INFECTION AND IMMUNITY, Mar. 2005, p. 1797–1810
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 3
Use of Genome-Wide Expression Profiling and Mutagenesis To Study
the Intestinal Lifestyle of Campylobacter jejuni
Alain Stintzi,1* Denver Marlow,2Kiran Palyada,1Hemant Naikare,1Roger Panciera,1
Lisa Whitworth,1and Cyril Clarke2
Department of Veterinary Pathobiology1and Department of Physiological Sciences,2College of Veterinary
Medicine, Oklahoma State University, Stillwater, Oklahoma
Received 1 March 2004/Returned for modification 18 June 2004/Accepted 20 September 2004
Campylobacter jejuni is the most common bacterial cause of diarrhea worldwide. To colonize the gut and
cause infection, C. jejuni must successfully compete with endogenous microbes for nutrients, resist host
defenses, persist in the intestine, and ultimately infect the host. These challenges require the expression of a
battery of colonization and virulence determinants. In this study, the intestinal lifestyle of C. jejuni was studied
using whole-genome microarray, mutagenesis, and a rabbit ileal loop model. Genes associated with a wide
range of metabolic, morphological, and pathological processes were expressed in vivo. The in vivo transcrip-
tome of C. jejuni reflected its oxygen-limited, nutrient-poor, and hyperosmotic environment. Strikingly, the
expression of several C. jejuni genes was found to be highly variable between individual rabbits. In particular,
differential gene expression suggested that C. jejuni extensively remodels its envelope in vivo by differentially
expressing its membrane proteins and by modifying its peptidoglycan and glycosylation composition. Further-
more, mutational analysis of seven genes, hspR, hrcA, spoT, Cj0571, Cj0178, Cj0341, and fliD, revealed an
important role for the stringent and heat shock response in gut colonization. Overall, this study provides new
insights on the mechanisms of gut colonization, as well as possible strategies employed by Campylobacter to
resist or evade the host immune responses.
Campylobacter is the most common nonviral etiological
agent of infectious enteritis in humans and has been implicated
in 14.2% of the 76 million food-borne illnesses reported an-
nually in the United States (21). Campylobacter infections vary
from mild diarrhea to severe abdominal pain (49). Rarely, they
result in the development of Guillain-Barre ´, syndrome, which
is the primary cause of acute neuromuscular paralysis in the
United States (49). Because of the extremely high number of
cases of food-borne infections reported yearly worldwide, the
development of new strategies to fight these infections is ur-
gently needed and will depend on developing an understanding
of host-pathogen interactions. The complete genomic se-
quence of Campylobacter jejuni NCTC 11168 was released in
2000 (27), providing new opportunities for the investigation of
Despite the high incidence of Campylobacter-mediated diar-
rhea, the microbial factors that govern gut colonization and
pathogenesis are poorly understood in comparison with other
enteric pathogens. In a complete infection cycle, Campy-
lobacter cells are transferred from contaminated foods to the
stomach, the intestinal tract and, finally, to the feces, allowing
their transmission to a new host (6, 13). During this stressful
journey through the gastrointestinal tract, Campylobacter en-
counters and must adapt to life-threatening environmental
conditions, such as the acidic pH of the stomach, the high
osmolarity of the gastrointestinal tract, intestinal gases, reac-
tive oxygen and nitrogen compounds, changes in nutrient avail-
ability, and low inorganic ion concentrations (13). For success-
ful colonization, Campylobacter cells must survive in the
intestinal tract, either as free-living microorganisms in the mu-
cus layer, attached to the epithelium, or intracellularly in epi-
thelial cells (6). Campylobacter determinants involved in colo-
nization and pathogenesis include flagella, host cell adherence
and invasion, and toxin production (6). Nonmotile and aflagel-
lated Campylobacter were shown to be affected in their ability
to colonize the gastrointestinal tract as well as to invade the
epithelial cells (25, 56). Many suspected adhesins have been
identified, such as lipo-oligosaccharide (20), flagella (56), or
surface-exposed proteins (CadF and PEB1) (23, 29). Host cell
invasion has been extensively studied and is thought to be an
important step in Campylobacter infection (6). Indeed, biopsies
of humans diagnosed with C. jejuni enteritis revealed the pres-
ence of intracellular Campylobacter cells (47). Interestingly,
toxin production has recently been proposed to modulate the
host immune response, allowing the bacterium to escape the
immune surveillance (9).
To identify new potential virulence factors, we analyzed C.
jejuni lifestyle in the gut using microarray technology. The C.
jejuni NCTC 11168 genome-wide expression profile was as-
sessed during host colonization and pathogenic development,
using a mammalian model of gastroenteritis, the rabbit ileal
loop (RIL) model. In addition, mutants were constructed by
deleting genes of interest identified by our microarray analysis,
and they were assessed for their ability to survive in the gas-
trointestinal tract of rabbits.
MATERIALS AND METHODS
Bacterial strains, plasmids, and preparation of inocula. The bacterial strains
and plasmids used in this work are listed in Table 1. The C. jejuni NCTC 11168
* Corresponding author. Mailing address: Department of Veteri-
nary Pathobiology, College of Veterinary Medicine, Oklahoma State
University, Stillwater, OK 74078. Phone: (405) 744-4518. Fax: (405)
744-5275. E-mail: email@example.com.
strain was acquired from the National Collection of Type Culture (NCTC) in
Spring 2000. Campylobacter strains were cultured in Mueller-Hinton (MH) me-
dium or on MH agar plates at 37°C in a microaerophilic chamber (Don Whiteley,
West Yorkshire, England). Chloramphenicol-resistant mutants were maintained
on MH medium supplemented with 20 ?g of chloramphenicol/ml.
C. jejuni inocula were prepared by microaerobic culture (84% N2, 5% O2, and
11% CO2) in MH medium at 37°C with agitation using a stirrer. The bacterial
growth was monitored by measuring the optical density at 600 nm (OD600). At
early-mid-log phase (OD600of approximately 0.3) the bacterial culture was split
in two, and one half was used to produce purified total RNA from C. jejuni grown
in vitro, while the other half was used to inoculate the RILs. Bacteria were
collected by centrifugation (10 min, 6,000 ? g), washed once with sterile phos-
phate-buffered saline (PBS) buffer, and resuspended in PBS buffer at a concen-
tration of approximately 6.6 ? 1010CFU/ml.
For the in vitro growth experiments, the C. jejuni wild-type and mutant strains
were grown in MH biphasic medium at 37°C under microaerophilic conditions
(83% N2, 4% H2, 8% O2, and 5% CO2).
RIL and isolation of C. jejuni total RNA. Rabbits were checked upon arrival to
see if they carried Campylobacter by taking cloacal swabs for culture. Ileal loops
were prepared according to published methods (3, 8). Briefly, New Zealand
White rabbits (?2 kg; females) were anesthetized, a laparotomy was performed,
and two 20-cm sections of ileum with intact mesenteric blood supply were ligated
per animal. Each loop was inoculated with approximately 1011mid-log-phase C.
jejuni in 1.5 ml of PBS buffer. The size of the inoculum was confirmed by
bacterial enumeration on MH agar plates. Loops of two rabbits were injected
with sterile PBS buffer and served as control animals. After replacing the intes-
tinal loops in their appropriate position in the abdominal cavity, the abdominal
wall and skin were closed in standard fashion and the rabbits were allowed to
recover from anesthesia. The rabbits were anesthetized again 24 or 48 h after the
inoculation, the intestinal loops were excised intact, and the animals were then
RNA turnover in the samples was quickly stopped by submerging the entire
loops into 10 ml of RNAlater solution (Ambion, Austin, Tex.). The loops were
first weighed in order to evaluate fluid accumulation. The contents of each loop
as well as the mucus layer were recovered into 20 ml of a 50% solution of
RNAlater in PBS buffer and centrifuged at low speed to remove epithelial cells
(5 min at 1,000 ? g). Thereafter, Campylobacter bacteria were separated from the
intestinal microflora by filtration through 0.8-?m-pore-size filters. Campylobacter
cells were pelleted by centrifugation, and total RNA was isolated using a hot
phenol-chloroform protocol, as previously described (40). Traces of genomic
DNA were removed by two or three consecutive treatments with DNase I
Amp-grade enzyme (Invitrogen, Carlsbad, Calif.). The absence of contaminating
genomic DNA was confirmed by PCR. RNA was further purified two to five
times using a QIAGEN RNeasy mini kit (QIAGEN, Valencia, Calif.), and the
concentration of RNA was determined using the RiboGreen RNA quantitation
reagent (Molecular Probes, Eugene, Oreg.).
Microarray construction and hybridizations. DNA microarrays were prepared
using PCR-amplified fragments of each annotated open reading frame from C.
jejuni NCTC 11168, as previously described (40, 41). Twenty micrograms of total
RNA from each growth condition (in vitro and in vivo) was converted to cDNA
using 2 pmol of C. jejuni 3?-specific primers (set of 1654 3? primers used for the
PCR amplification of C. jejuni open reading frames) and coupled to monoreac-
tive fluors (Cy3 and Cy5), according to previously described procedures (40).
Data collection and analysis. Microarray slides were scanned at 532-nm (Cy3)
and 635-nm (Cy5) wavelengths with a laser-activated confocal scanner (ScanAr-
ray 3000) at 10-?m resolution, generating two TIFF images. Fluorescence in-
tensities of each spot were collected using the GenePix Pro 3.0.5 software (Axon
Instruments, Foster City, Calif.) after manual optimization of spot registration
and exported to OriginPro 7 spreadsheets (OriginLab Corporation, Northamp-
ton, Mass.). The analysis of the fluorescence data was conducted as follows: (i)
the spots were filtered and excluded based on slide abnormalities or low signal
(corresponding to spots flagged bad or not found); (ii) after background sub-
traction, all spots with fluorescent mean intensities below three times the stan-
dard deviation of the background in both channels were removed from the final
data analysis; and (iii) the fluorescence intensity in each wavelength was log2
transformed and normalized using locally weighted linear regression (lowess)
performed by the MIDAS software (available from The Institute for Genomic
For the microarray analysis, Campylobacter RNA was isolated 48 h postinfec-
tion from five RIL rabbits. Each cDNA sample was individually cohybridized
with cDNA obtained from in vitro growth (mid-log-phase bacteria) on microar-
ray slides. The microarray hybridization was repeated up to three times depend-
ing on the amount of RNA purified from each rabbit, yielding between two and
six measurements per gene per rabbit (each gene was spotted in duplicate on
each slide). The microarray data were statistically analyzed using the significant
analysis of microarray (SAM) algorithm, which was specifically developed for
genomic expression data mining (the Microsoft Excel add-in software is available
at http://www-stat.stanford.edu/?tibs/SAM/) (46). Briefly, SAM uses the stan-
dard deviation of repeated gene expression measurements to assign a score to
each gene. It then estimates, for a particular score, a false discovery rate by
permutations of the data. This SAM analysis ascertains that genes identified as
differentially expressed do not arise from a random fluctuation of the large
quantity of data generated (46). To identify genes whose expression differed
significantly between in vivo and in vitro growth, we performed a one-class
response analysis by considering the five rabbits as one class. We applied a false
discovery rate of 0.11% and a delta value of 0.9. To identify genes with variable
expression between rabbits, we performed a multiple-classes analysis by treating
each rabbit as one class. We applied a false discovery rate threshold of 1.64% and
a delta of 0.19. The microarray data of SAM-positive genes were extracted
into a text output file using the Samster software (available at http://falkow
.stanford.edu/whatwedo/software/software.html). Finally, the ratios of the fluo-
rescence intensities of all replicate spots from the hybridization of RNA derived
from each rabbit were averaged and used for further analysis. The data gener-
ated by this study are available online at http://www.cvm.okstate.edu/research
C. jejuni mutant construction. Knockout mutants of C. jejuni NCTC 11168
were constructed by independently mutating six genes: hrcA, Cj0571, spoT, hspR,
Cj0341, and fliD. The same inactivation strategy was used for the hrcA, Cj0571,
spoT, and hspR mutants. Briefly, the gene to be mutated was amplified by PCR
from C. jejuni NCTC 11168 chromosomal DNA, which was extracted using a
standard protocol (35). The PCR product was cloned into pUC19, using a unique
restriction site (Table 2), and deletions of 42, 600, 627, and 72 bp were made by
inverse PCR in hrcA, Cj0571, spoT, and hspR, respectively. The chloramphenicol
resistance cassette (Camr) was PCR amplified from pRY111 (55) using primers
with appropriate restriction sites and cloned into the deletion site. Specific
primers used for the first PCR amplification and the following inverse PCR are
listed in Table 2. Recombinant plasmids carrying the Camrgene in the same
orientation as the genes of interest were selected by DNA sequencing and
transformed into C. jejuni NCTC 11168 using standard protocols (55). Transfor-
mants were identified on MH agar plates containing 20 ?g of chloramphenicol/
ml. The identity of the mutants was confirmed by PCR analysis using a combi-
nation of primer sets annealing within the mutated gene and the Camrgene.
The Cj0341 and fliD mutants were isolated from a library of random mutants
generated using the EZ::TN pMOD-3?R6K?ori/MCS? transposon (Epicen-
TABLE 1. Bacterial strains and plasmids
Strain or plasmidRelevant characteristic(s)a
E. coli strain
endA1 hsdR17 (rK
thi-1 recA1 gyrA rel A1
C. jejuni strains
C. jejuni NCTC 11168
Cloning and suicide vector, Ampr
cam resistance gene
pUC19 carrying ?Cj0571::cam
pUC19 carrying ?hspR::cam
pUC19 carrying ?hrcA::cam
pUC19 carrying ?spoT::cam
acam, chloramphenicol resistance gene; Ampr, ampicillin resistant.
1798STINTZI ET AL.INFECT. IMMUN.
tre), in which the Camrcassette from pRY111 has been cloned (A. Stintzi,
unpublished data). The mutant library was constructed following the manufac-
turer’s recommendation, and the Cj0341 and fliD mutants were identified during
the first trial to assess the randomness of the library. The insertion site of the
transposon in the Cj0341 and fliD mutants was identified using a single-primer
PCR procedure and DNA sequencing of the resulting amplification product, as
described by others (16). The Cj0341 and fliD mutations were confirmed by PCR
amplification using a primer that anneals within the mutated gene and another
primer that anneals within the Camrgene.
In vivo survival assays using a pool of mutants. The ability of the C. jejuni
mutants to survive within the RIL was assessed. Each mutant was grown indi-
vidually in MH broth to mid-log phase, harvested by centrifugation (10 min,
6,000 ? g), washed, and resuspended in PBS buffer. The mutants were mixed to
constitute the input pool by combining 5 ? 109CFU of each mutant with 7.5 ?
1010CFU of the wild-type strain, C. jejuni NCTC 11168, in 5 ml of PBS buffer.
One milliliter of this suspension was used to prepare genomic DNA following
standard protocols. The remaining bacterial suspension was used to equally
inoculate four ileal loops from two rabbits (1 ml per loop). At 48 h postinfection,
loop contents were harvested in PBS buffer and centrifuged at low speed to
remove debris and epithelial cells. The supernatant was immediately processed
to prepare genomic DNA, constituting the recovered pool of genomic DNA.
Each loop was processed individually.
The difference in relative abundance of each mutant between the input and
recovered pools was evaluated by quantitative PCR, which was performed using
an ABI Prism 7700 DNA analyzer (Applied Biosystems, Foster City, Calif.) and
the QuantiTect SYBR Green PCR kit (QIAGEN), according to the following
protocol: 500 ng of genomic DNA was added to 25 ?l of 2? QuantiTect SYBR
Green PCR solution and a 0.3 ?M concentration of each specific primer in a
50-?l final volume. The HotStar Taq DNA polymerase was activated by heating
the reaction mixture at 95°C for 15 min. PCR amplification was performed by 40
cycles of denaturation at 94°C for 15 s, annealing at 56°C for 30 s, and extension
at 72°C for 45 s. The specificity of the PCR was confirmed by melting curve
analysis of the PCR product following the manufacturer’s recommendations
(Applied Biosystems). Genomic DNA of each mutant was specifically amplified
from both pools by using a combination of two primers, with one of them
annealing within the mutated gene and the other within the chloramphenicol
resistance cassette. The sequences of the specific primer sets are available online
at http://www.cvm.okstate.edu/research/Facilities/CampyLab. The relative abun-
dance of each mutant was then normalized to the DNA pool by using the cydA
or argD gene. The competitive ratio of the relative abundance of each mutant
between the input and recovered pools was obtained using the comparative
threshold cycle (??CT) method, as recommended by Applied Biosystems. The
abundance of each mutant was assessed twice per loop, and the mean CTvalue
for each ileal loop was used for further analysis. The CTvalue corresponds to the
PCR threshold cycle at which the fluorescence detected is significantly higher
than the baseline value. The ratio of the mutant in the input to that in the
recovered pool was calculated as follows: ratio input/recovered ? 2???CT, where
??CT? ?CT, recovered? ?CT, input, and ?CT, recovered or inputis obtained by
subtracting the mean CTvalue of the specific gene from the mean CTvalue of the
reference gene (cydA or argD) in the genomic DNA from the input or recovered
pool. Given that the wild-type strain was inoculated at a higher level than the
mutants, the population as a whole (the mutants plus the wild-type strain) should
have a growth similar to the wild-type strain alone. The same assumption is
usually made for the analysis of data generated by signature tag mutagenesis
(10). Consequently, the normalized competitive ratio of input versus recovered
C. jejuni NCTC 11168 should be approximately equal to 1. A competitive input/
recovered ratio above 1 indicated that the mutant was attenuated in vivo, while
a competitive ratio below 1 indicated that the mutant survived better in vivo than
the wild type. The data were statistically analyzed using the Student t test, and a
P value below 0.01 was considered significant.
In vitro and in vivo competition experiments. C. jejuni wild-type and mutant
strains were grown in biphasic MH medium to mid-log phase, centrifuged, and
resuspended in PBS buffer to an OD600of ?1.8 (approximately 1010CFU/ml).
Two milliliters (each) of the wild-type and the mutant strains was mixed at a 1:1
ratio. Four ileal loops (from two rabbits) were injected (each) with 1 ml of this
suspension. The initial 1:1 mixture of mutant and wild-type strains was confirmed
by plating serial dilutions of this mixed culture on MH agar with and without
chloramphenicol (20 ?g/ml). At 48 h postinfection, the loops were recovered as
described above. Their content as well as the mucus layer were collected and
homogenized in 10 ml of PBS buffer. Serial dilutions of bacteria recovered from
each rabbit’s loop were plated on karmali-agar plates (Campylobacter agar base
[Oxoid CM935] supplemented with the Campylobacter-selective karmali supple-
ments [Oxoid SR167E]) and karmali-agar plates containing chloramphenicol (20
?g/ml). Plates were incubated at 37°C for 3 days before the colonies were
counted. The titer of the mutant was obtained from the CFU recovered on
karmali-agar plates containing chloramphenicol, and the titer of the wild-type
bacteria was calculated by subtracting the number of mutants from the total
number of bacteria recovered on karmali-agar plates without antibiotic. Finally,
the in vivo competitive index was calculated for each loop and corresponded to
the ratio of the mutant to the wild-type strain.
For the in vitro competition assays, overnight cultures of the wild-type and
mutant strains were mixed in a 1:1 suspension into fresh MH medium. This
suspension was used to inoculate three replicate biphasic MH cultures. The
bacterial growth was monitored by measuring the OD600over time. The titer of
each strain was determined in the inoculum and at early stationary phase, by
plating on MH agar and MH agar containing chloramphenicol (20 ?g/ml). The
in vitro competitive index was calculated for three independent growth experi-
ments and is defined as the ratio of mutant to wild-type.
Student’s t test was used to statistically analyze the data from the in vivo and
in vitro competition assays.
Real-time quantitative RT-PCR analysis. The relative expression of nine
genes (flgE2, Cj0178, katA, spoT, ahpC, fliD, Cj0571, cydA, and Cj0366) was
confirmed by real-time quantitative reverse transcription-PCR (RT-PCR), as
previously described (40), using the QuantiTect SYBR Green RT-PCR kit (QIA-
TABLE 2. Primers used in this study
PrimerDNA sequence from 5? to 3? (restriction site)a
For gene cloning
Cj0571-01 ............................................................................................................ATGCGAATTCATGCAAGAAAATTTCATACGC (EcoRI)
Cj0571-02 ............................................................................................................ATGCCTGCAGTCCCGTTGTAGCATCTTTTG (PstI)
For inverse PCR
Cj0571-03 ............................................................................................................ATGCGGATCCGCTTAATTTTCCCAAAGCAAA (BamHI)
Cj0571-04 ............................................................................................................ATGCGGATCCAGAACTGAAAATACGGCTAGAAGA (BamHI)
aThe restriction sites used for cloning are highlighted in bold and indicated in parentheses.
VOL. 73, 2005 CAMPYLOBACTER GENE EXPRESSION DURING IN VIVO GROWTH1799
GEN) according to the manufacturer’s recommendations. The relative expres-
sion of each gene was normalized to either the 16S or 23S RNA, and the extent
of its induction was obtained using the comparative threshold cycle (??CT)
method, as described above. The primers used are available online at http://www
Necropsy and histopathology. Four rabbits were used to evaluate the patho-
logical changes in the RIL model caused by C. jejuni NCTC 11168. The loops
were prepared as described above for the transcriptional profiling experiments.
The loops from two rabbits were inoculated with 1011CFU of mid-log-phase
C. jejuni NCTC 11168, while the loops of the two other rabbits were injected with
sterile PBS buffer. At 48 h postinfection, the rabbits were anesthetized, the loops
were recovered, and the animals were then euthanized with an overdose of
Beuthanasia D (?0.25 ml/kg of body weight). A 0.5-cm middle section of each
loop was immediately excised, flushed with formalin, linearly opened, placed flat
on a card, fixed in buffered 10% formalin, embedded in paraffin, section at 5 ?m,
and stained with hematoxylin and eosin. Specimens were examined for evidence
of inflammation, villus epithelial cell attenuation, and crypt dilatation or hyper-
RESULTS AND DISCUSSION
RIL model for campylobacteriosis. The RIL model was ini-
tially chosen because of its documented ability to accurately
model the histopathological lesions associated with human
Campylobacter gastroenteritis (8). In addition, Campylobacter
cells can be collected in a number sufficient for the investiga-
tion of in vivo genome-wide transcript abundance. The model
was created by surgical ligation of 20-cm sections of the ileum,
resulting in the cessation of the normal peristaltic flux, thereby
facilitating Campylobacter gut colonization. The strain of C.
jejuni NCTC 11168 used in our study is helically shaped, fully
motile, and colonizes the gastrointestinal tract of chicks (26).
Therefore, this strain is phenotypically different from the se-
quenced C. jejuni NCTC 11168 strain recently described by
Gaynor et al. (11), which was described to be straight rod-
shaped, nonmotile, and a poor colonizer of chicks. To explore
the feasibility of this model to study Campylobacter lifestyle in
the gut by transcriptome profiling, we undertook a pilot study
involving four rabbits: RIL were sampled at 24 or 48 h postin-
oculation (using two rabbits per time point). To note, the C.
jejuni strain was passaged three times in vitro before its inoc-
ulation in the ileal loops. C. jejuni NCTC 11168 colonized the
rabbit gut at bacterial concentrations of 105CFU/loop and 107
to 109CFU/loop, at 24 and 48 h postinoculation, respectively.
The initial decrease in bacterial population from 1011to 105
CFU per loop during the first 24 h after inoculation reflects the
challenges of surviving in a hostile environment, while the
subsequent bacterial growth from 105to 109CFU/loop at 48 h
suggests the successful adaptation and colonization of C. jejuni
in the rabbit intestinal tract. Considering that 105cells would
not generate sufficient amounts of RNA to perform microarray
hybridization, we decided to harvest the bacterial cells at 48 h
postinfection in the present study.
Intestinal distension resulting from accumulation of gas and
fluid (the first signs of diarrhea) was qualitatively observed at
48 h postinfection in all infected rabbits but not in the control
animals injected with the PBS buffer. Two infected rabbits and
two control animals were used to evaluate pathological
changes. Fluid accumulation was quantitatively estimated by
weighing the intestinal content of the eight loops, which indi-
cated an increase of 0.09 ? 0.05 g of content/g of ileal tissue in
the infected loops compared with control loops. This difference
was found to be statistically significant (P ? 0.05 using a paired
t test). In contrast to the observations of Everest et al. (8),
histopathological analysis of the ileal tissues did not reveal any
severe pathology. This lack of damages likely reflects the in-
ability of C. jejuni NCTC 11168 to invade epithelial cells or to
exert morphologically evident cytotoxic effects on intestinal
epithelial cells. In fact, the strain of C. jejuni NCTC 11168 used
in our study is poorly invasive into human epithelial INT407
cells (30). Consequently, the transcriptome profile presented
in this work reflects noninvasive Campylobacter lifestyle in the
intestine during survival, colonization, and the initial stages of
In vivo expression profiling validation. While microarrays
provide a powerful approach for the investigation of gene
expression, the performance of these expression studies in vivo
is technically challenging. To date, expression profiling exper-
iments have been limited mainly to in vitro environments. In
the present study, in vivo colonization of the intestinal tract by
C. jejuni was investigated by conducting transcriptional expres-
sion profiling experiments during growth and survival within
the natural gut environment. We utilized microarrays contain-
ing spotted PCR products representing approximately 98% of
the annotated open reading frames of C. jejuni NCTC 11168
(40). The challenge of recovering intact C. jejuni mRNA from
the intestine to ensure acquisition of an accurate and specific
transcriptome profile was addressed by excising the entire in-
testinal loops and immediately submerging them in RNA sta-
bilization solution to block RNA turnover. In order to mini-
mize RNA degradation and/or changes in the gene expression
level, loops were immediately processed for RNA extraction
and quantitative histopathological traits were not recorded.
The content of each loop, including the mucus layer, was re-
covered in RNA stabilization solution, and C. jejuni was puri-
fied by filtration through 0.8-?m filters. This physical separa-
tion removed most of the endogenous microflora; more than
80% of the bacterial population was estimated to be consti-
tuted of C. jejuni. The yield of RNA recovered was between 12
and 55 ?g per loop. The total RNA extracted from each rab-
bit’s two loops were combined. Twenty micrograms of RNA
was reverse transcribed using C. jejuni-specific 3?-end primers
and fluorescently labeled with the Cy5 dye, which fluoresces
red. The relative abundance of transcripts was monitored by
competitive hybridization with RNA extracted from bacteria
grown in vitro to mid-log phase and labeled with the green
fluorescent Cy3 dye. To address any potential cross-hybridiza-
tion with RNA extracted from the remaining natural intestinal
microflora, RNA was also purified from ileal loops of rabbits
which had been injected with PBS buffer only. The yield of
total RNA purified from the uninfected loops was between 1
and 3 ?g per loop. The total RNA harvested from two unin-
fected loops was combined, reversed transcribed, labeled with
Cy5, and hybridized to the C. jejuni microarray. As shown in
Fig. 1, this RNA did not cross-hybridize with genes from C.
jejuni. In addition to physical enrichment of C. jejuni by filtra-
tion, the use of 3?-specific primers to synthesize cDNA further
enhanced the specificity of the assay. A similar approach was
employed by Talaalt and coworkers to amplify mycobacterial
RNA from a mixture containing mammalian RNA (43).
Global gene expression analysis and validations. C. jejuni
NCTC 11168 was inoculated into five rabbits and colonized the
loops of these rabbits at a bacterial concentration of 3.108
1800STINTZI ET AL.INFECT. IMMUN.
CFU/loop (rabbit 1), 2.108CFU/loop (rabbit 2), 5.108CFU/
loop (rabbit 3), 4.107CFU (rabbit 4), and 4.108CFU/loop
(rabbit 5). Campylobacter RNA samples were extracted from
each rabbit 48 h postinfection and individually hybridized to
the microarray slides up to three times, depending on the
amount of RNA purified from each loop. Specifically, rabbits
1, 4, and 5 yielded two measurements per gene, rabbit 2 yielded
six measurements per gene, and rabbit 3 yielded four measure-
ments per gene. The data were quantified, normalized, and
reported as the ratio of gene expression of C. jejuni grown in
the rabbits to that of C. jejuni grown in vitro. To limit the
number of genes falsely identified as differentially expressed,
we performed a statistical procedure. This test consisted in
applying the SAM algorithm to our microarray data. This sta-
tistical method has been shown to be more reliable than a
standard t test or the use of a fold change threshold and is
relatively conservative in declaring a significant change in gene
expression (46). A one-class-response SAM analysis, using the
five rabbits as one group, identified 348 genes as being differ-
entially expressed between in vivo and in vitro growth with a
false discovery rate of 0.11%. All SAM-selected genes exhib-
ited expression ratios greater than 1.5. As demonstrated in our
previous study using the same microarray platform, a 1.5-fold
differential expression is technically and biologically significant
(40). This Campylobacter microarray platform has previously
been shown to generate data with a high level of concordance
with quantitative RT-PCR (40). However, in order to address
the reliability of the microarray data generated in this study,
the change in transcript abundance in rabbit 4 between in vitro
and in vivo growth was confirmed for nine genes (flgE2,
Cj0178, katA, spoT, ahpC, fliD, Cj0571, cydA, and Cj0366) by
real-time quantitative RT-PCR. The flgE2 gene was found to
be 4-fold up-regulated, Cj0178 was 200-fold up-regulated, katA
was 130-fold up-regulated, spoT was 61-fold up-regulated,
ahpC was 4-fold up-regulated, fliD was 5-fold down-regulated,
Cj0571 was 2.5-fold up-regulated, cydA was 350-fold up-regu-
lated, and Cj0366 was 300-fold up-regulated. Similarly to our
previous study, while the quantitative RT-PCR confirmed the
trend in differential gene expression observed with the mi-
croarray analysis, a quantitative difference in the fold change
was observed between these two technologies. This difference
reflects a lower dynamic range for the microarray experiments
compared to that of quantitative real-time RT-PCR, as previ-
ously reported by others (53). Notably, very few genes were
found to be differentially regulated more than 20-fold by the
microarray analysis, while the real-time RT-PCR found several
genes up-regulated more than 100-fold. This observation high-
lights the semiquantitative nature of microarray experiments
and the low dynamic range of this technology (4). Further-
more, this technical limitation appears to be amplified in situ-
ations where a gene exhibits a very low expression level under
only one of the growth condition, which is the case of in vivo
genome-wide expression analysis. As a consequence, the fold
change in gene expression presented in this study should be
significantly underestimated. Nevertheless, differentially ex-
pressed genes were readily identified by statistical analysis.
Overall, 185 genes were found to be induced in vivo in all five
rabbits. Among them, 177 exhibited more than twofold differ-
ential expression, with 91 of them showing more than fourfold
differential expression. Of the 199 genes found to be repressed
in vivo, the expression level of 153 genes was reduced twofold
and the expression level of 32 genes was reduced more than
Multiple-class response SAM analysis (considering each rab-
bit as one group) as well as two-class unpaired data SAM
analysis (considering each rabbit as one group and comparing
each rabbit with each of the others) revealed some gene ex-
pression variability between rabbits. Multiple-class response
SAM analysis identified 170 genes differentially expressed be-
tween rabbits with a false discovery rate of 1.64%. Importantly,
very few genes were found to be antagonistically expressed
between rabbits. Indeed, the trend of differential expression
remained essentially the same, while only the amplitude of
change in transcript abundance varied. To confirm the vari-
ability of gene expression between rabbits and to rule out the
possibility of intrinsic noise, we compared the expression mea-
surements of these 170 genes within each rabbit and between
rabbits (using the microarray data from rabbits 2 and 3). A
high level of concordance with a correlation coefficient higher
than 0.9 was obtained between replicate microarray hybridiza-
tions of RNA isolated from the same rabbit (Fig. 2A and B),
whereas a very weak correlation was observed between hybrid-
izations of RNA samples originating from two different rabbits
(Fig. 2C). The variability of gene expression between rabbits
was further confirmed by quantitative real-time RT-PCR for
flgE2 and Cj0178, which encode the flagellar hook subunit
protein and a putative outer membrane ferric-siderophore re-
ceptor, respectively. These were found to be differentially ex-
pressed between rabbits 3 and 4. The microarray analysis in-
dicated that the flgE2 gene was overexpressed in rabbit 4 and
down-regulated in rabbit 3, while the expression of Cj0178 was
essentially unaffected in rabbit 3 and up-regulated in rabbit 4.
By using the same RNA preparation as the one used for the
microarray hybridization, quantitative RT-PCR confirmed the
differential expression of both genes. The expression of flgE2
was found to be down-regulated approximately 70-fold in rab-
bit 3 and up-regulated 4-fold in rabbit 4, compared with in vitro
growth. Cj0178 was found to be equally expressed in rabbit 3
FIG. 1. Detection of C. jejuni transcriptome in vivo. (A and B) The
RIL 48 h postinoculation with C. jejuni or PBS buffer, respectively. The
arrows indicate intestine distended with gas and fluid accumulation.
Total RNA was extracted from the intestinal contents, reverse tran-
scribed using C. jejuni-specific 3? primer, and labeled with the Cy5 dye.
This labeled cDNA was cohybridized to the microarray with Cy3-
labeled cDNA, obtained from in vitro-grown bacterial RNA.
VOL. 73, 2005 CAMPYLOBACTER GENE EXPRESSION DURING IN VIVO GROWTH1801
and overexpressed 200-fold in rabbit 4 relative to in vitro
The observed variability in gene expression patterns is un-
clear but likely reflects both physiological and intrinsic varia-
tions in the rabbits. This hypothesis is in agreement with the
observed difference in colonization level (1 log) and the vari-
ation in the amount of fluid accumulation (?55%) between
rabbits. Obviously, the gastrointestinal environment cannot be
controlled and is likely to vary from one rabbit to another,
leading to variations in C. jejuni colonization and gene expres-
sion profiles. In addition, it is unknown if the rabbits used in
this study had previously encountered C. jejuni. If it was the
case, an immune response would likely take effect by 48 h
postinfection and might also result in the observed gene ex-
pression variability. Recently, Boyce et al. reported the ge-
nome-wide expression profile of Pasteurella multocida recov-
ered from blood of infected chickens 20 h after inoculation (2).
Although blood has questionable pathological relevance be-
cause it is not the site of infection of P. multocida, similarly to
our study those authors observed a variable bacterial gene
expression profile between infected hosts (2). More recently,
Xu et al. characterized the transcriptome of Vibrio cholerae
during intestinal growth 8 h postinfection using the RIL model
(54). In contrast to our study, V. cholerae gene expression was
similar in the three rabbits tested. All together, these data
highlight the complexity of studying genome-wide gene expres-
sion in vivo.
Campylobacter lifestyle in the gut. Overall, the expression of
482 genes was found to be significantly altered in vivo. Based
on their expression profiles, genes can be grouped into two
major categories: (i) genes exhibiting similar differential ex-
pression in all five rabbits tested (348 genes) (Fig. 3), and (ii)
genes with variable expression between rabbits (170 genes)
(Fig. 4). It should be noticed that 36 genes belong to both
categories. These genes exhibit similar expression alteration in
vivo in all five rabbits but different change amplitudes between
To elucidate further the intestinal lifestyle of Campylobacter,
we grouped genes by functional annotations and mapped their
expression profiles to all known biological processes, thus al-
lowing the investigation of the overall physiological status of C.
jejuni grown in vivo. This approach revealed the involvement of
a wide range of metabolic, morphological, and pathological
processes (Fig. 3 and 4). Figures 3 and 4 list only the genes
found to be significantly differentially expressed between in
vivo and in vitro growth by SAM analysis. However, a biolog-
ical process was considered to play a role in Campylobacter
physiology in the intestine when the constituting genes were
found to be either up-regulated or equally expressed in vivo
compared to in vitro growth.
Energy and central intermediary metabolism. The in vivo
transcriptome pattern of C. jejuni was consistent with the ox-
ygen-limited environment found in the intestine. The expres-
sion of genes encoding the key enzymes in the oxidative phos-
phorylation pathway was decreased dramatically in all five
rabbits. These genes encode NADH dehydrogenase (nuoG,
nuoL, and nuoH) and succinate dehydrogenase (sdhABC). Re-
cently, fumarate, nitrate, nitrite, and N- or O-oxides have been
shown to constitute alternative terminal electron acceptors,
allowing C. jejuni to carry out respiration under oxygen-re-
FIG. 2. Scatter plots showing the relationship between the log2
value of the gene expression ratio obtained from hybridization exper-
iments with bacterial cDNA derived from the same rabbit (A and B) or
from two different rabbits (C). The solid lines represent the linear
regression fit of the data.
1802 STINTZI ET AL.INFECT. IMMUN.
FIG. 3. Global view of genes with similar expression patterns between rabbits grouped by functional categories according to the Sanger Center
C. jejuni genome database. Each row represents one gene, and each column represents the expression profile in one rabbit (the mean fold change
in the expression ratio of the technical replicates). The column label corresponds to the rabbit numbering. An increasing red intensity denotes a
gene for which expression was significantly increased in vivo compared to in vitro growth, and an increasing green intensity indicates a gene for
which expression was significantly decreased in vivo compared to in vitro growth. A gray color indicates missing data. Genes with unknown
functions are not represented.
VOL. 73, 2005 CAMPYLOBACTER GENE EXPRESSION DURING IN VIVO GROWTH 1803
stricted conditions in vitro (36). However, the genes encoding
the reductases involved in this alternative respiratory pathway
were all down-regulated in vivo. In contrast, the genes encod-
ing for the cytochrome bd oxidase (cydAB) were expressed in
vivo but were not or were only slightly expressed in vitro. The
differential expression of the cydA gene was confirmed by
quantitative real-time PCR. The expression of cydA was found
to be 350-fold higher in vivo compared to in vitro growth.
Although the CydAB oxidase catalyzes the oxidation of mena-
quinone using oxygen as an electron acceptor, this enzymatic
complex has been shown in Escherichia coli to possess a high
affinity for oxygen, allowing the bacterium to carry out respi-
ration under limited oxygen tension (5). In addition, the ex-
pression of E. coli cydAB is known to be induced under limiting
oxygen conditions (5). Similarly, the CydAB complex could
facilitate C. jejuni respiration in the oxygen-limited environ-
ment of the intestine. Interestingly, formate dehydrogenase
(encoded by fdhABCD) was the only enzyme identified by the
microarray analysis to be overexpressed or equally expressed in
vivo relative to that in vitro and capable of transferring elec-
trons to the menaquinone pool. Other genes encoding enzymes
with similar activity were found to be down-regulated in vivo.
The FdhABCD enzyme participates in the respiratory chain of
many bacterial species, enabling these organisms to respire
FIG. 4. Global view of genes with a variable expression pattern between rabbits. Each row represents one gene. Columns 1, 2, 3, 4, and 5
represent the expression profiles in rabbits 1, 2, 3, 4, and 5, respectively. For each rabbit, the microarray data correspond to the mean fold change
in the expression ratio of the technical replicates. Red and green denote transcripts for which abundance was increased or decreased in vivo
compared to in vitro growth, respectively. The red and green intensities are proportional to the increase or decrease, with maximal fold changes
in transcript abundance of 3 and 0.33, respectively. A gray color denotes missing data.
1804 STINTZI ET AL.INFECT. IMMUN.
using formate as an alternative terminal electron donor under
anaerobic conditions (33). In C. jejuni, the formate dehydro-
genase, together with the CydAB complex, could allow the
bacterium to carry on oxygen respiration even under extreme
The expression of the genes encoding enzymes involved in
gluconeogenesis, the citric acid cycle, and the pentose phos-
phate pathway, were all down-regulated in vivo, except for
fructose biphosphate aldolase (fba). Down-regulation of these
genes is consistent with the oxygen-deprived intestinal environ-
ment and the up-regulation of the carbon storage regulator,
csrA. In E. coli, CsrA has been shown to repress the expression
of genes involved in glycogen catabolism, gluconeogenesis, gly-
colysis, and motility (34). This enzyme likely performs a similar
function in C. jejuni.
Macromolecular synthesis and processing. Genes encoding
proteins involved in the synthesis and modification of macro-
molecules, in particular the ribosomal proteins (with the ex-
ception of the rpsA gene) and aminoacyl tRNA synthetases,
were among the most highly up-regulated in vivo. The signif-
icance of this contradictory expression of rpsA (which encodes
the ribosomal protein S1) and other genes from the same
functional group is puzzling and requires confirmation by an
alternative method and further investigation. In E. coli, the
ribosomal protein S1 has been shown to be essential for cell
viability, to promote the efficiency of translation, and to act as
a repressor for its own synthesis (37). Depletion of the protein
S1 resulted in a stringent response consistent with amino acid
starvation and an increased production of ppGpp (37). There-
fore, the down-regulation of the rpsA expression would suggest
the induction of a stringent response in C. jejuni during intes-
Biosynthesis of cofactors. Another group of genes expressed
in vivo encodes proteins involved in the biosynthesis of the
cofactors, biotin (bioABCD), riboflavin (ribADFH), thiamine
(thiCDEGHJL), pantothenate (panBC), coenzyme A (accB
and acs), and folic acid (folCD). These genes were found to be
either up-regulated or equally expressed in vivo relative to in
vitro growth (with the exception of two genes, thiG and D, from
the thiamine biosynthetic pathway, which were found to be
down-regulated). The expression of these genes suggests that
these cofactors are unavailable in the intestine. As a conse-
quence, and because biotin, riboflavin, thiamine, and panto-
thenate are produced only by microbes and higher plants, these
biosynthetic pathways could constitute an ideal target for drug
development. This evidence that biotin is unavailable in the
intestine is corroborated by the up-regulation of V. cholerae
biotin biosynthetic genes during intraintestinal growth (54) and
by the inability of a V. cholerae biotin biosynthesis mutant to
colonize the gastrointestinal tract of mice (22).
Virulence and colonization determinants. Suspected viru-
lence and colonization factors of Campylobacter include motil-
ity and chemotaxis, host cell adherence and invasion, toxin
production, lipo-oligosaccharide and surface structure biosyn-
thesis, oxidative stress defense, iron acquisition, and heat shock
response (49). In contrast to genes encoding proteins involved
in general metabolism or bacterial physiology, the expression
levels of many genes related to virulence and/or colonization
factors were highly variable among infected rabbits. The most
notable among genes with flexible expression were those cod-
ing for proteins involved in flagellum biosynthesis. Motility is
known to be an essential requirement for C. jejuni to colonize
the host gut and ultimately cause disease (49). Considering
that the flagellin subunit is the immunodominant antigen rec-
ognized during human or animal infection, it is assumed that
the gene encoding this protein is expressed in vivo (28). How-
ever, our microarray data suggest that there is considerable
interanimal expression variability among genes belonging to
the flagellum locus. Most of the genes belonging to the flagel-
lum locus were found to be down-regulated in four rabbits
(and at a different level), while they were slightly up-regulated
in one rabbit. This variability may allow the bacterium to evade
the host immune system by shutting down flagellum produc-
tion once colonization is accomplished. In support of this hy-
pothesis, C. jejuni flagella have recently been proposed to be
necessary for passage through the gastrointestinal tract of
chickens, but not for persistence in the chickens’ cecum (51). A
similar effect on the expression of flagellar genes was also
recently demonstrated in Salmonella enterica during macro-
phage intracellular growth (7).
Another functional category of genes expressed in vivo re-
lates to iron-responsive genes which encode proteins involved
in iron metabolism and oxidative stress defense. Several of the
genes encoding iron acquisition systems were found to be ei-
ther up-regulated or equally expressed between in vivo and in
vitro growth. These genes code for a putative ferric-sid-
erophore transporter system (Cj0178 and Cj0173c-Cj0175c), a
putative iron transporter (p19 and Cj1658), and the three
TonB-ExbB-ExbD energy-transducing complexes. The genes
encoding a putative heme outer membrane transporter
(ChuABCD) were found to be only slightly expressed in vivo,
suggesting that heme does not constitute the main iron source
in the gut. The genes encoding the components of the ferric-
enterobactin uptake permease (ceuBCDE) were found to be
up-regulated in vivo; however, the cfrA gene encoding the
ferric-enterobactin receptor appeared to not be expressed. As
a microaerophilic bacterium, C. jejuni must deal with free
oxygen radicals and other reactive molecules generated by
normal aerobic metabolism and host defenses against micro-
bial attack (49). The expression of most genes known to be
associated with the C. jejuni oxidative stress response was
found to be increased during gastrointestinal growth (49). To
note, the up-regulation of these genes is in agreement with an
iron-limited environment. Theses genes include sodB (super-
oxide dismutase), ahpC (alkyl hydroperoxide reductase), tpx
(probable thiol peroxidase), and katA (catalase). Consistent
with the expression of these genes in vivo, a mutation of C. coli
sodB has been reported to impede colonization of chick gut
(31). Furthermore, a mutation in katA sensitizes C. jejuni to
hydrogen peroxide and reduces its intracellular survival in
macrophages (49). In Helicobacter pylori, the catalase KatA has
been shown to be required for persistent colonization in the
mouse model (14). These results highlight the iron-restricted
conditions in the rabbit intestine. The importance of iron me-
tabolism for successful host colonization has been established
for most pathogens (32) and should also be an essential factor
for C. jejuni colonization in the intestine. Indeed, a fur mutant
of C. jejuni, as well as cfrA, ceuE, and Cj0178 mutants, was
recently shown to be significantly affected in the ability to
colonize the gastrointestinal tract of chicks (26).
VOL. 73, 2005 CAMPYLOBACTER GENE EXPRESSION DURING IN VIVO GROWTH1805
Interestingly, C. jejuni possesses a system of general protein
glycosylation which has been proposed recently to play an
important role in C. jejuni pathogenesis (42). Indeed, mutation
of genes encoding the glycosyltransferases, pglB (also named
wlaF) and pglE (also named wlaK), affected their ability to
adhere to and invade human intestinal cells, as well as to
colonize the gastrointestinal tract of mice (42). In our study,
the expression of three genes, pglB, pglE, and pglG, which
belong to this functional category, was found to vary between
growth conditions. The expression of pglB and pglG was highly
induced in vivo (in four rabbits), while pglE expression was
either similar or repressed in vivo compared with that under in
vitro growth. The expression of the other genes from the gly-
cosylation cluster (pglH, pglA, wlaJ, and pglF) was found to be
similar under both growth conditions. It is not clear why ex-
pression of the genes belonging to the same biological pathway
varied. However, a similar difference in expression between the
pgl genes was observed previously in Campylobacter in re-
sponse to temperature up-shift (40) and iron starvation (26).
Considering the absence of data on the functional role of each
pgl gene in protein glycosylation, the significance of their ex-
pression profiles is difficult to assess.
The genes encoding the recently discovered multidrug efflux
pump in C. jejuni, cmeABC, were found to be highly up-regu-
lated in vivo. Expression of cmeB was confirmed by real-time
RT-PCR to be up-regulated by approximately 300-fold in vivo
compared with that under in vitro growth. This tripartite mul-
tidrug efflux transporter is composed of an outer membrane
protein, CmeC (Cj0367c), a periplasmic fusion protein, CmeA
(Cj0365c), and an inner membrane efflux transporter, CmeB
(Cj0366c) (18). Interestingly, this efflux system has been shown
to contribute greatly to bile resistance and to be required for
the colonization of the chick’s gastrointestinal tract (18, 19).
Given the presence of a high concentration of bile salts in the
gut, the up-regulation of these genes could contribute signifi-
cantly to the survival of Campylobacter in the host by allowing
the bacterium to resist the harmful effects of these salts.
Another important functional set of genes with variable ex-
pression between rabbits encodes proteins involved in pepti-
doglycan biosynthesis. Specifically, murB (a putative UDP-N-
acetylenolpyruvoyl glucosamine reductase), murC (UDP-N-
acetylmuramate-alanine ligase), and pbpC (penicillin-binding
protein) were among the genes from this category that were
the most differentially regulated. The differential expression of
this category of genes suggests that there may be a modifica-
tion of the murein sacculus in vivo, probably in response to the
high osmolarity of the intestinal environment. Likewise, Staph-
ylococcus aureus modifies its peptidoglycan layer under condi-
tions of high osmolarity (50). While the activation of genes
involved in peptidoglycan synthesis may constitute a repair
mechanism necessary for the bacterial adaptation to environ-
mental stress, the modification of the peptidoglycan structure
may have broad implications for the stiffness and elasticity of
the cell surface, thereby conditioning the bacterium to its eco-
Among the other genes annotated or previously character-
ized as virulence- or colonization-associated factors, Cj1279c
(putative fibronectin domain-containing lipoprotein) and sev-
eral genes involved in the heat shock response were found to
be significantly up-regulated in vivo. Heat shock proteins are
induced in response to various stresses and act by repairing and
preventing damage caused by the accumulation of unfolded
proteins. The importance of the heat shock response for
Campylobacter intestinal tract colonization has previously been
demonstrated (49). Genes identified as heat shock proteins
(40) and induced in vivo include clpB (ATP-dependent CLP
protease ATP-binding subunit), dnaK (heat shock protein),
grpE (heat shock protein), hrcA (putative heat shock regula-
tor), and htpG (hsp90 family heat shock protein). The up-
regulation of these genes, together with the previous identifi-
cation of ClpB as a B-cell antigen in human disease (45),
suggests that these proteins play an important role in bacterial
growth within the gastrointestinal tract.
Mutational analysis. In order to study further the C. jejuni
lifestyle in the intestinal tract of rabbits, we constructed knock-
out mutations of genes identified by our microarray analysis
and investigated the ability of these mutants to survive in the
RIL by comparison with the parent strain, C. jejuni NCTC
11168. Seven genes were selected and individually mutated.
The main goal of this mutational analysis was to disrupt phys-
iological functions that appeared to be important for the col-
onization of the ileal loop. In particular, the microarray data
suggested an important role for the genes involved in the heat
shock response, the stringent response, iron metabolism, and
the biogenesis of the flagellum in the intestinal lifestyle of C.
jejuni. Therefore, the heat shock response was disrupted by
mutagenesis of its two transcriptional regulators, hrcA and
hspR. The stringent response was disrupted by mutagenesis of
the spoT gene, which encodes the guanosine-3?,5?-bis(diphos-
phate) 3?-pyrophosphohydrolase. The iron metabolism was
disrupted by mutagenesis of the Cj0178 gene, which encodes a
ferric-siderophore outer membrane receptor. This gene has
been previously shown to be induced under iron restriction and
is highly up-regulated in vivo. The flagellum biogenesis was
disrupted by mutagenesis of the fliD gene, which encodes the
flagellar hook-associated protein. A mutant in the fliD gene
has been shown to be nonmotile (12). The fliD gene was found
to be down-regulated in all five rabbits. Interestingly, among
the genes encoding transcriptional regulators, Cj0571 was the
only one from this category found to be significantly up-regu-
lated in all five rabbits by the microarray analysis, suggesting an
important role for this protein in vivo. Consequently, this gene
was also mutated. Finally, a mutant in Cj0341 was chosen to be
tested in the ileal model as an experimental control. The ex-
pression of this gene was found to be off under in vivo and in
vitro growth conditions. Therefore, this mutant should not be
affected in its ability to colonize the ileal loop.
The seven mutants and the parent strain were pooled to-
gether and inoculated into four ileal loops constructed in two
different rabbits. After 48 h postinoculation, the ileal contents
were recovered and directly processed for chromosomal DNA
purification. Then, the relative amount of each mutant was
evaluated by quantitative real-time PCR, as described in Ma-
terials and Methods. The competitive ratio of the number of
cells at the time of the inoculation (in the input pool) to the
number of cells recovered 48 h postinoculation was normalized
to the entire bacterial population in the pool for each mutant
(Fig. 5) so that the competitive ratio of the whole population
was equal to 1. Considering that the wild-type strain was
present in excess, compared with each mutant in the input
1806 STINTZI ET AL.INFECT. IMMUN.
pool, and assuming that it represents the major proportion of
the population in the recovered pool, it should have a compet-
itive input/recovered ratio equal to approximately 1. There-
fore, any deviation of the competitive ratio from a value of 1
would indicate an effect of the mutation on the survival ability
of C. jejuni in the RIL. As expected, the competitive ratio of
the Cj0341 mutant was 0.9, indicating that this strain colonizes
the ileal loop as well as the wild type. Of the six other mutants,
one was unaffected (Cj0571), one had an advantage over the
others with respect to survival in and colonization of the RIL
(fliD; P ? 0.003), and four were significantly attenuated (hrcA,
Cj0178, spoT and hspR; P ? 0.002). The spoT and hspR mu-
tated strains were the most affected mutants, while the hrcA
and Cj0178 mutants were only slightly attenuated. Because
mutants are out-competed by many other strains during a
mixed infection, the in vivo phenotype of the affected mutants
was confirmed in a one-to-one competition assay. Each mutant
was independently mixed with the wild-type strain in equal
numbers and injected into four ileal loops (constructed in two
rabbits). The one-to-one ratio of the inocula was confirmed by
CFU determination. Forty-eight hours postinfection, the loop
contents were plated on selective medium for bacterial enu-
meration. Then, the competitive index was calculated as the
ratio of the mutant to the wild-type strain recovered from each
ileal loop. As shown in Fig. 6A, three out of the five mutants
were confirmed to be statistically affected in their colonization
ability (with a P value of ?10?4). In order to determine
whether the colonization phenotype of these mutants was spe-
cific for in vivo growth, an in vitro competition assay was
performed. An equal amount of each mutant and wild-type
strain was mixed in MH broth. The cultures were incubated at
37°C until late log phase (?30 h), after which serial dilutions
were plated on MH agar with or without chloramphenicol.
These experiments were performed in triplicate. The in vitro
competitive index was determined as described for the in vivo
competition assay (Fig. 6B). Four of the five mutants were
found to be statistically affected in their ability to out-compete
the wild-type strain during in vitro growth. Finally, in order to
determine whether the in vitro growth defect was caused by the
competition with the wild-type strain, the growth kinetic of
each mutant was independently determined (Fig. 7). All five
mutants were found to have a growth defect in vitro, with the
spoT, hspR, and fliD mutants being the most affected.
The competitive index of the hspR mutant in vitro and in
vivo was 1.5 ? 10?2and 7 ? 10?5, respectively. While this
mutant is affected in vitro, the 200-fold difference between the
in vitro and in vivo competitive indices indicates a significant in
vivo-specific growth defect. The attenuation of the hspR mu-
tant in vivo suggests a role for the heat shock regulatory net-
FIG. 5. Competitive colonization ability of seven mutants (hrcA,
Cj0178, Cj0571, spoT, Cj0341, hspR, and fliD). The strains were pooled
with the parent strain C. jejuni NCTC 11168 (constituting the input
pool) and inoculated into four RIL. Forty-eight hours postinoculation,
the intestinal contents were recovered and processed for chromosomal
DNA extraction. The number of bacteria was estimated by quantitative
real-time PCR for each mutant as described in Materials and Methods.
The normalized competitive ratio corresponds to the ratio of the
number of mutant cells to the total number of bacterial cells in the
input pool divided by the ratio of the number of mutants to the total
number of bacteria in the recovered pool. The data are the mean of
eight determinations (four biological replicates with two technical rep-
licates each), and the error bars represent the standard deviations.
FIG. 6. In vivo (A) and in vitro (B) competition assays. The in vivo competitive index is the ratio of the mutant to the wild-type strain recovered
in the ileal loop 48 h postinfection. Four loops were infected with a mixture of each mutant and the wild-type strain at a ratio of 1:1. The in vitro
competitive index is the ratio of the mutant to the wild-type strain in MH broth at late log phase. The in vitro competition assay was performed
in triplicate. The error bars indicate the standard deviations. *, statistical significance of P ? 0.001.
VOL. 73, 2005 CAMPYLOBACTER GENE EXPRESSION DURING IN VIVO GROWTH1807
work in Campylobacter gut colonization. In contrast, the hrcA
mutant was not significantly affected in its in vitro growth nor
in its ability to colonize the ileal loop. While the function of
hspR and hrcA in C. jejuni is essentially unknown, the products
of these genes were recently demonstrated to repress the tran-
scription of the major heat shock proteins in H. pylori (groESL,
hrcA, grpE, and dnaK) (38, 39). In addition, the HrcA-medi-
ated repression was shown to be dependent on the binding of
HspR to the promoter region (38). Interestingly, the transcrip-
tion of the cbpA-hspR-orf operon was found to be exclusively
regulated by HspR (38). Consequently, it is tempting to pro-
pose that the loss of HspR induces an increase in the abun-
dance of heat shock proteins. Given that heat shock proteins
are major immunodominant antigens, the overexpression of
these proteins would likely contribute to host resistance (52).
As a result, the hspR mutants should be less capable of colo-
nization and survival in the host. In addition, given the absence
of a colonization defect for the hrcA mutant, the amount of
these proteins would not be increased at a sufficient level in
this mutant to induce host resistance. In support of this hy-
pothesis, H. pylori hspR mutants and hspR-hrcA double mu-
tants were reported to have attenuated colonization efficiency
in wild-type mice, while they were unaffected in interleukin-
12-deficient mice (15). Furthermore, C. jejuni-infected patients
have been shown to develop a humoral response against the
heat shock protein DnaK (44).
In E. coli, spoT codes for a bifunctional enzyme able to
catalyze the biosynthesis and the degradation of hyperphos-
phorylated guanine [(p)ppGpp] (1). In most eubacteria,
(p)ppGpp has been shown to accumulate in response to strin-
gent conditions, such as amino acid starvation, triggering the
down-regulation of genes encoding the transcription and trans-
lation apparatus (1). As shown in Fig. 6, the spoT mutant
exhibited a competitive index of 9 ? 10?6in vivo. In contrast
to this in vivo result, the in vitro competitive index was only
0.06, suggesting a significant in vivo-specific defect. The de-
creased ability of the C. jejuni spoT mutant to colonize the host
gut suggests an important role for the stringent response in
vivo, likely allowing Campylobacter to deal with periods of
nutrient starvation or other environmental stresses in the in-
While the function of Cj0178 has not been characterized,
this protein exhibits high homology with ferric-siderophore
outer membrane receptors. In addition, the expression of
Cj0178 is Fur regulated and induced in response to iron star-
vation (26). Consequently, Cj0178 is probably required for the
acquisition of iron from an uncharacterized siderophore. The
Cj0178 mutant exhibits a competitive index of 0.05 in vivo and
0.37 in vitro. Given that C. jejuni NCTC 11168 does not seem
to produce any siderophore (48), the growth defect of this
mutant in vitro is unclear. The significant attenuation of the
Cj0178 mutant in vivo is in agreement with its overexpression
in our microarray experiment and might suggest an important
role for this iron acquisition system in gut colonization.
Interestingly, while the fliD mutant had a significant growth
defect in vitro (exhibiting a competitive index of 0.1), it colo-
nized the ileal loop as well as the wild-type strain (exhibiting a
competitive index of 1.3). The fliD gene encodes a putative
flagellar hook-associated protein. In H. pylori, fliD is an essen-
tial component in the assembly of a functional flagellum and is
FIG. 7. Growth kinetics of C. jejuni NCTC 11168 and five mutants,
?hrcA (A), ?hspR (B), ?spoT (C), ?Cj0178 (D), and ?fliD (E). Bi-
phasic MH cultures were incubated at 37°C under microaerophilic
conditions. The growth kinetics were performed in triplicate, and the
error bars represent the standard deviations.
1808 STINTZI ET AL.INFECT. IMMUN.
required for colonization of the gastric mucosa of mice (17).
Similarly to H. pylori, the C. jejuni fliD mutant is nonmotile and
aflagellated (12). While the survival of the fliD mutant in the
gut is in disagreement with the essential role of the flagellum in
the colonization of the gastrointestinal tract, it is consistent
with the characteristics of the RIL animal model. Indeed, the
physical ligation of the rabbit intestinal tract likely favors the
survival of mutants affected in their ability to adhere to the
mucus or the intestinal epithelial cells. In addition, considering
that flagellin is the major immunodominant antigen during
infection (24), the loss of the flagellum structure should pro-
mote evasion of the immune system. Consequently, a fliD mu-
tant will have an advantage over the wild type in vivo and thus
compete better during in vivo than in vitro growth.
Concluding remarks. This genome-wide expression profiling
study revealed important elements of the Campylobacter life-
style during host intestinal tract colonization. In addition to the
genes discussed above that have known or potential functions,
many other genes of unknown function were also found to be
differentially expressed between in vivo and in vitro growth and
therefore constitute many new directions for future investiga-
tions. The transcriptome pattern of C. jejuni in vivo was con-
sistent with that expected in an environment that is oxygen
limited, hyperosmotic, nutrient restricted, and containing re-
active oxygen compounds. Interestingly, the comparison of the
C. jejuni transcriptomes between different rabbits revealed
gene expression variability during the course of an infection.
This flexibility in gene expression is probably essential for
Campylobacter to adapt to the changing environment of the
gut. Furthermore, the genes encoding proteins involved in
flagellum biogenesis were found to be differentially expressed
between rabbits. They were up-regulated in one rabbit and
down-regulated (at a different level) in all others. While the
up-regulation of these genes is in agreement with the role of
the flagellum in gut colonization, the decreased expression of
these genes probably reflects a bacterial strategy to evade the
host response. Finally, regulation of both the heat shock re-
sponse and the stringent response were found to be necessary
for efficient colonization of the host gastrointestinal tract.
The project described was supported by National Institutes of
Health grant numbers AI055612 and RR15564.
We are grateful to all the staff from Oklahoma University and the
OSU microarray core facilities. We thank I. Turcot for providing
helpful comments on the manuscript.
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