INFECTION AND IMMUNITY, Sept. 2007, p. 4342–4350
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 9
The Capsule Encoding the viaB Locus Reduces Interleukin-17 Expression
and Mucosal Innate Responses in the Bovine Intestinal Mucosa during
Infection with Salmonella enterica Serotype Typhi?
Manuela Raffatellu,1Renato L. Santos,1Daniela Chessa,1R. Paul Wilson,1Sebastian E. Winter,1
Carlos A. Rossetti,2Sara D. Lawhon,2Hiutung Chu,1Tsang Lau,1Charles L. Bevins,1
L. Garry Adams,2and Andreas J. Ba ¨umler1*
Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Ave.,
Davis, California 95616-8645,1and Department of Veterinary Pathobiology, College of Veterinary Medicine,
Texas A&M University, College Station, Texas 77843-44672
Received 28 September 2006/Returned for modification 16 November 2006/Accepted 15 June 2007
The viaB locus contains genes for the biosynthesis and export of the Vi capsular antigen of Salmonella enterica
serotype Typhi. Wild-type serotype Typhi induces less CXC chemokine production in tissue culture models
than does an isogenic viaB mutant. Here we investigated the in vivo relevance of these observations by
determining whether the presence of the viaB region prevents inflammation in two animal models of gastro-
enteritis. Unlike S. enterica serotype Typhimurium, serotype Typhi or a serotype Typhi viaB mutant did not
elicit marked inflammatory changes in the streptomycin-pretreated mouse model. In contrast, infection of
bovine ligated ileal loops with a serotype Typhi viaB mutant resulted in more fluid accumulation and higher
expression of the chemokine growth-related oncogene alpha (GRO?) and interleukin-17 (IL-17) than did
infection with the serotype Typhi wild type. There was a marked upregulation of IL-17 expression in both the
bovine ligated ileal loop model and the streptomycin-pretreated mouse model, suggesting that this cytokine is
an important component of the inflammatory response to infection with Salmonella serotypes. Introduction of
the cloned viaB region into serotype Typhimurium resulted in a significant reduction of GRO? and IL-17
expression and in reduced fluid secretion. Our data support the idea that the viaB region plays a role in
reducing intestinal inflammation in vivo.
Salmonella enterica serotype Typhi causes a severe systemic
infection in humans known as typhoid fever. In contrast, non-
typhoidal Salmonella serotypes, such as S. enterica serotype
Typhimurium, cause a localized infection in humans manifest-
ing as gastroenteritis (41, 64). The different clinical presenta-
tions of infections with serotype Typhi and serotype Typhi-
murium point to important differences during the interaction
of these pathogens with their human host. One such difference
is the host response elicited in the intestinal mucosa. Gastro-
enteritis is a typical diarrheal disease characterized by a mas-
sive neutrophil influx in the terminal ileum and colon and a
predominance of neutrophils in the stool samples of patients
(9, 17, 26). In contrast, typhoid fever is not a typical diarrheal
disease, and the intestinal pathology is characterized by a pre-
dominantly mononuclear infiltrate (i.e., macrophages and den-
dritic cells) (17, 23, 29, 30, 46).
The nature of these differences is poorly understood, partly
because the strict adaptation of serotype Typhi to its human
host severely limits our ability to study host-pathogen interac-
tions in vivo. While higher primates (i.e., chimpanzees) are
susceptible to infections, nonprimate vertebrates and even
lower primates (i.e., rhesus macaques) are resistant to serotype
Typhi (11). As a result, differences between infections with
serotypes Typhi and Typhimurium have mostly been explored
with tissue culture models. Although the in vivo relevance of
results from tissue culture assays remains to be established in
many cases, these studies have revealed marked differences
during the interactions of serotypes Typhi and Typhimurium
with host cells. Microarray analysis shows that unlike serotype
Typhimurium, serotype Typhi does not trigger a classical
proinflammatory gene expression program in intestinal epithe-
lial cell lines (62). Serotype Typhimurium can trigger the mi-
gration of neutrophils across monolayers of polarized colonic
epithelial T84 cells, but serotype Typhi is not able to elicit this
response (25). Furthermore, stimulation of macrophage-like
THP-1 cells with serotype Typhi results in markedly reduced
interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-?)
expression compared to stimulation with serotype Typhi-
murium (19, 35).
Recent studies show that the viaB locus is required to
prevent serotype Typhi from eliciting proinflammatory re-
sponses in tissue culture models. The viaB locus encodes the
production of the Vi polysaccharide capsular antigen (3, 18,
22) and is located on a 134-kb serotype Typhi DNA region,
termed SPI7, that is absent from the serotype Typhimurium
chromosome (32). Deletion of the viaB locus from serotype
Typhi results in increased expression of the CXC chemokine
IL-8 after infection of human colonic epithelial cell lines
(35, 43) and in increased IL-8 and TNF-? expression after
infection of human macrophage-like cells (19, 35). These in
vitro data suggest that the presence of the viaB locus may be
in part responsible for the reduced propensity of serotype
* Corresponding author. Mailing address: Department of Medical
Microbiology and Immunology, School of Medicine, University of Cal-
ifornia at Davis, One Shields Ave., Davis, CA 95616-8645. Phone:
(530) 754-7225. Fax: (530) 754-7240. E-mail: email@example.com.
?Published ahead of print on 25 June 2007.
by Renee Tsolis on August 22, 2007
Typhi to rapidly elicit neutrophilic inflammation in the in-
testinal mucosa, which is characteristic of infections with
serotype Typhimurium (36). However, the assumption that
the viaB locus prevents intestinal inflammation in vivo has
not been tested experimentally with an appropriate animal
Two animal models used for the study of serotype Typhi-
murium-induced intestinal inflammation are bovine ligated il-
eal loops and streptomycin-pretreated mice. Mice are normally
not well suited for the study of serotype Typhimurium-induced
gastroenteritis, because these animals develop a systemic in-
fection without diarrhea and neutrophils are scarce in intesti-
nal infiltrates (44, 63). Pretreatment of mice with streptomycin
drastically increases their susceptibility to oral infection with
serotype Typhimurium (5) by promoting bacterial intestinal
overgrowth (34), which triggers a neutrophil influx in the ce-
cum (4). Streptomycin-pretreated mice can thus be used as a
model for the study of serotype Typhimurium-induced neutro-
phil recruitment in the cecal mucosa (4, 7, 13, 14). Natural or
experimental infection of calves with serotype Typhimurium
results in an enteric disease with clinical and pathological fea-
tures that parallel the disease in humans. Serotype Typhi-
murium causes a localized infection in calves, with the most
severe pathological changes being restricted to the intestine
(51, 59). Animals develop diarrhea and intestinal inflammation
characterized by a severe diffuse inflammatory infiltrate com-
posed predominantly of neutrophils (51, 59). Bovine ligated
ileal loops have been used successfully for the study of fluid
accumulation (a surrogate of diarrhea), neutrophil recruit-
ment, and cytokine responses following serotype Typhimurium
infection (10, 37–40, 42, 52, 53, 55, 56, 63, 65, 66). The goal of
this study was to evaluate the role of the viaB locus in these two
animal models of serotype Typhimurium-induced intestinal
MATERIALS AND METHODS
Bacterial strains, plasmids, and culture conditions. Serotype Typhimurium
strain IR715 is a fully virulent, nalidixic acid-resistant derivative of isolate
ATCC 14028 (48). Serotype Typhimurium strain ZA21 is a derivative of
IR715 carrying mutations in sipA, sopA, sopB, sopD, and sopE2 (66). Strain
EHW26 is a nonflagellated derivative of ATCC 14028 (fliC fljB mutant),
which has been described previously (35). Serotype Typhi strain Ty2 was
obtained from the American Type Culture Collection (ATCC 19430). Strain
STY2, a derivative of Ty2 carrying a deletion of the viaB region (?tviABCDE
vexABCDE::Km) (35). Plasmid pHP45? (33), carrying a streptomycin resis-
tance gene, was introduced into serotype Typhi strains by electroporation
prior to infection of streptomycin-pretreated mice. Cloning of the tviABCDE
vexABCDE genes (viaB locus) was performed with Escherichia coli strain
DH5? (12). The viaB region was amplified by PCR using the primers 5?-CG
CAACACACGGAGTATCACC-3? and 5?-TCGCCTACCAGCACAAAGC
G-3? for the upstream segment and the primers 5?-AAGTGCTGGAAGAA
CAGGTCG-3? and 5?-ACTAGTGTGAATACTTAGGCTGGGGTG-3? for
the downstream segment. The resulting 7.255-kb and 7.113-kb PCR products
were cloned into the vector pCR2.1 (Invitrogen), and amplification of the
correct fragments was confirmed by sequence analysis. The upstream region
was cloned into the SpeI and EcoRI sites of the low-copy-number vector
pWSK29 (54) to give rise to the plasmid pDC3. The downstream fragment
was then cloned into the EcoRI and KpnI sites of the plasmid pDC3 to give
rise to the plasmid pDC5. The tviA gene and its 600-bp promoter region were
amplified by PCR using the primers 5?-GGTACCCAGTATGACGTTCTG-3?
and 5?-CGAATTCTTGTCCGTGTTTTAC-3? and cloned into the EcoRI and
KpnI sites of the plasmid pWSK29 to give rise to the plasmid pTVIA1.
Strains were cultured aerobically at 37°C in Luria-Bertani (LB) broth supple-
mented with antibiotics, as appropriate, at the following concentrations: carben-
icillin, 100 mg/liter (LB?Cb); chloramphenicol, 30 mg/liter (LB?Cm); tetracy-
cline, 20 mg/liter (LB?Tc); kanamycin, 60 mg/liter (LB?Km); streptomycin, 100
mg/liter (LB?Str); nalidixic acid, 50 mg/liter (LB?Nal). For infection of bovine
ligated ileal loops, each strain was grown overnight at 37°C in 4 ml of LB broth
in a roller. A volume of 0.04 ml of this overnight culture was used for inoculation
of 4 ml of LB broth, and bacteria were grown at 37°C for 3 h in a roller.
Subsequently, this culture was used as the inoculum, and the numbers of CFU
were determined by plating serial 10-fold dilutions on LB plates.
To determine the generation time (g), bacteria were cultured aerobically and
the increase in bacterial numbers over time was monitored by measuring the
optical density at 600 nm (OD600). The slope (m) of the logarithmic increase
[plotted as the change in log2(OD600) over time] in bacterial numbers was
calculated by linear regression by the program Excel (Microsoft). The number of
bacteria (Nt) at a given time (t) is proportional to the number of bacteria at time
zero (N0) and the number of doublings, which can be calculated from the
generation time (g) by the formula 2t/g. After logarithmic conversion, this con-
nection can be described as log2(Nt) ? log2(N0) ? t/g. The slope, m, of the
logarithmic increase in bacterial numbers thus equals 1/g, which provides a
simple means of calculating the generation time.
Flow cytometry. Flow cytometry to detect expression of the Vi capsule was
performed as described previously (20, 35). DNA was labeled with propidium
iodide, and Vi antigen was detected by labeling of cells with rabbit anti-Vi serum
(1:250 dilution) (BD) and goat anti-rabbit fluorescein isothiocyanate (FITC)-
conjugate (1:250 dilution) (Jackson ImmunoLabs). For each sample, the fluo-
rescence of 10,000 particles (bacterial cells) was measured (FACSCalibur; Bec-
ton Dickinson). The gate for detection of Vi expression was set such that cells
expressing the capsule (IR715[pDC5] or Ty2) were considered positive for ex-
pressing the Vi antigen when their FITC fluorescence intensity exceeded that of
all but a small fraction (less than 2%) of the control population of an isogenic
noncapsulated strain (IR715 or STY2, respectively).
Streptomycin-pretreated mouse model. To study inflammation in the cecum,
8-week-old streptomycin-pretreated C57BL/6 mice were orally infected with
Salmonella serotypes as described previously (4). In brief, groups of four mice
were inoculated intragastrically with streptomycin (0.1 ml of a 200 mg/ml solu-
tion in phosphate-buffered saline [PBS]). Mice were inoculated intragastrically
24 h later either with sterile LB broth or with bacteria (0.2 ml containing
approximately 1 ? 109CFU/ml). At 24 h, 48 h, and 72 h after infection, groups
of four mice were euthanized and samples of the cecum were collected for
isolation of mRNA and histopathological analysis. Salmonella serotypes were
enumerated in the cecal contents, the mesenteric lymph node, and the spleen by
plating serial 10-fold dilutions of homogenates on selective agar plates
(LB?Carb or LB?Nal).
Bovine ligated ileal loop model. Four male Holstein calves, 4 to 5 weeks of age,
weighing 45 to 55 kg were used. They were fed milk replacer twice a day and
water ad libitum. The calves were clinically healthy before the experiment and
were culture negative for fecal excretion of Salmonella serotypes. Detection of
Salmonella serotypes in fecal swabs was performed by enrichment in tetrathio-
nate broth (Difco) followed streaking on brilliant green agar (BBL) and XLT4
Bovine ligated ileal loop surgery has been described previously (1, 39). In brief,
the calves were fasted for 24 h prior to surgery. Anesthesia was induced with
Propofol (Abbott Laboratories, Chicago, IL), followed by placement of an en-
dotracheal tube and maintenance with isoflurane (Abbott Laboratories, Chicago,
IL) for the duration of the experiment. A laparotomy was performed, the ileum
was exposed, and loops with lengths ranging from 6 to 9 cm were ligated, leaving
1-cm loops between them. The loops were infected by intralumenal injection of
3 ml of either sterile LB broth or a suspension of bacterial strains in LB broth
containing approximately 1 ? 109CFU. The loops were then replaced into the
abdominal cavity until collection at the indicated time points. Each bacterial
strain was tested in four different animals.
After surgical removal of loops, the fluid accumulated in the loops was mea-
sured and samples for bacteriological culture, histopathological analysis, and
mRNA isolation were collected. Intestinal samples for bacteriological analysis
were obtained with 3.5-mm biopsy punches and incubated in PBS containing 0.1
mg/liter gentamicin for 90 min. Tissue samples were then homogenized in PBS,
serially diluted, and plated on LB agar plates containing antibiotics appropriate
for determining CFU. Data on bacterial CFU were normalized to the length of
the ligated loop and the CFU present in the inoculum prior to statistical analysis.
Tissue collected for extraction of mRNA was snap frozen.
Histopathology. Tissue samples were fixed in formalin, processed according to
standard procedures for paraffin embedding, sectioned at 5 ?m, and stained with
hematoxylin and eosin. Inflammatory changes were scored from 0 to 3 according to
the following criteria: 0, no inflammation; 1, mild inflammatory changes, character-
VOL. 75, 2007 viaB LOCUS AND IL-17 EXPRESSION 4343
by Renee Tsolis on August 22, 2007
ized by multifocal intravascular margination and mild perivascular infiltration of
neutrophils in the lamina propria and submucosa; 2, moderate inflammatory
changes, characterized by moderate multifocal to coalescent or diffuse infiltration of
neutrophils in the lamina propria and submucosa, associated with mild to moderate
neutrophils in the lamina propria and submucosa, associated with moderate to
severe edema and/or multifocal hemorrhage and epithelial loss. Although the same
scoring system was adopted for both calves and mice, lesions scored as 2 and 3
tended to be more severe in calves, in which necrosis and erosion/ulceration of the
mucosa were often observed. Therefore, this scoring system is intended for compar-
species. Scores of 0, 1, 2, and 3 corresponded to averages of 3.3, 16.9, 46.7, and 144,9
neutrophils per high-magnification microscopic field (10 fields per animal were
counted and then averaged within each score). Such quantification of neutrophils in
calves was not performed due to the higher numbers of neutrophils and more-severe
Tissue culture assay. J774.A1 cells (ATCC TIB-67) were grown in Dulbecco’s
modified Eagle medium with glucose (4.5 g/liter) supplemented with 10% calf
serum (Gibco). Cells were seeded on a 24-well plate at a density of 2.5 ? 105
cells/well overnight. The next day, cells were infected with bacteria at a multi-
plicity of infection (MOI) of 10 for gene expression analysis and at an MOI of 0.1
for detection of protein levels in the supernatant. After 1 h, the cells were washed
and incubated with 0.1 mg/ml gentamicin for 1 h. Subsequently, the medium was
replace to contain 0.025 mg/ml gentamicin for the remaining time of the exper-
iment. The level of TNF-? in the supernatant was then quantified at 24 h
postinfection by enzyme-linked immunosorbent assay (ELISA) (eBioscience)
according to the instructions provided by the manufacturer. RNA extraction and
real-time PCR analysis were performed 6 h after infection, as described below.
Real-time PCR. For analysis of changes in gene expression after Salmonella
infection, tissue samples of murine cecum and bovine ileum were collected,
immediately snap-frozen in liquid nitrogen at the site of surgery, and stored at
?80 °C until processing. RNA was then extracted from snap-frozen tissue with
TriReagent (Molecular Research Center) according to the instructions of the
manufacturer. Next, 1,000 ng of RNA from each sample was retrotranscribed in
a 0.05-ml volume (Taqman reverse transcription reagent; Applied Biosystems),
and 0.004 ml of cDNA was used for each real-time reaction. Real-time PCR was
performed with SYBR green (Applied Biosystems) and the 7900HT Fast Real-
Time PCR System. The data were analyzed by the comparative CTmethod
(Applied Biosystems). Increases in cytokine expression in infected mice were
calculated relative to the average level of the respective cytokine in four control
animals from the corresponding time point after inoculation with sterile LB
broth. Increases in cytokine expression in calves were calculated for each in-
fected loop relative to a loop (collected at the same time point from the same
animal) that was inoculated with sterile LB broth. A list of genes analyzed in this
study with the respective primers is provided in Table 1.
Statistical analysis. For statistical analysis of ratios (i.e., increases in cytokine
expression or data expressed as percentages), data were transformed logarith-
mically prior to performance of a statistical analysis. A parametric test (paired
Student’s t test for ligated loop samples and Student’s t test for murine samples)
was used to calculate whether differences in increases or percentages between
treatment groups were statistically significant. Significance levels for Pearson’s
correlation analyses were determined with Instat software (GraphPad, San
Infection of streptomycin-pretreated mice with serotype
Typhi. To investigate whether the deletion of the viaB locus
would result in increased intestinal inflammation during sero-
type Typhi infection in vivo, we infected streptomycin-pre-
treated mice with serotype Typhi (Ty2), a serotype Typhi viaB
mutant (STY2), or serotype Typhimurium (IR715), a pathogen
that has been shown previously to cause inflammation in this
model (4). Each strain was used to inoculate groups of mice
(n ? 12 [per group]) that had been pretreated with strepto-
mycin 24 h earlier. As a negative control, streptomycin-pre-
treated mice were inoculated with sterile LB broth (n ? 12).
For each treatment, a subgroup of four mice was euthanized at
24, 48, and 72 h after infection.
Serotype Typhimurium was retrieved from the mesenteric
lymph node at significantly (P ? 0.05) higher numbers than
either the serotype Typhi wild type or viaB mutant at 48 and
72 h after infection. The serotype Typhi wild type (Ty2) was
recovered on average in significantly (P ? 0.05) higher num-
bers than was the viaB mutant (STY2) from the mesenteric
lymph nodes at 72 h after infection. Serotype Typhimurium
was isolated from the spleen starting at 48 h after infection,
and a high bacterial load, indicative of a severe systemic infec-
tion, was observed in this organ by 72 h after infection. In
contrast, serotype Typhi was not recovered in large numbers
from the spleen at any time point.
Gross pathological changes were not observed at any time
point in ceca from mice infected with serotype Typhi strains or
with sterile LB broth. In contrast, at 48 and 72 h after inoculation
the ceca from mice infected with serotype Typhimurium were
with whitish discoloration and a gelatinous appearance indicative
of severe edema. Histopathological analysis of hematoxylin- and
eosin-stained sections from the ceca revealed that at 48 and 72 h
after infection, only serotype Typhimurium induced a strong in-
flammatory response (Fig. 1A), which was characterized by mod-
erate to severe diffuse neutrophil infiltrate in the lamina propria
and submucosa, associated with severe edema, particularly in the
submucosa, and multifocal epithelial detachment of the luminal
To further characterize the inflammatory response in the ce-
cum, we analyzed the expression of cytokines in cecal tissue by
real-time PCR. Expression of keratinocyte-derived chemokine
(KC) and macrophage inflammatory protein 2 (MIP-2), two neu-
trophil chemoattractants related to the human GRO proteins (6,
28, 31, 45, 50, 58), were strongly upregulated 48 and 72 h after
TABLE 1. Primers used for real-time PCR
SpeciesGene Primer pair
4344 RAFFATELLU ET AL.INFECT. IMMUN.
by Renee Tsolis on August 22, 2007
infection with serotype Typhimurium but not with serotype Typhi
(data not shown). Expression of IL-17 (also known as IL-17A), a
cytokine contributing to neutrophil recruitment in response to
bacterial infection in the mouse lung (15, 16, 24, 60, 61), was
increased in all infected mice by 24 h after inoculation. However,
was considerably higher in the ceca of mice infected with serotype
Typhimurium (Fig. 1B). TNF-? expression was significantly (P ?
0.014) higher in mice 72 h after infection with the serotype Typhi
viaB mutant than in animals infected with the serotype Typhi wild
type (Fig. 1C).
The viaB locus reduces TNF-? expression in murine mac-
rophages. The only significant difference in the host response
of streptomycin-pretreated mice associated with the viaB locus
was an induction of TNF-? expression observed 72 h after
infection with the viaB mutant but not with the serotype Typhi
wild type. To model expression of TNF-? in vitro, a murine
macrophage-like cell line (J774.A1 cells) was infected with
serotype Typhi strains and cytokine production was monitored
by real-time PCR and ELISA. The serotype Typhi viaB mutant
elicited significantly higher TNF-? expression in J774.A1 cells
than did the serotype Typhi wild type (Fig. 2). We constructed
low-copy-number plasmids carrying the whole viaB region
(plasmid pDC5) or the cloned tviA regulatory gene (pTVIA1)
and introduced each into the serotype Typhi viaB mutant by
electroporation. Introduction of the cloned viaB locus, but not
of the cloned tviA regulatory gene, complemented the pheno-
type of the viaB mutant. These data suggested that reduced
TNF-? expression in murine macrophages depends on the
presence of capsule biosynthesis genes. TviA, the positive reg-
ulator encoded within the viaB region, was not sufficient to
complement this phenotype. Deletion of the serotype Typhi
flagellin gene (fliC) did not significantly reduce TNF-? expres-
sion elicited by the viaB mutant. These data suggested that
pathogen-associated molecular patterns, in addition to flagel-
lin, contributed to TNF-? expression elicited by the serotype
Typhi viaB mutant in murine J774.A1 macrophages.
Introduction of the cloned viaB locus into serotype Typhi-
murium reduces inflammatory responses in bovine ligated il-
eal loops. Overall, the lack of an acute inflammatory reaction in
response to infection of streptomycin-pretreated mice with sero-
type Typhi wild type and the viaB mutant suggested that this
animal model may not be well suited for the study of serotype
Typhi-specific virulence factors. To study the role of the viaB
locus during infection of cattle, we introduced the cloned viaB
region (pDC5) into serotype Typhimurium (IR715) by electropo-
ration. Expression of the Vi capsular antigen in serotype Typhi-
murium strain IR715(pDC5) was detected by slide agglutination
that serotype Typhimurium carrying pDC5 expressed the Vi an-
tigen on its surface at levels similar to those detected in the
serotype Typhi wild type (Fig. 3A and B). Introduction of pCD5
into S. Typhimurium did not reduce its generation time during
growth in LB broth (Fig. 3C).
Bovine ligated ileal loops were infected with the serotype
Typhimurium wild-type strain (IR715) and its capsulated deriva-
FIG. 1. Host responses in the cecum of streptomycin-pretreated
mice in response to infection with serotype Typhi wild type (open
squares), serotype Typhi viaB mutant (open triangles), or serotype
Typhimurium wild type (closed squares). (A) Average histopathology
score determined by blinded examination of sections from tissues col-
lected at the indicated time points after infection by a veterinary
pathologist. Expression of IL-17 (B) and TNF-? (C) in the cecum of
streptomycin-pretreated mice in response to infection was determined
by real-time PCR. Data are expressed as increases in mRNA levels
relative to those in the ceca of control animals inoculated with sterile
LB broth and collected at the same time point. The probability (P) that
differences are statistically significant is indicated. The graphs show
geometric means of increases in expression ? standard deviations
FIG. 2. TNF-? expression elicited by serotype Typhi strains in
J774.A1 macrophage-like cells determined by real-time PCR (A) and
ELISA (B). Bars represent averages from three independent experi-
ments ? standard errors. The probability (P) that differences are
statistically significant is indicated.
VOL. 75, 2007viaB LOCUS AND IL-17 EXPRESSION4345
by Renee Tsolis on August 22, 2007
tive [IR715(pDC5)]. Recovery of bacteria from the ileal mucosa
at 2 h and 8 h after infection showed that plasmid pDC5 was
maintained by serotype Typhimurium for the duration of the
experiment (data not shown). To evaluate the magnitude of the
inflammatory response, expression of the CXC chemokine
GRO? and the cytokine IL-17 were detected in the ileal mucosa
by real-time PCR at 2 h after infection. GRO? expression was
induced approximately sevenfold in loops infected with the sero-
type Typhimurium wild type (IR715) compared to mock-infected
GRO? expression, suggesting that the presence of the Vi capsule
attenuated the inflammatory response to serotype Typhimurium
By 2 h after infection of loops with serotype Typhimurium
(IR715), IL-17 expression was increased, on average, 56-fold
compared to loops inoculated with sterile LB broth. Similarly,
strain IR715(pDC5) (serotype Typhimurium expressing the Vi
capsule) elicited approximately 14-fold upregulation of IL-17
expression compared to control loops inoculated with sterile
LB broth (Fig. 4B).
At 8 h after infection, fluid accumulation elicited by each
strain was measured. Capsulated bacteria [IR715(pDC5)] trig-
gered reduced fluid accumulation compared to the serotype
Typhimurium wild-type strain (IR715) (Fig. 4C). However,
these differences were not statistically significant. A milder
inflammatory response and reduction of neutrophil influx was
observed in the histopathological analysis of sections from the
ileal mucosa infected with IR715(pDC5) compared to the se-
rotype Typhimurium wild-type strain (Fig. 5).
Previous studies have implicated flagellin as a major contribu-
tor to serotype Typhimurium-induced inflammation in bovine
ligated ileal loops (42). To investigate whether the anti-inflam-
matory effect of the viaB locus was solely dependent on flagella,
plasmid pDC5 was introduced into a serotype Typhimurium fliC
FIG. 3. Vi antigen expression in serotypes Typhimurium and
Typhi. (A) Cells of serotype Typhimurium (IR715) (left) and of a
serotype Typhimurium strain carrying a plasmid encoding the viaB
locus (pDC5) (right) were labeled with the DNA stain propidium
iodide (PI) (x axis) and with rabbit anti-Vi serum/goat-anti rabbit FITC
conjugate (?-Vi) (y axis), and fluorescence intensities were determined
for 10,000 particles. The gate for detection of Vi expression was set
such that cells expressing the capsule [serovar Typhimurium (pDC5)]
were considered positive for expressing the Vi antigen when their
FITC fluorescence intensity exceeded that of all but a small fraction
(less than 2%) of the control population (serovar Typhimurium wild
type). (B) Cells of serotype Typhi (Ty2) (right) and of a serotype Typhi
?viaB mutant (STY2) (left) were labeled as described above. The gate
for detection of Vi expression was set such that cells expressing the
capsule (serovar Typhi wild type) were considered positive for express-
ing the Vi antigen when their FITC fluorescence intensity exceeded
that of all but a small fraction (less than 2%) of the control population
(serovar Typhi ?viaB). In panels A and B, numbers within the gated
area indicate the fractions of cells (in percentages) expressing the Vi
antigen. (C) Growth curve of the serotype Typhimurium wild type and
derivatives carrying pDC5 or pTVIA1. The generation time (in min-
utes) calculated for each strain during logarithmic growth is indicated.
FIG. 4. Host responses in the bovine terminal ileum during infec-
tion of ligated loops with the indicated strains of serotype Typhi-
murium (open bars) and serotype Typhi (hatched bars). Expression
levels of GRO? (A) and IL-17 (B) were determined by real-time PCR
using tissue collected 2 h after infection. Data are shown as increases
of cytokine expression relative to control loops inoculated with sterile
LB broth. Bars represent geometric means ? standard errors.
(C) Fluid accumulation was quantified at 8 h after infection. Data are
expressed as percentages of the response elicited by the serotype
Typhimurium wild type (wt). Bars represent geometric means ? stan-
dard errors. The probability (P) that differences are statistically signif-
icant is indicated.
4346 RAFFATELLU ET AL.INFECT. IMMUN.
by Renee Tsolis on August 22, 2007
fljB mutant (EHW26). Compared to infection with the fliC fljB
mutant (EHW26), infection with the fliC fljB mutant carrying
pDC5 elicited significantly (P ? 0.05) less GRO? expression (Fig.
6). The fliC fljB mutant carrying pDC5 also elicited less IL-17
expression, less fluid accumulation, and a milder inflammatory
response; however, these differences were not statistically signif-
icant. Although these data do not rule out the possibility that the
viaB locus reduces inflammatory responses triggered by flagellin,
the fact that the viaB locus significantly reduced GRO? expres-
effect mediated by this DNA region cannot be exclusively attrib-
uted to an inhibition of flagellin-mediated responses.
Deletion of the viaB region results in increased inflamma-
tory responses elicited during serotype Typhi infection of
bovine ligated ileal loops. Serotype Typhi does not elicit
marked inflammation in bovine ligated ileal loops (37). To
investigate whether the absence of the viaB locus would in-
crease the response to serotype Typhi infection in calves, loops
were infected with the serotype Typhi wild type (Ty2) and a
viaB mutant (STY2). The serotype Typhi wild type (Ty2) elic-
ited significantly less GRO? expression (P ? 0.018) and IL-17
expression (P ? 0.045) than did the serotype Typhimurium
wild type (Fig. 4). The serotype Typhi viaB mutant elicited
significantly more GRO? expression (P ? 0.02) and IL-17
expression (P ? 0.049) than did the wild type (Ty2) in ligated
ileal loops. Interestingly, at 2 h after infection, the serotype
Typhi viaB mutant (STY2) elicited IL-17 expression (approx-
imately 38-fold upregulation compared to mock-infected
loops) at levels similar to those elicited by the serotype Typhi-
murium wild type (P ? 0.1). Loops infected with the serotype
Typhi viaB mutant (STY2) contained amounts of fluid at 8 h
after infection similar to those infected with the serotype
Typhimurium wild-type strain (P ? 0.09). The amount of fluid
elicited by the serotype Typhi viaB mutant (STY2) was signif-
icantly higher than that elicited by the serotype Typhi wild type
(Ty2) (P ? 0.005). Furthermore, the serotype Typhi viaB mu-
tant triggered more inflammation than did its capsulated par-
ent (Ty2) (Fig. 5).
In summary, data from the bovine ligated ileal loop model
show that the presence of the viaB region significantly reduces
the inflammatory responses to serotype Typhi or serotype
Typhimurium infection in vivo.
IL-17 levels correlate with the severity of inflammatory re-
sponses elicited during infection with Salmonella serotypes.
Previous studies of inflammation in the respiratory tract sug-
gest that IL-17 contributes to neutrophil recruitment by stim-
ulating other cells to produce CXC chemokines (15, 16, 24, 60,
61). The assumption that IL-17 significantly contributes to
neutrophil recruitment in the intestine would predict that there
should be a positive correlation between the expression levels
of IL-17 and the expression levels of neutrophil chemoattrac-
tants (CXC chemokines) in infected tissue. To test this pre-
diction, we compared the expression levels of IL-17 and of
CXC chemokines in the bovine ileal mucosa. The expression
levels of IL-17 and GRO? determined for individual loops
collected at 2 h after infection of the bovine ileal mucosa with
serotype Typhi or serotype Typhimurium strains were com-
pared (Fig. 7A). This analysis revealed a positive correlation
between expression of IL-17 and GRO? (R2, 0.66; P, 0.001).
The ?-defensins are a group of inducible antimicrobial pep-
tides that contribute to host defense at several mucosal sur-
faces, including the enteric mucosa. IL-17 has been shown to
induce expression of ?-defensin in human bronchial epithelial
cells (21). Comparison of the expression levels of IL-17 and
FIG. 5. Histopathological changes in the bovine terminal ileum in
response to infection of ligated ileal loops. An average histopathology
score was determined by blinded examination of sections from tissue
collected at 8 h after infection with serotype Typhimurium (open bars)
or serotype Typhi (hatched bars) strains by a veterinary pathologist.
FIG. 6. Host responses in the bovine terminal ileum during infec-
tion of ligated loops with nonflagellated serotype Typhimurium strains.
Expression levels of IL-17 (A) and GRO? (B) and were determined by
real-time PCR using tissue collected 2 h after infection. Data are
shown as increases of cytokine expression relative to control loops
inoculated with sterile LB broth. The probability (P) that differences
are statistically significant is indicated. (C) Fluid accumulation was
quantified at 8 h after infection. Data are expressed as percentages of
the response elicited by the serotype Typhimurium wild type (wt). Bars
represent geometric means ? standard errors. The probability (P) that
differences are statistically significant is indicated. (D) Histopatholog-
ical changes in the bovine terminal ileum in response to infection of
ligated ileal loops. An average histopathology score was determined by
blinded examination of sections from tissue collected at 8 h after
VOL. 75, 2007viaB LOCUS AND IL-17 EXPRESSION4347
by Renee Tsolis on August 22, 2007
bovine enteric ?-defensin (Fig. 7B) revealed a significant pos-
itive correlation (R2, 0.60; P, ?0.0001).
Humans infected with serotype Typhimurium develop a
massive neutrophil influx in the intestine, but this host re-
sponse does not develop in patients infected with serotype
Typhi. It has been proposed that serotype Typhi does not elicit
neutrophil influx in the human intestine because it possesses
the capsule-encoding viaB locus, a DNA region that is absent
from the serotype Typhimurium genome (35, 36). This hypoth-
esis is based on in vitro data showing that deletion of the viaB
locus from serotype Typhi causes an upregulation of IL-8 ex-
pression during infection of human cell lines (T84, THP-1) and
human colonic tissue explants (35, 43). To investigate the in
vivo relevance of these findings, we investigated whether the
presence of the viaB locus influences the intestinal inflamma-
tory response in two animal models of serotype Typhimurium-
induced neutrophil influx.
Bovine ligated ileal loops are well suited for the study of
cytokine expression, neutrophil influx, and fluid accumulation
in response to serotype Typhimurium infection (10, 37, 39, 40,
52, 53, 55–57, 63, 65, 66). However, infection of bovine ligated
ileal loops with serotype Typhi strain Ty2 does not result in
profound inflammatory changes or fluid accumulation (37).
We show that introduction of the capsule-encoding viaB locus
into serotype Typhimurium resulted in reduced inflammatory
cytokine production, reduced severity of histopathological
changes, and reduced fluid accumulation in the bovine ligated
ileal loop model. Remarkably, deletion of the viaB locus from
serotype Typhi strain Ty2 resulted in increased inflammation
in the calf intestine and in fluid secretion at levels that were
similar to those elicited by serotype Typhimurium (35).
The ability to invade the intestinal epithelium with the in-
vasion-associated type III secretion system (T3SS-1) is criti-
cally important for the induction of inflammation and fluid
accumulation by serotype Typhimurium in vivo (38, 66). Ex-
pression of the Vi antigen reduces the invasiveness of serotype
Typhi for epithelial cells in vitro (2, 27). These data suggest
that one possible mechanism by which the viaB locus may
prevent inflammatory responses in vivo is by reducing bacterial
invasion. Although this possibility cannot be ruled out, we did
not find a significant correlation between the numbers of cap-
sulated or noncapsulated bacteria recovered from gentamicin-
treated tissue and the magnitude of proinflammatory cytokine
expression (R2, 0.18; P, 0.12) (Fig. 7C). Furthermore, deletion
of the viaB locus results in increased IL-8 expression during
serotype Typhi infection of colonic epithelial T84 cells regard-
less of whether T3SS-1 is functional or inactivated by a muta-
tion in invA (35). These data suggest that the viaB locus can
reduce inflammatory responses by a T3SS-1-independent
mechanism. A T3SS-1-independent mechanism by which the
Vi antigen may inhibit inflammatory responses in vivo is by its
interference with Toll-like receptor (TLR) recognition (19,
35). Human epithelial kidney 293 (HEK293) cells produce
IL-8 in response to infection with a serotype Typhi viaB mutant
only when they are transfected with TLR5 or TLR4/MD2/
CD14. IL-8 production by TLR5 or TLR4/MD2/CD14-trans-
fected HEK293 cells is significantly reduced during infection
with serotype Typhi wild type (35), supporting the idea that the
viaB region may interfere with bacterial recognition by TLRs
expressed on host cells.
Oral infection of streptomycin-pretreated mice with sero-
type Typhimurium triggers the development of a neutrophil
influx in the cecum (typhlitis) (4). Host responses to infection
with serotype Typhimurium in the cecum of streptomycin-
pretreated mice are similar to those elicited in germfree mice
(47). Oral infection of germfree mice with serotype Typhi
strain Ty2 results in substantial bacteria growth in the cecum,
but bacteria are not recovered from the liver or spleen (8).
Similarly, we recovered serotype Typhi strain Ty2 in large
numbers from the cecum of streptomycin-pretreated mice,
while animals were able to contain growth at systemic sites of
infection (data not shown). Furthermore, in contrast to sero-
type Typhimurium, serotype Typhi caused very few inflamma-
tory changes in the cecum of streptomycin-pretreated mice.
These results are similar to those obtained by infecting strep-
tomycin-pretreated mice with another strictly human -adapted
pathogen, S. enterica serotype Paratyphi A, which colonizes the
FIG. 7. Correlation of IL-17 expression levels in bovine ligated ileal
loops with expression of enteric ?-defensin (A), GRO? expression
levels with expression levels of IL-17 (B), or GRO? expression levels
with bacterial tissue loads (C). (A) Expression levels of GRO? and
IL-17 in individual ligated loops (circles) collected 2 h after inoculation
with serotype Typhimurium or serotype Typhi strains were determined
by real-time PCR. (B) Expression levels of enteric ?-defensin and
IL-17 in individual liagated loops (circles) collected 2 h after inocula-
tion with serotype Typhimurium or serotype Typhi strains were deter-
mined by real-time PCR. (C) Bacterial numbers recovered from tissue
2 h after inoculation with the serotype Typhimurium wild type (closed
circles), the serotype Typhimurium wild type carrying plasmid pDC5
(open circles), the serotype Typhi wild type (open squares), or the
serotype Typhi viaB mutant (closed squares) were correlated with
GRO? expression levels in each respective loop. The R2and P values
for each correlation are indicated.
4348RAFFATELLU ET AL.INFECT. IMMUN.
by Renee Tsolis on August 22, 2007
cecum in large numbers but does not trigger pronounced in-
flammation in the cecum (49).
The only significant difference between inflammatory re-
sponses elicited by capsulated and noncapsulated serotype
Typhi strains was a 10-fold upregulation in TNF-? expression
in ceca from mice infected with the viaB mutant at 72 h after
infection. A viaB-mediated reduction in TNF-? expression was
also observed during serotype Typhi infection of murine mac-
rophage-like cells (J774). Deletion of the viaB region from
serotype Typhi increases TNF-? expression during infection of
a human macrophage-like cell line (differentiated THP-1 cells)
with serotype Typhi (19). In this model, TNF-? expression
depends on the presence of the TLR4 adaptor protein CD14
In conclusion, this report describes the first evidence for a
role of the capsule-encoding viaB operon in downregulating
intestinal inflammation in vivo. In addition, our data implicate
a new cytokine, IL-17, as a contributor to gastroenteritis elic-
ited by serotype Typhimurium.
We thank Josely F. Figueiredo, Sangeeta Khare, and Tamara Gull
for assistance with calf surgeries.
These studies were supported by USDA/NRICGP grant 2002-
35204-12247 (L.G.A.) and by Public Health Service grants AI060933
(S.D.L.), AI040124 (A.J.B.), AI044170 (A.J.B.), AI065534 (A.J.B.),
AI032738 (C.L.B.), and AI050843 (C.L.B.).
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Editor: J. L. Flynn
4350RAFFATELLU ET AL.INFECT. IMMUN.
by Renee Tsolis on August 22, 2007