pVBY9 DNA as template. This recombinant plasmid containing the cdt operon and the portion of the
upstream and downstream regions (45). Lane 1, RNA plus RNase prior to cDNA synthesis; lane 2, RNA minus RNase. (C) RT-PCR-based
detection of the transcriptional termination of the cdt opron. Lanes: 1, cDNA with cdtCF/TM1; 2, cDNA with cdtCF/TM2; 3, pVBY9 with
cdtCF/TM1; 4, pVBY9 with cdtCF/TM2. A 1-kb ladder (Invitrogen) is located on the left (M).
3562GE ET AL.INFECT. IMMUN.
detected by Q-PCR in jejunum (four of five), ileum (four of
five), cecum (five of five), and colon (four of five) of the male
SW mice (Fig. 2A). Colonization with WT H. hepaticus per-
sisted in the cecum and colon through 16 wpi in all dosed male
mice, but at this later time point, WT H. hepaticus was not
detected in one of three or two of three male jejunal or ileal
samples, respectively (Fig. 2B). At 8 wpi, WT H. hepaticus was
detected by Q-PCR in all dosed female mice with positive
results from jejunum (three of five), ileum (four of five), cecum
(four of five), and colon (four of five). For each intestinal site
that was positive for H. hepaticus at 8 wpi, there were no
significant differences in colonization levels between male and
female mice (P ? 0.22). At 16 wpi in female mice, WT H.
hepaticus persisted in the cecum and colon but was detected
proximal to the cecum only in the jejunum of three of five
female mice. Colonization of WT H. hepaticus in the cecum
and colon of male mice significantly increased between 8 and
16 wpi (P ? 0.015, cecum; P ? 0.03, colon), whereas coloni-
zation in the cecum and colon of female mice trended lower
through 16 wpi (P ? 0.3, cecum; P ? 0.15, colon). Consistent
with previous reports describing the cecum and colon as the
natural niche for H. hepaticus (13), cecal samples contained the
highest level of H. hepaticus colonization. Interestingly, at 16
wpi, the levels of H. hepaticus colonization in the cecum and
colon from male mice were approximately 1,000-fold higher
than those from female mice (P ? 0.0079).
Male mice at 8 wpi were colonized with HhcdtBm7 in the
jejunum, ileum, and cecum at comparable levels to those for
WT H. hepaticus (P ? 0.05 for all comparisons), whereas Hh-
cdtBm7 was less efficient in colonizing the colon (three out of
five) compared to WT H. hepaticus (five out of five). Hhc-
dtBm7 was not detected in the jejunum, ileum, cecum, or colon
from any female mice dosed 8 weeks earlier, and the isogenic
mutant could not be detected by Q-PCR or culture in either
male or female mice at 16 wpi.
Culture was performed to confirm viability of H. hepaticus in
jejunum, ileum, cecum, and colon from SW mice infected with
WT H. hepaticus or HhcdtBm7. The results for the samples
from the mice at 8 wpi supported those by Q-PCR. At 16 wpi,
the cecum (five out of five) and colon (five out of five) of WT
H. hepaticus-dosed male mice as well as the cecum (one out of
five) of WT H. hepaticus-dosed female mice, which contained
the relatively high levels of H. hepaticus by Q-PCR, were H.
hepaticus positive by culture. In contrast, 15 samples (Fig. 2B),
13 of which contained relatively lower levels of H. hepaticus by
Q-PCR, were negative for H. hepaticus by culture. These data
indicate that Q-PCR is more sensitive for determining the
status of H. hepaticus colonization in the murine intestine than
Histopathology. Gross and histopathological evaluation of
the intestines of all mice did not reveal significant lesions.
Serum IgG and mucosal IgA responses to H. hepaticus an-
tigens. Mice dosed with WT H. hepaticus or its cdtB-deficient
mutant HhcdtBm7 had seroconverted to H. hepaticus antigens
by 8 wpi (P ? 0.0001) (data not shown). At 16 wpi, male mice
infected with WT H. hepaticus had developed a progressively
higher Th1-associated IgG2a response compared to female
mice (P ? 0.009) (Fig. 3). The IgG2a responses of both males
and females infected with WT H. hepaticus were significantly
higher than mice of either sex dosed with the HhcdtBm7 mu-
tant (P ? 0.002). Male and female mice colonized with WT H.
hepaticus had similar Th2-associated IgG1 responses at 16 wpi
(P ? 0.40) and, like the IgG2a responses, were significantly
higher than mice dosed with the HhcdtBm7 mutant (P ?
0.002). Levels of mucosal IgA specific for H. hepaticus were
significant only for mice infected with WT H. hepaticus (P ?
0.0006; control data not shown). IgA responses of mice dosed
with HhcdtBm7 were equivalent to background levels pro-
duced by uninfected control mice (P ? 0.26). There was a
trend for female mice infected with WT H. hepaticus to pro-
duce more IgA than male mice (P ? 0.09).
Cytokine production in intestinal tissues. Given that H.
hepaticus CDT plays an important role in colonization of SW
mice and females were more resistant to H. hepaticus coloni-
zation than males, mRNA for three proinflammatory cytokines
(IL-6, TNF-?, IFN-?) and one anti-inflammatory Th2-type
cytokine (IL-10) were analyzed using Q-PCR in samples from
FIG. 2. Q-PCR-based detection of H. hepaticus in four segments of
the mouse intestinal tract. Symbols: F, female mice; M, male mice;
WT, WT H. hepaticus dosed; cdtB inact., F HhcdtBm7-dosed cdtB
inactivated); *, P values for the compared groups. The levels of H.
hepaticus in the respective samples are expressed as its genomic copies
per ?g of mouse DNA. Ileal and jejunal tissues from two male mice of
all the groups at 16 wpi were lost during processing. Therefore, the
16-wpi data on the colonization levels and cytokine profiles in the
ileum and jejunum of the male mice were based on three mice of each
group. The samples negative for H. hepaticus by culture are indicated
VOL. 73, 2005ROLE OF CDT IN H. HEPATICUS COLONIZATION3563
jejunum, ileum, cecum, and colon. At 8 wpi, infection with WT
H. hepaticus significantly increased the level of ileal and cecal
IFN-? transcripts in the female but not in male mice (Fig. 4, P
? 0.0079). In contrast, male mice dosed with WT H. hepaticus
expressed more ileal IL-10 transcripts than female mice (Fig. 4,
P ? 0.0079). Both female and male mice dosed with Hhc-
dtBm7 did not express altered levels of ileal IL-10 and IFN-?;
however, the level of IFN-? mRNA was significantly higher in
the colon (Fig. 5, P ? 0.0079) and trended to be higher in the
cecum without statistical significance (Fig. 4, P ? 0.15) from
the male mice infected with HhcdtBm7 compared with unin-
fected male mice. There were no significant differences in
expression of IL-6 or TNF-? mRNA in the jejunum, ileum,
cecum, and colon among all comparable groups (data not
At 16 wpi, both female and male mice infected by WT H.
hepaticus had significantly down-regulated IL-10 mRNA ex-
pression compared to the control mice in the colon (Fig. 5;
P ? 0.015, female; P ? 0.0079, male). In addition, infection
with HhcdtBm7 down-regulated the IL-10 mRNA expression
only in males (P ? 0.0079) but not in females (P ? 0.9) that
had eliminated colonization prior to 8 wpi. At 16 wpi, there
were no significant differences in the levels of IL-10 or IFN-?
transcripts in the ileum and cecum among all the comparable
groups (data not shown).
Many gram-negative pathogenic bacteria, including Acti-
nobacillus actinomycetemcomitans, Campylobacter spp., E. coli,
H. ducreyi, enterohepatic Helicobacter species, Salmonella en-
terica serovar Typhi, and Shigella species, produce CDT, a
heat-labile cytotoxin that causes cell cycle arrest, subsequent
cell distension, and eventual cell death in cultured mammalian
cells (40). It has been reported that CDT activity was not
required for colonization of C. jejuni and H. hepaticus in im-
munodeficient mice, including scid, NF-?B-deficient, and IL-
10?/?mice (14, 32, 44). However, the C. jejuni cdtB mutant
was eliminated by 4 months p.i. from C57BL/129 mice, indi-
cating that CDT is necessary for persistent colonization of C.
FIG. 3. IgG1, IgG2a, IgA antibody responses to the H. hepaticus
antigens. The sera for measuring IgG1 and IgG2a were collected from
the mice orally dosed with either WT H. hepaticus or HhcdtBm7 (cdtB
inactivated) for 16 wpi. The IgA antibody response to H. hepaticus was
measured in fecal extracts from mice orally dosed with either WT H.
hepaticus or HhcdtBm7 (cdtB inactivated) by 16 wpi. *, P values for the
compared groups. OD, optical density.
FIG. 4. mRNA levels of IL-10 and IFN-? in the respective intesti-
nal segments. The mice were orally dosed with either Brucella broth,
WT H. hepaticus, or HhcdtBm7 (cdtB inactivated) and necropsied at 8
wpi. Symbols: *, P values for the compared groups; C, the controls;
WT, WT H. hepaticus-dosed; cdtB inact., HhcdtBm7 (the cdtB inacti-
vated); F, female mice; M, male mice. The levels of the IL-10 and
IFN-? transcripts were expressed as their copy numbers per 106of the
GAPDH transcripts in the same samples.
3564GE ET AL.INFECT. IMMUN.
jejuni in immunocompetent mice (14). In this study, we dem-
onstrated that CDT is critical for persistent colonization of H.
hepaticus in outbred SW mice. Loss of HhcdtBm7 colonization
in female mice by 8 wpi and male mice by 16 wpi was supported
by the serological data indicating that mice infected with the
CDT-deficient mutant developed significantly lower Th1-asso-
ciated IgG2a, Th2-associated IgG1 and IgA responses than
those mice colonized by WT H. hepaticus. Results from our
pilot study indicated that HhcdtBm7 could be eliminated from
female mice as early as 2 wpi. Inability of HhcdtBm7 to per-
sistently colonize does not appear to be due to a polar effect of
the mutation, because the H. hepaticus cdtA, cdtB, and cdtC
genes constitute a single operon and the insertion of minitrans-
poson in the cdtB did not affect the transcription of HH1450
immediately downstream of the cdt operon. Recently, Young
et al. (44) reported that CDT-deficient H. hepaticus mutants
induced less severe typhlocolotis than WT H. hepaticus, but its
colonization levels were not different from WT H. hepaticus in
C57BL/6 IL-10?/?mice. The contrasting results of our study
may be explained by use of outbred, immunocompetent SW
mice compared to IL-10-deficient mice. We hypothesize that
successful colonization of CDT-deficient bacterial mutants in
immunodeficient mice results from the inefficient immune host
responses. The molecular mechanisms underlying the role of
CDT in colonization are unclear at present. H. hepaticus CDT
may suppress IFN-? responses to helicobacter infection, per-
haps by inhibiting T- and B-cell function as demonstrated for
A. actinomycetemcomitans CDT (34–36). This notion is sup-
ported by clearance of HhcdtBm7 by 8 wpi in female SW mice,
which was associated with a significantly elevated IFN-? ex-
pression in the ileum; in contrast, the elevated ileal IFN-?
expression was not observed in male mice which were colo-
nized with the mutant at least through 8 wpi. In addition, the
subsequent elimination of the CDT isogenic mutant from the
male mice by 16 wpi may be due to the gradually increased
expression of IFN-? in the cecum and colon. The importance
of IFN-? in protecting hosts from bacterial infection has been
demonstrated in other mouse models. Increased IFN-? expres-
sion in Yersinia-specific T cells and activated macrophages in
Peyers patches and mesenteric lymph nodules was shown to be
essential for clearing Y. enterocolitica infection from C57BL/6
mice, whereas BALB/c mice that did not respond with elevated
Yersinia–associated IFN-? expression were susceptible to Yer-
sinia infection (1, 2).
In H. hepaticus, 953 of the predicted open reading frames
(50.8%) have orthologs in C. jejuni (38). C. jejuni colonizes the
cecum and large intestine as primary sites but also colonizes
proximal distal small intestine in chicks and mice (3, 10, 28).
Importantly, a recent study reported that the C. jejuni coloni-
zation in the jujenum of humans was associated with immuno-
proliferative small intestinal disease with characteristic lym-
phoepithelial lesions caused by infiltration of CD20-positive (a
B-cell marker) centrocyte-like lymphocytes (27). Given the fact
that H. hepaticus causes typhlitis in A/J mice (13, 42), these
studies prompted us to characterize the status of H. hepaticus
colonization in the small intestine of mice. WT H. hepaticus
persistently colonized the jejunum of both male and female
mice but in the ileum was eliminated by 16 wpi from the female
mice, whereas HhcdtBm7 colonized the jejunum and ileum of
male SW mice only through 8 wpi. H. hepaticus colonization in
ileum of female SW mice was associated with the up-regulation
of ileal IFN-? mRNA at 8 wpi. The levels of ileal IFN-?
mRNA between the control and WT H. hepaticus-dosed fe-
male mice were not significantly different at 16 wpi when H.
hepaticus was eliminated from this niche. These results suggest
that the colonization of H. hepaticus in this intestinal niche at
an early time postinfection may play an important role in the
up-regulation of IFN-? expression.
Our data indicated that female SW mice are more resistant
to both WT H. hepaticus and HhcdtBm7 infection than male
mice. Gender effect on the colonization by WT H. hepaticus
and HhcdtBm7 in SW mice appears to be associated with
different IFN-? responses in the intestine by 8 wpi between
female and male mice. In the ileum and cecum of female mice,
but not males, after being infected with WT H. hepaticus the
level of IFN-? was significantly increased. This result is con-
sistent with the recent finding that the IFN-? transcripts were
increased in the cecum of female A/JCr mice compared to
males after H. hepaticus infection for 1 month (29). However,
elevated cecal IFN-? in female A/JCr mice persisted through 3
months postinfection, and cecal IL-10 and TNF-? mRNA lev-
els were also increased. These differences may be ascribed to
different genetic backgrounds between inbred A/JCr and the
outbred SW mice. Although the difference in IFN-? expression
between female and male SW mice may explain the sex-de-
pendent colonization outcomes for WT H. hepaticus and
HhcdtBm7 in SW mice, we cannot rule out potential additional
host and other intestinal microbial factors that could contrib-
ute to the observed sex effect on H. hepaticus colonization.
Also, colonization of H. hepaticus was associated with the
down-regulation of IL-10 production in the colon of SW mice
by 16 wpi. The role of IL-10 in the development of H. hepati-
cus–driven intestinal diseases in mice has been well docu-
mented. H. hepaticus colonization in IL-10-deficient mice can
FIG. 5. mRNA levels of IL-10 and IFN-? in the colon at 16 wpi.
Symbols: *, P values for the compared groups; C, sham dosed; WT,
WT H. hepaticus-dosed; cdtB inact., HhcdtBm7 dosed (the cdtB inac-
tivated); F, female mice; M, male mice. The levels of the IL-10 and
IFN-? transcripts were expressed as their copy numbers per 106of the
GAPDH transcripts in the same samples.
VOL. 73, 2005ROLE OF CDT IN H. HEPATICUS COLONIZATION3565
significantly accelerate severity of typhlocolits, suggesting that
IL-10 is required for suppressing progression of intestinal pa-
thology (23, 24, 44). In 129/RAG2?/?mice, H. hepaticus col-
onization induces severe typhlocolitis and colon tumors (9, 21,
22, 30). Adoptive transfer of CD4?CD45RBloCD25?T-regu-
latory cells from wild-type mice to 129/RAG2?/?mice both
before and after infection with H. hepaticus inhibits this pa-
thology (8, 9, 22, 30). In contrast, this protective effect was
abrogated when Treg cells from IL-10?/?mice were used,
indicating that IL-10 plays a pivotal role in this process (9, 22,
30). Hence, the down-regulation of IL-10 production in the
murine colon by H. hepaticus colonization may promote H.
hepaticus-induced colonic carcinoma in 129/RAG2?/?mice (8,
9). This effect also occurred in the colon of male mice which
eliminated HhcdtBm7 by 16 wpi but was not noticed in the
colon of female mice which eliminated the mutant by 2 wpi,
indicating that the colonization of H. hepaticus and not CDT
activity led to down-regulation of colonic IL-10 expression. We
speculate that additional virulence factors (e.g., secretory pro-
teins) from H. hepaticus suppress the IL-10-producing cell pop-
ulations in the colon. Further investigation into the interplay
between the H. hepaticus, immune cells, cytokines, and epithe-
lia in this lower bowel niche will provide a valuable model to
dissect molecular mechanisms controlling colon carcinoma in
In summary, we demonstrated that H. hepaticus CDT and
sex differences have significant impact on colonization by H.
hepaticus in outbred SW mice. The elevated IFN-? production
in the murine intestine is associated with loss of initial (in
females) and persistent (in males) colonization of the CDT-
deficient H. hepaticus mutant as well as the reduction of WT H.
hepaticus colonization levels in SW females. H. hepaticus-me-
diated down-regulation of IL-10 in the colon of SW mice may
play a role in H. hepaticus-induced colitis and colon carcinoma
observed in other mouse models. These results provide new
insights into H. hepaticus colonization in male and female SW
mice, including the important roles of CDT and host immune
We thank David Schauer and Vincent Young for providing plasmid
pVBY9 and Kathleen Cormier, Erinn Stefanich, and Jeff Bajko for
histologic processing of tissues.
This study was supported by NIH grants R01 CA67529 (J.G.F.), R01
AI50952 (J.G.F.), P01 CA26731, and P30ES02109.
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Editor: J. T. Barbieri
VOL. 73, 2005ROLE OF CDT IN H. HEPATICUS COLONIZATION 3567