INFECTION AND IMMUNITY, Dec. 2005, p. 7817–7826
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 12
Host Restriction of Salmonella enterica Serotype Typhi Is Not Caused
by Functional Alteration of SipA, SopB, or SopD
Manuela Raffatellu,1,2Yao-Hui Sun,1,2R. Paul Wilson,1,2Quynh T. Tran,2,3Daniela Chessa,1,2
Helene L. Andrews-Polymenis,2Sara D. Lawhon,3Josely F. Figueiredo,3Rene ´e M. Tsolis,1,2
L. Garry Adams,3and Andreas J. Ba ¨umler1,2*
Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Ave.,
Davis, California 95616-86451; Department of Medical Microbiology and Immunology, College of Medicine,
Texas A&M University System Health Science Center, 407 Reynolds Medical Building, College Station,
Texas 77843-11142; and Department of Veterinary Pathobiology, College of Veterinary Medicine,
Texas A&M University, College Station, Texas 77843-77673
Received 30 December 2004/Returned for modification 10 February 2005/Accepted 25 August 2005
Salmonella enterica serotype Typhi is a strictly human adapted pathogen that does not cause disease in
nonprimate vertebrate hosts, while Salmonella enterica serotype Typhimurium is a broad-host-range pathogen.
Serotype Typhi lacks some of the proteins (effectors) exported by the invasion-associated type III secretion
system that are required by serotype Typhimurium for eliciting fluid secretion and inflammation in bovine
ligated ileal loops. We investigated whether the remaining serotype Typhi effectors implicated in enteropatho-
genicity (SipA, SopB, and SopD) are functionally exchangeable with their serotype Typhimurium homologues.
Serotype Typhi elicited fluid accumulation in bovine ligated ileal loops at levels similar to those elicited by a
noninvasive serotype Typhimurium strain (the sipA sopABDE2 mutant) or by sterile culture medium. However,
introduction of the cloned serotype Typhi sipA, sopB, and sopD genes complemented the ability of a serotype
Typhimurium sipA sopABDE2 mutant to elicit fluid secretion in bovine ligated ileal loops. Introduction of the
cloned serotype Typhi sipA, sopB, and sopD genes increased the invasiveness of a serotype Typhimurium sipA
sopABDE2 mutant for human colon carcinoma epithelial (HT-29 and T84) cells and bovine kidney (MDBK)
cells. Translational fusions between the mature TEM-1 ?-lactamase reporter and SipA or SopD demonstrated
that serotype Typhi translocates these effectors into host cells. We conclude that the inability of serotype Typhi
to cause fluid accumulation in bovine ligated ileal loops is not caused by a functional alteration of its SipA,
SopB, and SopD effector proteins with respect to their serotype Typhimurium homologues.
Salmonella enterica serotype Typhi is strictly adapted to hu-
man hosts, in whom it causes a systemic disease known as
typhoid fever which results in some 600,000 deaths annually
(27). Salmonella enterica serotype Typhimurium is the caus-
ative agent of enterocolitis, an infection of humans and cattle
that normally remains localized to the intestine and the mes-
enteric lymph nodes. The hallmark of intestinal inflammation
during serotype Typhimurium infection of humans or cattle is
a massive neutrophilic infiltrate into the intestinal mucosa,
with necrosis of the upper mucosa and pseudomembrane for-
mation (9, 17, 26, 42, 50). The massive neutrophil influx into
the intestines of humans and cattle infected by serotype Ty-
phimurium is accompanied by diarrhea (developing between
12 h and 2 days after infection), and neutrophils are commonly
found in stool samples (18, 38, 42, 50). In contrast, only one-
third of typhoid fever patients develop diarrhea (later than 5 to
9 days after infection), and the intestinal infiltrate, as well as
the fecal leukocyte population, is composed predominantly of
mononuclear cells (18, 22, 30, 31, 39).
While the mechanisms by which serotype Typhimurium elic-
its a neutrophilic influx into the intestinal mucosae of humans
and cattle are beginning to be elucidated (52), comparatively
little is known about the pathogenesis of serotype Typhi infec-
tion or the reason why diarrhea is an insignificant symptom
during typhoid fever. One limitation to studying the pathogen-
esis of typhoid fever is the absence of a good animal model,
because serotype Typhi is strictly human adapted, causing dis-
ease only in higher primates (e.g., chimpanzees) (12). Mice
infected with serotype Typhimurium develop a systemic ty-
phoid-like disease, which is commonly used to model serotype
Typhi infections in humans (45). However, an obvious short-
coming of this mouse model is the fact that serotype Typhi-
murium does not cause typhoid fever in humans, suggesting
that genetic differences between serotype Typhi and serotype
Typhimurium are critically important for the disease outcome.
The evolution from a host generalist, such as serotype Ty-
phimurium, to a host-restricted variant, such as serotype Typhi,
may have occurred by acquisition of new genetic material
through horizontal gene transfer, by genome degradation (i.e.,
loss of genetic information by deletion or pseudogene forma-
tion), or by a combination of both mechanisms (5). Whole-
genome sequencing has revealed that genome degradation is
an extensive phenomenon in host-restricted Salmonella sero-
types. There are approximately 210 pseudogenes in the ge-
nome of serotype Typhi (strains CT18 and Ty2) and 173 pseu-
dogenes in the genome of Salmonella enterica serotype
Paratyphi A, another strictly human adapted serotype (10, 24,
32). In contrast, the genome of the broad-host-range serotype
Typhimurium contains only 39 pseudogenes (25). Thus, it is
* Corresponding author. Mailing address: Department of Medical
Microbiology and Immunology, School of Medicine, University of
California at Davis, One Shields Ave., Davis, CA 95616-8645. Phone:
(530) 754-7225. Fax: (530) 754-7240. E-mail: email@example.com.
possible that attenuation of neutrophilic infiltration and diar-
rhea during serotype Typhi infection may be due to loss of
function rather than to gain of function.
The type III secretion system (T3SS-1) encoded by Salmo-
nella pathogenicity island 1 (SPI1) mediates invasion of intes-
tinal epithelial cells by serotype Typhimurium (15). The
T3SS-1 of serotype Typhimurium is furthermore required for
eliciting the production of neutrophil chemoattactants (51), a
massive influx of neutrophils (1, 42, 48, 54), and fluid accumu-
lation in bovine ligated ileal loops (1, 48, 54). Finally, the
T3SS-1 is essential for causing diarrhea and lethal morbidity
during oral infection of calves with serotype Typhimurium (42,
43, 54). The main function of the T3SS-1 is the translocation of
proteins, termed effectors, into the cytosol of a host cell (14).
Six T3SS-1-secreted effectors, SipA, SopA, SopB, SopD, SopE,
and SopE2, act in concert to elicit fluid accumulation and
neutrophil infiltration during infection of bovine ligated ileal
loops with serotype Typhimurium (53, 54). The gene encoding
one of these effectors, SopE, is carried by a prophage that is
present only in the genomes of serotype Typhimurium clonal
isolates (defined by phage typing) that have caused recent
epidemics among cattle in Europe (29). Acquisition of sopE by
phage-mediated horizontal gene transfer increases the ability
of serotype Typhimurium to elicit fluid accumulation in bovine
ligated ileal loops, suggesting that acquisition of T3SS-1-se-
creted effectors contributes to host adaptation (53).
By analogy, it has been speculated that loss of T3SS-1-se-
creted effectors by genome degradation may account for the
inability of serotype Typhi to elicit infiltration of neutrophils
and for its low propensity to cause diarrhea (24). In support of
this idea, some effector genes contributing to fluid secretion
and infiltration of neutrophils during serotype Typhimurium
infection in calves are pseudogenes (sopE2 and sopA) in the
genome of serotype Typhi strain CT18 (3, 32, 40). Similarly,
sopA is a pseudogene in the genomes of serotype Paratyphi A
strain ATCC 9150 and serotype Typhi strain Ty2, and sopE is
absent from the serotype Paratyphi A genome (10, 24). The
sipA, sopD, and sopB genes are present in the serotype Typhi
genome, but the effectors encoded differ by three (SipA), four
(SopD), or five (SopB) amino acid substitutions from their
serotype Typhimurium homologues, which could potentially
alter their function. The presence of pseudogenes (sopA and
sopE2) and of genes carrying nonsynonymous substitutions
(sipA, sopB, and sopD) in the serotype Typhi genome may thus
be in part responsible for the reduced ability of serotype Typhi
to cause fluid secretion in the intestine.
To formally test this hypothesis, we cloned the sipA, sopB,
and sopD genes, which are intact in all typhoidal serotypes
whose genome has been sequenced, and expressed them in a
serotype Typhimurium strain carrying mutations in sipA, sopA,
sopB, sopD, and sopE2 (the sipA sopABDE2 mutant). We then
compared the functionality of sipA, sopB, and sopD genes
cloned from serotype Typhi and serotype Typhimurium in a
calf model of infection and in cultured human epithelial cells
derived from a colon carcinoma.
MATERIALS AND METHODS
Bacterial strains, tissue culture cells, and culture conditions. T3SS-1 effector
genes were cloned using Escherichia coli strain DH5?, which has been described
previously (16). Serotype Typhimurium and serotype Typhi strains used in this
study are listed in Table 1. For construction of a strain carrying a mutation in
invA, an internal part of the gene was amplified from serotype Typhimurium with
a primer pair published previously (35) and introduced into suicide vector
pEP185.2 (20) to give rise to plasmid pINV5. Plasmid pINV5 was conjugated
into serotype Typhi strain AJB70, and an exconjugant was selected and termed
STY1. Strains were cultured aerobically at 37°C in Luria-Bertani (LB) broth
supplemented with antibiotics as appropriate at the following concentrations:
carbenicillin, 100 mg/liter; chloramphenicol, 30 mg/liter; tetracycline, 20 mg/liter;
kanamycin, 60 mg/liter; or nalidixic acid, 50 mg/liter. For invasion assays with
tissue culture cells or bovine ligated ileal loops, each strain was grown overnight
at 37°C in 4 ml of LB broth in a roller. A 0.04-ml volume 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 an inoculum, and the
numbers of CFU were determined by plating serial 10-fold dilutions on LB
The human colon carcinoma cell line HT-29 has been described previously
(13) and was obtained from ATCC. HT-29 cells were grown in McCoy’s 5a
medium with 1.5 mM L-glutamine (Gibco) supplemented with 10% fetal calf
serum. Bovine kidney epithelial (MDBK) cells were obtained from ATCC and
were grown in Eagle minimal essential medium supplemented with 10% horse
serum, 2 mM L-glutamine, and Earle’s balanced salt solution adjusted to contain
1.5 g/liter sodium bicarbonate, 0.1 mM nonessential amino acids, and 1.0 mM
sodium pyruvate. For invasion assays, cells were seeded at 2.5 ? 105/well in
24-well plates and the invasion assay was performed on the following day. For
fluorescence microscopy, MDBK cells were seeded at 3 ? 104/well in a 96-well
plate and the assay was performed on the following day. The human colon
carcinoma cell line T84 has been described previously (11) and was obtained
from ATCC. T84 cells were grown in Dulbecco’s modified Eagle medium–F12
medium (Gibco) containing 1.2 g/liter sodium bicarbonate, 2.5 mM L-glutamine,
15 mM HEPES, and 0.5 mM sodium pyruvate (Gibco) supplemented with 10%
fetal calf serum. To polarize T84 cells, 0.5 ml of medium containing 4 ?
105cells/well was seeded on the apical compartment in 12-mm Transwell plates
with 0.4-?m-pore-size polycarbonate membranes (Corning Costar), and 1.5 ml of
medium was added to the basolateral compartment. The medium was changed
every other day, and the transepithelial electrical resistance was measured after
a week. When the cells reached a transepithelial electrical resistance of at least
1,500 ?/cm2, they were incubated overnight in fresh medium, and the invasion
assay was performed the following day.
Construction of plasmids. The primers used for amplification of the sipA,
sopB, and sopD genes from serotype Typhi strain SARB63 by PCR were the
same as those used previously to amplify the homologous genes from serotype
and amplification of the correct genes was confirmed by sequence analysis.
A DNA fragment containing the serotype Typhi sopD gene and its promoter
region was cloned into the SacI site of the low-copy-number vector pWSK29
(approximately 6 copies per cell) (47) to give rise to plasmid pMR16. The
serotype Typhi sipA gene is located in the sipBCDA operon and was amplified
without its promoter and cloned directionally behind the lac promoter of
pWSK29 by using BamHI and XbaI to give rise to plasmid pMR30. The serotype
Typhi sopB gene was amplified with its promoter but without a termination loop
and cloned into pWSK29 by using NotI and XbaI to give rise to plasmid pMR27.
The serotype Typhi sipA gene was introduced in the same orientation and
downstream of the sopB stop codon into plasmid pMR27 to give rise to plasmid
pPW8. The serotype Typhi sopB sipA genes were excised from pPW8 with NotI
and BamHI and were cloned into pMR16 to give rise to plasmid pMR25.
Plasmids derived from pWSK29 carrying the cloned sopD gene (pMR15), sopB
gene (pMR26), sipA gene (pMR29), or sopB sipA genes (pPW1) of serotype
Typhimurium have been described previously (34) (Table 1). The serotype Ty-
phimurium sopB sipA genes were excised from pPW1 with NotI and BamHI and
were cloned into pMR16 to give rise to plasmid pMR24.
To generate translational fusions between T3SS-1 effectors and the TEM-1
?-lactamase reporter, the following plasmids were constructed. A 3?Flag tag was
PCR amplified from plasmid pSUB11 (46) using primers Flag-F and Kan-R
(Table 2) and cloned into pCR2.1 to yield plasmid pTopoFlag. A 1.5-kb DNA
fragment encoding LacIQ and the trc promoter was PCR amplified from plasmid
pTrc99A (2) using primers LacIQ-F and Trc-R (Table 2). The PCR product was
digested with NdeI and SphI and introduced into pTopoFlag. A 1.56-kb DNA
fragment encompassing LacIQ, the trc promoter, and the 3?Flag tag was PCR
amplified from the resulting plasmid using primers LacIQ-F and Flag-R
(Table 2). The SphI- and SmaI-digested PCR product was cloned into plasmid
pBBR1MCS (21), yielding plasmid pBBR-Flag. A DNA fragment encoding the
TEM-1 ?-lactamase without the N-terminal signal peptide (6) was PCR ampli-
fied from pTrc99A using primers TEM-F and TEM-R (Table 2) and cloned into
7818RAFFATELLU ET AL.INFECT. IMMUN.
pCR2.1 to yield plasmid pTopoTEM1. An 800-bp SmaI-SacI-digested DNA
fragment of plasmid pTopoTEM1 was cloned into pBBR-Flag to yield plasmid
pFlagTEM1 (Table 1). The sipA gene from serotype Typhimurium 14028 was
PCR amplified without the stop codon by using the primer pair SipA-F–SipA-R
(Table 2). The 1.6-kb PCR product digested with NdeI and SalI was cloned into
plasmid pFlagTEM1 to give rise to plasmid pSipA/FT, encoding a SipA–Flag–
TEM-1 translational fusion whose expression is controlled by the isopropyl-?-D-
thiogalactopyranoside (IPTG)-inducible trc promoter. The sopD gene from se-
rotype Typhi strain SARB63 was PCR amplified without the stop codon by using
the primer pair SopD-F–SopD-R (Table 2). This PCR product was digested with
XhoI and cloned into plasmid pFlagTEM1 to give rise to plasmid pSopD/FT,
encoding a SopD–Flag–TEM-1 translational fusion under the control of the
sopD promoter. The gene encoding glutathione S-transferase (GST) was PCR
amplified from plasmid pGEX-4T-1 (Amersham) using primers GST-F and
GST-R (Table 2), digested with NdeI and XhoI, and cloned into plasmid
pFlagTEM1 to give rise to plasmid pGST/FT, encoding a GST–Flag–TEM-1
translational fusion under the control of the trc promoter (Table 1).
Construction of mutants. To generate a serotype Typhi sopB mutant, the
sopB::mudJ insertion in serotype Typhimurium strain ZA21 was transduced into
serotype Typhi strain AJB70 using bacteriophage P22 int. The insertional inac-
tivation of sopB in a transductant, termed STY5, was confirmed by Southern
hybridization using a sopB-specific DNA probe.
To generate a serotype Typhi sopD mutant, an internal fragment of the sopD
open reading frame was PCR amplified as described previously (54) and cloned
into pCR2.1 to generate plasmid pMR34. A 391-bp EcoRV-XbaI restriction
fragment of pMR34 was cloned into suicide vector pEP184.5 to give rise to
plasmid pMR35. The suicide plasmid pMR35 was introduced into serotype Typhi
strain AJB70 by conjugation. In one exconjugant, termed STY6, inactivation of
sopD by integration of pMR35 via homologous recombination was confirmed by
Southern hybridization using the labeled insert of pMR34 as a DNA probe.
Tissue culture assays. Invasion assays were performed using protocols for
gentamicin protection assays described previously (44). In brief, human colon
carcinoma cells were seeded as described above and infected with serotype
Typhimurium or serotype Typhi strains at approximately 1 ? 107CFU/well (the
multiplicity of infection was approximately 10). The bacteria were incubated for
1 h at 37°C under 5% CO2to allow for invasion. Each well was washed five times
with sterile phosphate-buffered saline (PBS) (2.7 mM KCl, 1.8 mM KH2PO4,
140 mM NaCl, 10 mM Na2HPO4, pH 7.4) to remove extracellular bacteria, and
medium containing gentamicin at a concentration of 0.1 mg/ml was added for a
90-min incubation at 37°C under 5% CO2. After three washes with PBS, the cells
were lysed with 0.5 ml of 1% Triton X-100, the lysates were transferred to sterile
tubes, and each well was rinsed with 0.5 ml of PBS. Tenfold serial dilutions were
plated to count the intracellular bacteria. Each experiment was repeated three
Protocols for detection of translocation of TEM-1 ?-lactamase fusion proteins
by fluorescence microscopy were based on a recent protocol (8). MDBK cells
were infected at a multiplicity of infection of approximately 500 with serotype
Typhi or serotype Typhimurium strains for 20 min. Cells were washed and
incubated for 3 h with medium containing gentamicin (0.05 mg/ml) to kill ex-
tracellular bacteria. Cells were washed and loaded with the fluorescent substrate
CCF2/AM (Invitrogen) for 1 h at room temperature using an enhanced loading
protocol according to the manufacturer’s manual. The fluorescence microscopy
analysis was performed with an Eclipse 300 microscope (Olympus, Japan)
TABLE 1. Bacterial strains and plasmids used in this study
Strain or plasmidRelevant genotypea
Nalidixic acid-resistant derivative of bovine wild-type isolate ATCC 14028
IR715 ?sipA ?sopA sopB::MudJ sopD::pEP182.5 sopE2::pSB1039
ATCC 14028 StrrphoN::Tn10dCm ?sipB
ATCC 14028 StrrphoN::Tn10dCm ?sipC
Plasmid carrying 3?Flag
Broad-host-range cloning vector
Plasmid carrying GST
Cloning vector for protein expression under control of trc promoter
TEM-1 without signal sequence in pCR2.1
3?Flag in pCR2.1
LacIQ, trc, and Flag in pBBR1MCS
Flag-tagged ?-lactamase-fused protein-expressing vector
Plasmid encoding SipA–TEM-1 fusion protein (STM)
Plasmid encoding SopD–TEM-1 fusion protein (STY)
Plasmid encoding GST–TEM-1 fusion protein
pWSK29 carrying the sopD gene (STM)
pWSK29 carrying the sopD gene (STY)
pWSK29 carrying the sopB gene (STM)
pWSK29 carrying the sopB gene (STY)
pWSK29 carrying the sipA gene (STM)
pWSK29 carrying the sipA gene (STY)
pWSK29 carrying the sipA sopBD genes (STM)
pWSK29 carrying the sipA sopBD genes (STY)
aSTY, serotype Typhi; STM, serotype Typhimurium.
VOL. 73, 2005SipA, SopB, AND SopD OF SEROTYPE TYPHI 7819
equipped with a CCF2 filter set (a 400-nm excitation filter, a 435-nm long-pass
emitter, and a 435-nm dichroic mirror) (Chroma Technology, Brattleboro, VT).
Analysis of protein secretion. Bacteria were grown under SPI1-inducing con-
ditions as described above. The cells were pelleted by centrifugation, and 2 ml of
supernatant was collected for each sample. The supernatants were then filtered
(pore size, 0.45 ?m), and the proteins were precipitated with 25% trichloroacetic
acid by high-speed centrifugation (14,000 ? g for 30 min). The pellet was washed
in cold acetone and resuspended in PBS. Four independent extractions for each
sample were added together to minimize differences in the protein recovery from
sample to sample. The proteins were then boiled in sodium dodecyl sulfate
(SDS) for 5 min, and an aliquot for each sample was separated by 10% SDS-
polyacrylamide gel electrophoresis.
Animal experiments. 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 Sal-
monella serotypes in fecal swabs was performed by enrichment in tetrathionate
broth (Difco) followed by streaking on brilliant green agar and XLT4 (Difco).
Bovine ligated ileal loop surgery has been described previously (36). In brief,
food was withheld from the calves for 24 h prior to the surgery. Anesthesia was
induced with Propofol (Abbot Laboratories, Chicago, IL), followed by placement
of an endotracheal tube and maintenance with isoflurane (Abbot Laboratories,
Chicago, IL) for the duration of the experiment. A laparotomy was performed,
the ileum was exposed, and loops ranging in length from 6 to 9 cm were ligated,
leaving 1-cm loops between them. The loops were infected by intraluminal
injection of 3 ml of a suspension of either sterile LB broth or bacterial strains in
LB broth containing approximately 1 ? 109CFU. The loops were placed back into
the abdominal cavity. Samples for bacteriologic culture were collected at 1 h after
infection by using a 3.5-mm biopsy punch and were incubated in PBS containing
0.1 mg/liter gentamicin for 90 min. Tissue samples were homogenized in PBS,
determination of 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. At 8 h
after infection, the fluid that had accumulated in loops was measured.
Statistical analysis. For analysis of percentage values, data were transformed
logarithmically. Geometric means were determined, and the statistical signifi-
cance of differences was calculated using parametric tests. A one-tailed paired
Student t test was used to determine whether introduction of plasmids into the
sipA sopABDE2 mutant resulted in a significant increase in the invasiveness of
the resulting strain.
Proteins secreted by serotype Typhimurium and serotype
Typhi strains. To study secretion of effectors, low-copy-num-
ber plasmids carrying sipA, sopB, sopD, or sipA sopA sopD of
serotype Typhi or serotype Typhimurium (Table 1) were in-
troduced into a serotype Typhimurium sipA sopABDE2 mutant
(ZA21). We did not include sopA and sopE2 in our investiga-
tion, because these genes are not intact in some serotype Typhi
isolates. Furthermore, the sopE gene was not investigated,
because it is absent from the serotype Typhimurium wild-type
isolate (ATCC 14028) from which the strains used in this study
were derived. Culture supernatants of serotype Typhimurium
strains IR715 (wild type), CAS152 (sipB mutant), and CAS108
(sipC mutant) and of serotype Typhi strains SARB63 (wild
type), AJB70 (wild type), and STY1 (invA mutant) were ana-
lyzed as controls. Proteins were isolated from culture superna-
tants and analyzed by SDS-polyacrylamide gel electrophoresis
to evaluate secretion of T3SS-1 effectors in the bacterial su-
pernatant (Fig. 1).
Bands with apparent sizes of 68 kDa (SipB) and 40 kDa
(SipC) were absent from the culture supernatants of serotype
Typhimurium strains CAS152 (sipB mutant) and CAS108
(sipC mutant), respectively. A large band (approximately
80 kDa) was visible in culture supernatants of wild-type sero-
type Typhimurium, the sipB mutant, the sipC mutant, and all
derivatives of the sipA sopABDE2 mutant carrying the cloned
sipA gene (pMR29, pMR30, pMR24, and pMR25). The
80-kDa band was also visible in the serotype Typhi wild-type
isolates (SARB63 and AJB70) but not in a serotype Typhi invA
mutant (STY1). The presence of a band of approximately
60 kDa in the culture supernatant of the sipA sopABDE2 mutant
carrying the cloned sopB gene (pMR27) suggested that this
protein represented SopB. Similarly, the presence of a band of
approximately 40 kDa in culture supernatants of the sipA
sopABDE2 mutant carrying the cloned sopD gene (pMR15) sug-
gested that this band represented SopD. We were not able to
visualize expression of SopD in strain ZA21(pMR16) or of SopB
sipA gene from either serotype Typhimurium [ZA21(pMR29)] or
serotype Typhi [ZA21(pMR30)] secreted SipB and SipC at mark-
edly reduced levels. The culture supernatants of strains carrying
the cloned serotype Typhimurium or serotype Typhi sopB sipA
sopD genes [ZA21(pMR24) and ZA21(pMR25), respectively]
had a similar appearance. Overall, lower levels of proteins ap-
peared to be secreted by serotype Typhi wild-type isolates
(SARB63 and AJB70) than by serotype Typhimurium strains,
with SipB being visible only as a faint band in serotype Typhi
TABLE 2. Primers used for construction of TEM-1 ?-lactamase fusion proteins
XhoI, Stop, SDb
aRestriction sites are underlined.
bSD, Shine-Dalgarno sequence.
7820RAFFATELLU ET AL.INFECT. IMMUN.
Serotype Typhi does not elicit fluid secretion in bovine li-
gated ileal loops. We next compared the ability of serotype
Typhi (SARB63) to cause fluid accumulation in bovine ligated
ileal loops with that of serotype Typhimurium strains IR715
(wild type) and ZA21 (sipA sopABDE2 mutant). The serotype
Typhimurium wild-type strain elicited significantly more fluid
accumulation at 8 h after infection than the serotype Typhi-
murium sipA sopABDE2 mutant (ZA21) or the serotype Typhi
wild-type strain (SARB63) (Fig. 2A). The amount of fluid that
had accumulated in loops infected with the serotype Typhi-
murium sipA sopABDE2 mutant (ZA21) or the serotype Typhi
wild-type strain (SARB63) was not significantly different from
that measured in loops infected with sterile LB broth. These
data suggested that serotype Typhi was unable to elicit fluid
accumulation in the calf, which is consistent with its host re-
striction to humans and its absence from the bovine reservoir.
The serotype Typhi sipA, sopB, and sopD genes elicit fluid
secretion in bovine ligated ileal loops when introduced into a
serotype Typhimurium sipA sopABDE2 mutant. Since SipA,
SopA, SopB, SopD, and SopE2 are essential for serotype Ty-
phimurium to elicit fluid secretion in bovine ligated ileal loops
(54), we investigated whether an altered function of T3SS-1
effectors in serotype Typhi may account for its inability to
cause this host response. We focused our analysis on SipA,
SopB, and SopD, because these effectors are present in both
serotype Typhi genomes for which the sequences have been
elucidated (10, 32). However, each of these serotype Typhi
effectors carries several amino acid substitutions compared to
its respective serotype Typhimurium homologue, which could
potentially alter their function during the host-pathogen inter-
action. We thus determined whether introduction of the
cloned serotype Typhi effector genes sipA, sopB, and sopD
(pMR25) would complement the ability of the serotype Typhi-
murium sipA sopABDE2 mutant (ZA21) to cause fluid secre-
tion in bovine ligated ileal loops at 8 h after infection. As a
positive control, the serotype Typhimurium sipA sopABDE2
mutant (ZA21) was complemented with the serotype Typhi-
murium effector genes sipA, sopB, and sopD (pMR24). Intro-
duction of either pMR24 or pMR25 resulted in a significant
(P ? 0.05) increase in fluid accumulation elicited by serotype
Typhimurium strain ZA21 (Fig. 2A). Furthermore, the amounts
of fluid secretion elicited by strains ZA21(pMR24) and
ZA21(pMR25) were not significantly different (P ? 0.367).
These results suggested that sipA, sopB, and sopD of serotype
Typhi function similarly to the homologous genes of serotype
Typhimurium in the bovine model of infection.
Invasion of the bovine intestinal mucosa by serotype Typhi
and serotype Typhimurium. We next determined whether se-
rotype Typhi would invade the bovine intestinal mucosa. At 1 h
and 8 h after infection of bovine ligated ileal loops with sero-
type Typhi (SARB63) or serotype Typhimurium strain IR715
(wild type) or ZA21 (sipA sopABDE2 mutant), tissue was col-
lected and extracellular bacteria were killed by gentamicin treat-
ment. The serotype Typhimurium wild-type strain (IR715) was
recovered at higher numbers than the serotype Typhi wild-type
strain (SARB63) (P ? 0.096 at 1 h; P ? 0.056 at 8 h) and the
serotype Typhimurium sipA sopABDE2 mutant (ZA21) (P ?
0.064 at 1 h; P ? 0.041 at 8 h) (Fig. 2B). Introduction of the
cloned effector genes sipA, sopB, and sopD from serotype
Typhi (pMR25) increased recovery of the serotype Typhi-
murium sipA sopABDE2 mutant (ZA21) from the bovine ileal
mucosa at 1 h (P ? 0.133) and at 8 h (P ? 0.029) after
infection. Similarly, ZA21(pMR24) was more invasive in li-
gated ileal loops than ZA21 at both 1 h (P ? 0.008) and 8 h
FIG. 1. Profiles of proteins secreted by serotype Typhimurium and serotype Typhi strains into the culture supernatant. Arrows indicate the
positions of SipA, SipB, SipC, SopB, and SopD. The molecular sizes of marker proteins are given on the left. The serotypes (Typhimurium or
Typhi) and genotypes (wt, wild type; 21, sipA sopABDE2 mutant) of strains are given below the gel. The presence of plasmids carrying cloned
effector genes of serotype Typhimurium (TM) or serotype Typhi (TY) is indicated at the bottom. Lane 1, molecular weight marker; lane 2, IR715;
lane 3, ZA21; lane 4, CAS152; lane 5, CAS108; lane 6, ZA21(pMR29); lane 7, ZA21(pMR30); lane 8, ZA21(pMR26); lane 9, ZA21(pMR27); lane
10, ZA21(pMR15); lane 11, ZA21(pMR16); lane 12, ZA21(pMR24); lane 13, ZA21(pMR25); lane 14, STY1; lane 15, AJB70; lane 16, SARB63.
VOL. 73, 2005SipA, SopB, AND SopD OF SEROTYPE TYPHI7821
(P ? 0.051) after infection. However, some of these differ-
ences were not statistically significant, due mainly to large
differences in the numbers of bacteria recovered from the
mucosae of different animals. We thus reasoned that cultured
epithelial cells would provide a more powerful tool for analyz-
ing differences in the invasiveness of bacterial strains.
The serotype Typhi effectors SipA, SopB, and SopD partially
complement the invasion defect of a serotype Typhimurium
sipA sopABDE2 mutant. To further investigate whether the
serotype Typhi effector genes sipA, sopB, and sopD have func-
tions comparable to those of their serotype Typhimurium ho-
mologues, we performed invasion assays with HT-29 cells, an
epithelial cell line derived from a human colon carcinoma. The
serotype Typhimurium wild-type strain (IR715) was recovered
from HT-29 cells in approximately 640-fold-higher numbers on
average (P ? 0.05) than the sipA sopABDE2 mutant (ZA21)
(Fig. 3A). The sipA sopABDE2 mutant (ZA21) complemented
by introduction of the cloned sipA, sopB, and sopD genes from
serotype Typhi (pMR25) was recovered from HT-29 cells at
approximately 240-fold-higher numbers on average (P ? 0.05)
than its parent (ZA21). Similarly, introduction of the cloned
serotype Typhimurium sipA, sopB, and sopD genes (pMR24)
into the sipA sopABDE2 mutant (ZA21) resulted in recovery
from HT-29 cells of approximately 140-fold-higher bacterial
numbers (P ? 0.05) compared to the isogenic parent strain
(ZA21) (Fig. 3A). Strains ZA21(pMR24) and ZA21(pMR25)
invaded HT-29 cells at significantly lower levels (P ? 0.05)
than wild-type serotype Typhimurium (IR715). Thus, the sipA,
sopB, and sopD genes from both serotypes partially comple-
mented a serotype Typhimurium sipA sopABDE2 mutant for
invasion of HT-29 cells.
To further compare the functions of sipA, sopB, and sopD of
serotype Typhimurium with those of serotype Typhi, each ef-
fector gene was introduced individually into the serotype Ty-
phimurium sipA sopABDE2 mutant (ZA21). Complementa-
tion of the sipA sopABDE2 mutant for invasion of HT-29 cells
by introduction of either the cloned sipA, sopB, or sopD gene
of serotype Typhimurium confirmed the results obtained in a
previous study (34). The level of invasion of the sipA sopABE2
mutant complemented with either sipA or sopD from serotype
FIG. 2. Fluid accumulation (A) and bacterial invasion (B) in bo-
vine ligated ileal loops. (A) Fluid accumulation elicited by serotype
Typhi or serotype Typhimurium strains 8 h after infection of bovine
ligated ileal loops. Data are geometric means (bars) ? standard devi-
ations. Statistical significances of differences between treatment groups
(brackets) are given at the top right. (B) Bacterial recovery from tissue
(3.5-mm biopsy punch) collected 1 h after infection of ligated ileal
loops with serotype Typhi or serotype Typhimurium strains and sub-
sequently incubated with gentamicin to kill extracellular bacteria. CFU
recovered from each animal was expressed as a percentage of CFU
recovered from a ligated ileal loop of the same animal infected with
the wild-type serotype Typhimurium strain (IR715). Data are geomet-
ric means (bars) ? standard deviations from experiments performed
with three animals.
FIG. 3. Invasion of HT-29 cells by serotype Typhimurium strains.
(A) Complementation with plasmids carrying the cloned sipA, sopB,
and sopD genes from either serotype Typhi (pMR25) or serotype
Typhimurium (pMR24). Data are geometric means (bars) ? standard
deviations. (B) Complementation with plasmids carrying individual
effector genes from serotype Typhi or serotype Typhimurium. Data are
geometric means (bars) ? standard errors. Statistical significances of
differences between treatment groups (brackets) are given at the top.
Ty, serotype Typhi; Tm, serotype Typhimurium; wt, wild type.
7822 RAFFATELLU ET AL.INFECT. IMMUN.
Typhi was in each case similar to that of the sipA sopABDE2
mutant complemented with the corresponding gene from se-
rotype Typhimurium (Fig. 3B). However, introduction of the
serotype Typhimurium sopB gene significantly (P ? 0.014)
increased the invasiveness of the sipA sopABDE2 mutant, while
introduction of the serotype Typhi sopB gene did not (P ?
0.079). Although the invasiveness of the sipA sopABDE2 mu-
tant was influenced by the presence or absence of effector
genes, we did not observe a correlation between expression
levels of effectors (Fig. 1) and invasiveness (Fig. 3B).
The data discussed above (Fig. 3) demonstrated that intro-
duction of the cloned serotype Typhi sipA gene into a serotype
Typhimurium sipA sopABDE2 mutant resulted in increased
invasiveness. However, these data did not provide information
regarding the functionality of the serotype Typhi sopB and
sopD genes. We have shown previously that the serotype Ty-
phimurium sopB and sopD genes are required for invasion of
polarized T84 cells (34). We thus determined whether intro-
duction of the cloned serotype Typhi sopB and sopD genes
would complement a serotype Typhimurium sipA sopABDE2
mutant for invasion of polarized T84 cells. The serotype Ty-
phimurium sipA sopABDE2 mutant (ZA21) became signifi-
cantly more invasive when transformed with plasmids carrying
individual serotype Typhi effector genes, including sipA (P ?
0.017), sopB (P ? 0.018), and sopD (P ? 0.005) (Fig. 4). The
finding that each of the serotype Typhi effector genes could
enhance the invasiveness of the serotype Typhimurium sipA
sopABDE2 mutant suggested that SopB, SopD, and SipA from
serotype Typhi are functional.
The serotype Typhi T3SS-1 mediates invasion of human and
bovine epithelial cells. Serotype Typhi was recovered at num-
bers similar to those of a noninvasive serotype Typhimurium
mutant (ZA21) from the bovine ileal mucosa (Fig. 2B). We
further investigated whether this low recovery of serotype
Typhi from bovine tissue was due to a general defect in its
ability to invade epithelium. To this end, the invasiveness of
two serotype Typhi wild-type isolates (SARB63 and AJB70)
and a derivative of AJB70 carrying an insertion in the invA
gene (STY1) was compared in human HT-29 cells (Fig. 5A).
Both wild-type serotype Typhi isolates invaded HT-29 cells at
levels similar to that of wild-type serotype Typhimurium
(Fig. 3 and 5A). Serotype Typhi strain AJB70 (wild type) was
recovered from HT-29 cells in approximately 13,500-fold-
higher bacterial numbers on average (P ? 0.05) than its iso-
genic invA mutant (STY1) (Fig. 5A). These data confirmed
that the serotype Typhi T3SS-1 is fully functional during inva-
sion of human epithelial cell lines.
We next investigated whether the inability of serotype Typhi
to invade the bovine ileal mucosa (Fig. 2B) was due to an
inability of this strictly human adapted pathogen to invade
bovine cells. The serotype Typhi wild-type strain AJB70 was
recovered from bovine kidney epithelial (MDBK) cells in sig-
nificantly higher numbers than its isogenic invA mutant
(STY1) (Fig. 5B), suggesting that serotype Typhi T3SS-1 me-
diates invasion of bovine cells. Furthermore, introduction of
the cloned serotype Typhi sipA, sopB, and sopD genes partially
complemented the invasiveness of the serotype Typhimurium
sipA sopABDE2 mutant (Fig. 5B), suggesting that the encoded
serotype Typhi effectors are functional in cells of bovine origin.
We next investigated whether the sopB and sopD genes
contribute to invasion of polarized T84 cells by serotype Typhi.
The serotype Typhi wild-type strain AJB70 was 2-fold more
invasive than a serotype Typhi sopB mutant (P ? 0.03) and
FIG. 4. Invasion of polarized T84 cells by the indicated strains of
serotype Typhimurium. Complementation with cloned effector genes
of serotype Typhi (Ty) is indicated below. Data are geometric means
(bars) ? standard deviations. Statistical significances of differences
between treatment groups (brackets) are indicated above.
FIG. 5. Invasion of HT-29 cells (A), MDBK cells (B), and polar-
ized T84 cells (C) by the indicated strains of serotypes Typhi and
Typhimurium. Data are geometric means (bars) ? standard devia-
tions. Statistical significances of differences between treatment groups
(brackets) are given at the top of panel C.
VOL. 73, 2005SipA, SopB, AND SopD OF SEROTYPE TYPHI7823
1.8-fold more invasive than a serotype Typhi sopD mutant (P ?
0.07) (Fig. 5C). Although serotype Typhi invaded HT-29 hu-
man colon epithelial cells equally well, it was less invasive for
polarized T84 human colon epithelial cells than serotype Ty-
phimurium (Fig. 3, 4, and 5C).
Translocation of T3SS-1 effectors into host cells by serotype
Typhi. To further investigate whether the T3SS-1 of serotype
Typhi is fully functional, we compared the abilities of serotype
Typhimurium and serotype Typhi to translocate effectors into
host cells in vitro. To this end we constructed fusions between
the serotype Typhimurium SipA protein and the TEM-1 ?-lac-
tamase reporter (SipA–TEM-1), between the serotype Typhi
SopD protein and ?-lactamase (SopD–TEM-1), and (as a neg-
ative control) between GST and ?-lactamase (GST–TEM-1).
Translocation was detected in living MDBK cells by using the
fluorescent ?-lactamase substrate CCF2/AM as described pre-
viously (8). The use of this technique allows identification of
cells loaded with CCF2 by virtue of their green fluorescence.
Cells in which CCF2 has been cleaved by ?-lactamase emit
blue fluorescence. Uninfected MDBK cells and MDBK cells
infected with serotype Typhi expressing GST–TEM-1 showed
green fluorescence (Fig. 6). In contrast, a fraction of cells
infected with serotype Typhi expressing SipA–TEM-1 or
SopD–TEM-1 emitted blue fluorescence, suggesting that fu-
sions between T3SS-1 effectors and TEM-1 are translocated
into host cells by serotype Typhi. The fraction of cells emitting
blue fluorescence after infection with serotype Typhimurium
strains expressing SipA–TEM-1 or SopD–TEM-1 was similar
to that observed with the corresponding serotype Typhi strains
(Fig. 6). However, no cells emitting blue fluorescence were
detected after infection of MDBK cells with serotype Typhi-
murium invA mutant strains expressing SipA–TEM-1 or
SopD–TEM-1, demonstrating that translocation of ?-lacta-
mase fusion proteins was dependent on the presence of a
functional T3SS-1. Collectively, these data showed that sero-
type Typhi is fully capable of translocating SipA and SopD into
host cells of bovine origin.
Evolution of a host-restricted serotype, such as serotype
Typhi, from a host generalist, such as serotype Typhimurium,
likely involved both gain of function (i.e., acquisition of new
genetic material through horizontal gene transfer) and loss of
function (i.e., genome degradation by deletion or by pseudo-
gene formation) (5). It can be speculated that during this
FIG. 6. Demonstration of translocation of T3SS-1 effectors into live MDBK cells by using TEM-1 fusions and fluorescence microscopy. MDBK
cells were infected with serotype Typhi or serotype Typhimurium strains (indicated above and below the micrographs) expressing different TEM-1
fusion proteins (SipA–TEM-1, SopD–TEM-1, or GST–TEM-1). After infection, MDBK cells were washed and loaded with CCF2/AM. The
uncleaved CCF2 ?-lactamase substrate emits green fluorescence. Translocation of ?-lactamase into MDBK cells is revealed by blue fluorescence
emitted by the cleaved CCF2 substrate.
7824RAFFATELLU ET AL.INFECT. IMMUN.
process serotype Typhi acquired genetic material that allowed
it to cause typhoid fever in humans. Furthermore, serotype
Typhi likely lost genetic material required for infection of
other host species by genome degradation. Although the func-
tions of the approximately 210 pseudogenes present in the
serotype Typhi genome are apparently not required for causing
disease in humans (10, 32), some of these genes may be essen-
tial during infection of other host species, such as cattle.
Similarly, some of the 479 open reading frames present in
the serotype Typhimurium genome but absent from the sero-
type Typhi genome may be required for interaction with the
bovine host (25). Thus, the inability of serotype Typhi to cause
disease in cattle may be due to genome degradation affecting
functions other than those related to its T3SS-1. This hypoth-
esis would explain why serotype Typhi effectively enters human
and bovine epithelial cells in vitro (Fig. 5) (23, 28, 49), while
the invasiveness of serotype Typhi for the bovine ileal mucosa
in vivo is similar to that of a noninvasive serotype Typhi-
murium mutant (the sipA sopABDE2 mutant) (Fig. 2B).
It is less obvious why invasion of the human intestinal mu-
cosa by serotype Typhi does not trigger the massive neutrophil
influx (18, 22, 30, 31, 39) that is the hallmark of human infec-
tions with serotype Typhimurium (18, 38, 42, 50). Here we
investigated the hypothesis that the low propensity of serotype
Typhi to cause diarrhea and neutrophil influx into the intesti-
nal mucosa may be due to a loss of function. Whole-genome
sequencing has revealed that some of the T3SS-1 effector
genes that contribute to the ability of serotype Typhimurium to
cause fluid accumulation and inflammation in bovine ligated
ileal loops (54) are pseudogenes in the genomes of human-
adapted serotypes causing typhoid fever (10, 24, 32). Based on
this observation, it has been speculated that inactivation of
T3SS-1 effector genes by genome degradation may account for
the low propensity of serotype Typhi to cause diarrhea in
humans (24). Here we show that the relevant T3SS-1 effector
genes that remained intact in the genomes of typhoidal Sal-
monella serotypes (i.e., sipA, sopB, and sopD) mediated fluid
accumulation when introduced into serotype Typhimurium,
despite the fact that serotype Typhi does not elicit fluid secre-
tion in bovine ligated ileal loops (Fig. 2A). Serotype Typhi was
able to translocate effector proteins into host cells (Fig. 6) and
to invade human epithelial cell lines in vitro by using its T3SS-1
(Fig. 5) (23, 28, 49). Thus, the inability of serotype Typhi to
cause fluid accumulation in bovine ligated ileal loops is not
caused by an absence of T3SS-1-secreted effectors that can
elicit this response.
Our data are not consistent with the hypothesis that serotype
Typhi has a lower propensity to elicit neutrophil influx than
serotype Typhimurium because the T3SS-1 is subject to ge-
nome degradation. Recent data suggest an alternative hypoth-
esis, namely, that the scarcity of neutrophils in intestinal infil-
trates of typhoid fever patients may be the result of acquisition
(rather than loss) of genetic material during the evolution of
host adaptation in the serotype Typhi lineage (33). Serotype
Typhi contains a 135-kb DNA region, termed SPI7, that is
absent from the serotype Typhi genome (32). The viaB locus
on SPI7 contains genes for the biosynthesis and export of the
Vi capsular antigen. Expression of the Vi antigen reduces the
production of neutrophil chemoattractants (e.g., interleukin-8)
during infection of human epithelial cells (33, 37) or macro-
phages (19, 33) in vitro and during infection of human colon
tissue explants with serotype Typhi (33). Thus, acquisition of
SPI7 by an organism ancestral to serotype Typhi may explain
why this pathogen elicits a different host response in the hu-
man intestine than nontyphoidal Salmonella serotypes, such as
We thank Andrea D. Humphries, Cagla Tu ¨kel, Carlos Rossetti,
Sangeeta Khare, and Tamara Gull for help with the calf surgery.
Work in A.J.B.’s laboratory is supported by USDA/NRICGP grant
2002-35204-12247 and Public Health Service grants AI040124,
AI044170, and AI065534. H.L.A.-P. is supported by Public Health
Service grant AI052250. S.D.L. is supported by Public Health Service
grant AI060933. J.F.F. is supported by CAPES, Brazil.
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Editor: D. L. Burns
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