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Infection and Immunity, October 2000, p. 5567-5574, Vol. 68, No. 10
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Salmonella enterica Serovar Typhimurium-Dependent
Regulation of Inducible Nitric Oxide Synthase Expression in Macrophages
by Invasins SipB, SipC, and SipD and Effector SopE2
Bobby J.
Cherayil,*
Beth A.
McCormick, and
Jacob
Bosley
Mucosal Immunology Laboratory, Combined
Program in Pediatric Gastroenterology and Nutrition, Massachusetts
General Hospital, Charlestown, Massachusetts 02129
Received 9 June 2000/Accepted 28 June 2000
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ABSTRACT |
When Salmonella enterica invades mammalian cells, it
activates signals leading to increased expression of inflammatory
mediators. One such mediator is nitric oxide (NO), which is produced
under control of the enzyme inducible NO synthase (iNOS).
Induction of iNOS in response to Salmonella infection has
been demonstrated, but the bacterial effector molecules that regulate
expression of the enzyme have not been identified. In the study
reported here, an analysis of Salmonella-dependent iNOS
expression in macrophages was carried out. Wild-type
Salmonella strains increased the levels of both iNOS
protein and mRNA in murine macrophage cell lines in an
invasion-independent fashion. Mutant strains lacking a functional pathogenicity island 1-encoded type III secretion system, as well as
strains lacking the invasins SipB, SipC, and SipD, were impaired in
iNOS induction. Complementation experiments indicated that all three of
the invasins were required for induction of iNOS expression. These
results suggested that an effector protein, translocated into
macrophages via the type III secretion system in a SipB-, SipC-, and
SipD-dependent manner, might be the ultimate mediator of iNOS
induction. In keeping with this idea, a mutant strain deficient in
SopE2, a recently described homolog of SopE, was found to be impaired
in the induction of iNOS expression. These observations suggest that
iNOS expression is regulated by signals activated by SopE2 (and
possibly SopE) and that the role of SipB, SipC, and SipD in this
process is to facilitate translocation of the relevant effector.
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INTRODUCTION |
Infections with Salmonella
enterica (most commonly serovar Typhimurium) constitute a
significant public health problem in most countries. In the United
States, it is estimated that between 800,000 and 4 million cases occur
every year, and of these about 500 are fatal (13). The
organism usually causes a self-limited, acute intestinal inflammation,
manifesting clinically with diarrhea and vomiting, but sometimes it can
lead to disseminated, potentially serious infections. One of the most
important characteristics of Salmonella virulence is the
ability of the organism to invade mammalian cells. After it is ingested
in contaminated food, Salmonella first invades the
epithelial cells of the intestine, and then, once it has crossed the
epithelial barrier, the macrophages of the lamina propria and Peyer's
patches (23). A crucial bacterial structure required for the
invasion process is a specialized secretory apparatus, the type III
secretion system, which is encoded at a locus known as
Salmonella pathogenicity island 1 (SPI1) (4). The
SPI1 type III secretion system is involved in the
signal-peptide-independent export of a set of proteins (referred to
here as effector proteins) that are ultimately translocated into the
cytosol of the host cells. The translocated effectors interact with
specific cellular proteins to induce dramatic cytoskeletal and membrane
rearrangements that lead to the active uptake of the bacteria by the
cell (14). Other effects include the induction of
apoptosis, the opening of chloride channels, and the activation
of cellular signal transduction pathways leading to the increased
expression of proinflammatory cytokines (4, 18, 20, 21,
31). Salmonella mutants that lack either a
functional SPI1 type III secretion system or certain of the effector
proteins that are secreted through it are defective in invasion and
have reduced virulence in animal models of disease (10, 21).
The effector proteins SipB, SipC, and SipD have been of particular
interest because of their involvement in invasion. They are secreted
into bacterial supernatants individually but are mutually
interdependent for translocation into the host cell (4). Mutant strains lacking any one of these proteins fail to translocate the others, resulting in an invasion-defective phenotype (4, 21). In addition, SipB, SipC, and SipD are also involved in the
translocation of several other effector proteins, including SopB, SopE,
SptP, and AvrA (9, 11, 15, 16, 40). While the roles of SipB
and SipD in invasion may be largely to act as chaperones during
translocation, SipC has been shown to have direct effects on actin
polymerization and bundling (17). Together with SipA, an
actin-binding effector, and SopE, a guanine nucleotide exchange factor
for Rho GTPases, SipC plays an important part in inducing the
cytoskeletal rearrangements required for invasion (14, 43).
During the course of cell invasion, bacterial lipopolysaccharide (LPS)
and some of the Salmonella effectors activate cellular signaling pathways, leading to changes in the expression of various genes, including those for inflammatory mediators such as interleukin-8 and tumor necrosis factor alpha (20). The increased
production of these cytokines is involved in the generation of an acute
inflammatory response that, on the one hand, helps in the clearance of
the organism and the induction of adaptive immunity and, on the other, contributes to tissue damage. Another factor that plays a similar double-edged role in the response to Salmonella is nitric
oxide (NO), a labile gas with antimicrobial, proinflammatory, and
immunomodulatory effects (28). NO is produced from arginine
in a reaction catalyzed by the enzyme nitric oxide synthase (NOS).
There are three isoforms of NOS, which differ in patterns of expression
and dependence on calcium. The main isoform expressed in macrophages is
NOS2, also known as iNOS because of its inducible expression and its calcium-independent activity. This isoform is active even at the trace levels of calcium found within resting cells and, once expressed, is capable of sustained, high-output NO production.
Increased expression of iNOS following infection with
Salmonella was recently demonstrated in intestinal
epithelial cell lines (33, 39). Since the organism is
susceptible to NO and its reaction products (6, 22), this
response could constitute an important innate defense mechanism. On the
other hand, the proinflammatory effects of NO could contribute to
pathological tissue damage. Elucidating how Salmonella
induces the expression of iNOS would thus shed light on an important
aspect of the host reaction to the organism. In the study reported
here, we have examined the induction of iNOS in macrophages following
infection with Salmonella in order to test the hypothesis
that one or more of the bacterial effector proteins may be involved in
regulating expression of the enzyme.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
The wild-type
S. enterica serovar Typhimurium strain SL1344, and the
isogenic mutant strains VV341, EE633, and EE638 have been described
previously (21, 41). The strains SB201
(invG::cam), and SB136
(invA::aphT), also isogenic with
SL1344, were provided by Jorgé Galan, Yale University. The SL1344
derivative GG5 (sopE::pMG1) was constructed by
integrating an ampicillin-resistant suicide plasmid, pMG1, into the
sopE locus of SL1344 (L. Guogas and C. Lee, unpublished results), and
was provided by Catherine Lee, Harvard Medical School. The wild-type
serovar Dublin strain 2229 and the isogenic mutant strains A1, AV1, B1,
SB2, and SE1 have been characterized elsewhere (11, 35, 40)
and were provided by Edouard E. Galyov, Institute for Animal Health,
Compton, United Kingdom, who also kindly gave us the SopE-deficient,
wild-type serovar Typhimurium strain F98 and its isogenic,
SopE2-deficient derivative, SE2.1, both of which have been described
previously (2), as well the serovar Dublin strain SC2, in
which the entire gene encoding SptP was removed (E. Galyov, unpublished
data). All bacteria were grown in Luria-Bertani (LB) medium, with
ampicillin added to 100 µg/ml for strains transformed with
-lactamase-encoding plasmids. To prepare bacteria for macrophage
infection, one to two colonies of the appropriate strains were
inoculated into 2 ml of LB medium and shaken at 37°C for several
hours until the culture reached saturation. Then, 10 µl of the
saturated culture was diluted into 10 ml of fresh medium, followed by
incubation overnight at 37°C without shaking in tightly capped, 12-ml tubes.
Cell culture.
The murine macrophage cell lines RAW264.7 and
J774 were maintained in Dulbecco modified Eagle medium (DMEM)
containing 10% heat-inactivated fetal calf serum, 50 U of penicillin
per ml, and 50 µg of streptomycin per ml at 37°C in an atmosphere
of 5% carbon dioxide. To prepare the cells for infection, they were detached from the tissue culture plates by incubating them for 10 min
in phosphate-buffered saline (PBS) containing 1 mM EGTA and 4 mM EDTA.
The cells were counted and then resuspended in medium without
antibiotics, and 0.5-ml aliquots (1.5 × 106 cells)
were seeded into the wells of a 24-well tissue culture plate. The cells
were allowed to adhere for 2 h before infecting them with
Salmonella strains.
Infections.
Aliquots (1 ml) of the overnight standing
cultures of Salmonella were centrifuged at 12,000 × g for 5 min, and the bacteria were washed twice with sterile
PBS. The final pellet was resuspended in antibiotic-free DMEM at a
density of ca. 2 × 108 bacteria/ml. Then, 50-µl
aliquots of the suspension (ca. 107 bacteria) were added to
the wells containing the RAW264.7 or J774 cells. The multiplicity of
infection (MOI) was therefore 5 to 10:1. After incubation at 37°C for
1 h, the culture supernatants were aspirated, and the cells were
washed twice with sterile PBS before being overlaid with medium
containing 100 µg of gentamicin per ml. The cells were incubated at
37°C for different times before preparing protein extracts or RNA.
Since wild-type strains of Salmonella invaded the cells more
efficiently than the mutant strains (see Results), there was a
significant difference in the numbers of the two types of bacteria surviving after overnight incubation. To minimize this difference, tetracycline was added to a final concentration of 10 µg/ml to cells
infected with wild-type strains of Salmonella 2 h after the end of the infection period. The addition of the antibiotic inhibited the multiplication of intracellular bacteria, as found by
others (5). In our experiments, addition of the drug reduced the number of wild-type bacteria surviving at the end of the experiment from 0.36 ± 0.06 × 106 to 0.28 ± 0.02 × 104, thereby ensuring that the numbers of
surviving wild-type and mutant Salmonella were comparable.
Tetracycline was always added to uninfected cells to control for any
effects the drug might have on iNOS expression.
Quantitation of bacterial invasiveness.
Invasiveness was
determined by means of a standard gentamicin protection assay
(21). In brief, after the completion of the infection
period, the cells were incubated in gentamicin-containing medium in
order to kill extracellular bacteria. Two hours later, the supernatant
medium was aspirated from the cultures, and the cells were washed twice
with sterile PBS. The cells were then lysed in 0.2 ml of sterile 1%
Triton X-100. Serial dilutions of the lysates were made, and aliquots
plated onto LB agar to determine the number of viable,
gentamicin-protected (intracellular) bacteria. The mean number and
standard deviation of intracellular bacteria, expressed as a percentage
of the original inoculum, were calculated from triplicate wells.
To quantitate bacterial attachment, the cells were lysed immediately
after completion of the infection period, and serial
dilutions of the
lysates were plated to obtain the number of "cell-associated"
bacteria. The number of attached bacteria was then calculated
by
subtracting the number of internalized bacteria (as determined
above)
from the cell-associated bacteria (
12).
Western blotting.
At different times after completion of
infection, the cells were washed with ice-cold Tris-buffered saline
(TBS; 10 mM Tris-Cl, pH 8.0; 150 mM NaCl) and then lysed in 0.2 ml of
1% NP-40 in TBS containing 10 µg of aprotinin and leupeptin per ml
and 2 mM phenylmethylsulfonyl fluoride. After incubation on ice for 15 min, the lysates were cleared by centrifugation at 12,000 × g for 15 min at 4°C. The protein concentrations of the cleared
lysates were estimated by the DC protein assay (Bio-Rad, Hercules,
Calif.) according to the manufacturer's instructions. Equal amounts of
protein, along with prestained molecular weight markers (Gibco-BRL,
Grand Island, N.Y.), were subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 9% acrylamide
gels and then transferred to a nitrocellulose membrane by semidry
blotting as previously described (3). The membrane was
blocked with 5% nonfat dry milk in TBS-Tween 20 (TBST; TBS containing
0.05% Tween 20) for 30 min at room temperature. The filter was then
divided in two, using the prestained markers as a guide. The upper half of the filter (68 kDa and above) was probed with an antibody specific for iNOS (NOS2; Santa Cruz Biotechnology, Santa Cruz, Calif.). The
lower half of the filter was probed with an antibody to the housekeeping protein elongation factor 1
(anti-EF1; Upstate Biotechnology, Lake Placid, N.Y.) to confirm equal loading of lanes.
The blot was then developed with horseradish peroxidase-conjugated second antibodies and enhanced chemiluminescence. Bands were
quantitated with NIH Image software after scanning the autoradiograms.
Northern blotting.
Total RNA was prepared from infected
cells using Trizol reagent (Gibco-BRL, Grand Island, N.Y.), according
to the manufacturer's instructions. Equal amounts of total RNA (15 to
20 µg per lane) were electrophoresed on a 1.2% formaldehyde-agarose
gel, photographed under UV transillumination, and then blotted to
nitrocellulose membrane according to a standard protocol
(34). After baking the membrane at 80°C for 1 h, it
was hybridized overnight at 65°C to an
[-32P]dCTP-labeled probe specific for murine iNOS,
washed, and exposed for autoradiography.
Construction of Sip expression plasmids and transformation into
Salmonella.
The reading frames encoding SipC and SipD were
amplified by PCR from the genomic DNA of wild-type
Salmonella (SL1344) using the following primers: SipC-5',
5'-CGCCGAAGCTTAATATGTTAATTAGTAATG-3'; SipC-3',
5'-GCGAATTCTTTCAGATTAAGCGCG-3'; SipD-5',
5'-GCGAATTCATCTATACGCCATCATG-3'; and SipD-3',
5'-AGAAGAGTGCGGCCGCCATTATTAATATCCTC-3'. Restriction sites
incorporated into the primers were used to clone the PCR products into
the bacterial expression vector pBH (Roche, Nutley, N.J.) downstream of
a trp-lac combination promoter and a ribosome binding site.
The SipC reading frame was cloned into the HindIII and
EcoRI sites of the vector to give the plasmid pBHSipC, while the SipD reading frame was cloned into the EcoRI and
NotI sites to give pBHSipD. To construct a plasmid
expressing both SipC and SipD, the SipD-encoding fragment was cloned
into the EcoRI and NotI sites of pBHSipC,
downstream of the SipC reading frame. The sequences of the SipC-3' and
SipD-5' primers were such that the resultant plasmid (pBHSipCD)
reproduced the sequence of the SipC and SipD encoding region of the
wild-type genomic locus, except for the introduction of the
EcoRI site (24). pBHSipC, pBHSipD, and pBHSipCD
were individually transformed into appropriate Salmonella strains by electroporation (32), and transformants were
selected on ampicillin-containing medium.
Analysis of Sip proteins in bacterial supernatants.
Salmonella strains (wild-type, mutant, and transformed) were
grown exactly as described above for the infections.
Isopropyl--D-thiogalactopyranoside (IPTG) was added to
the overnight standing cultures to a final concentration of 0.5 mM.
Incubation at 37°C was continued for a further 3 h, after which
the bacteria were spun down and the supernatants were filtered through
0.45-µm-pore-size filters. The filtered supernatants were mixed with
trichloroacetic acid (final concentration, 10%) and incubated on ice
for 30 min. The precipitated proteins were collected by centrifugation
at 15,000 × g for 15 min, washed with acetone, dried,
and dissolved in SDS-PAGE sample buffer. After separation on 9%
acrylamide gels, the proteins were stained with Gel Code Blue (Pierce
Chemical Co., Rockford, Ill.) according to the manufacturer's recommendations.
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RESULTS |
Induction of iNOS by Salmonella is sensitive to heat,
chemical fixation, and inhibition of protein synthesis.
RAW264.7
cells were infected at an MOI of 5 to 10:1 with the wild-type serovar
Typhimurium strain SL1344. Cell lysates were prepared about 18 h
after infection, and Western blotting was carried out to analyze iNOS
expression as well as expression of the housekeeping protein EF1. As
shown in Fig. 1A, lanes 1 and 2, infection with SL1344 resulted in a significant increase in iNOS
protein expression (an average of ca. ninefold over control, as
determined by quantitation of band intensities; Fig. 1B). This increase
could be detected as early as 4 h after infection (data not shown)
but was maximal at 18 h. The level of EF1 did not change
appreciably, confirming the equal loading of lanes.
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FIG. 1.
(A) Western blot of iNOS and EF1 expression in RAW264.7
cells infected with Salmonella. Lane 1, control; lane 2, SL1344; lane 3, heat-killed SL1344; lane 4, formaldehyde-fixed SL1344;
lane 5, 100 ng of LPS per ml; lane 6, heated LPS; lane 7, control; lane
8, 2229; lane 9, chloramphenicol-treated 2229. (B) Quantitation of mean
iNOS band intensities from three separate experiments similar to that
shown in panel A. Con, control; WT, wild-type Salmonella;
HK, heat-killed Salmonella; LPS, 100 ng of LPS per ml. Error
bars indicate the standard deviations.
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To determine if bacterial viability was required for iNOS induction,
the experiment was carried out with an equal number of
SL1344 organisms
which had been either heated to 80°C for 10 min
or fixed with 2%
formaldehyde for 20 min. Both of these treatments
significantly reduced
the level of iNOS induction (Fig.
1A, lanes
3 and 4, and Fig.
1B).
Treating the cells with 100 ng of LPS per
ml for 18 h resulted in
an increase in iNOS expression that was
appreciably lower than that
induced by
Salmonella (ca. fourfold
over that for the
control; Fig.
1, lane 5, and Fig.
1B). The LPS-induced
increase was
unaffected by heating the LPS to 80°C for 10 min
(Fig.
1, lane 6).
These results indicate that the
Salmonella had
to be viable
to induce iNOS expression. They also suggest that
it is unlikely that
the LPS in the bacterial cell wall is sufficient
to explain the
induction.
Similar results were obtained with a second wild-type
Salmonella strain (strain 2229, serovar Dublin; Fig.
1,
lanes 7 and
8), as well as with a second murine macrophage cell line,
J774
(data not
shown).
To determine if bacterial protein synthesis was required for iNOS
induction, the
Salmonella strains were treated for 1 h
at
37°C with 100 µg of chloramphenicol per ml before they were
added
to the cells. Chloramphenicol was also added to the cell culture
medium during the 1-h infection period. As shown in Fig.
1, lanes
7 to
9, for the Dublin strain 2229, this treatment significantly
reduced the
level of iNOS that was induced, suggesting that the
bacteria had to be
actively synthesizing protein for proper induction.
The same effect was
observed with the wild-type Typhimurium strain
SL1344 (data not
shown).
Hersh et al. have reported that infection of RAW264.7 with wild-type
Salmonella leads to apoptosis and that the mechanism
of cell death involves binding of SipB to caspase 1 (
18). We
did not observe any evidence of
Salmonella-induced
apoptosis in
our studies, either by trypan blue exclusion or by
examination
of nuclear morphology (data not shown). This difference may
be
attributable to the much lower MOIs used in our experiments (5
to
10:1 versus 100:1 in the studies of Hersh et al.) and to the
fact that
we used tetracycline to inhibit intracellular multiplication
of
wild-type
Salmonella.
Induction of iNOS by Salmonella requires an intact
SPI1-encoded type III secretion system and specific effector
proteins.
The requirement for bacterial viability and protein
synthesis led us to inquire whether the secretion of the
Salmonella effector proteins via the SPI1 type III system
might be involved in iNOS induction. To evaluate this possibility, we
examined mutant strains of Salmonella that were deficient in
components of this type III secretion system or various effector
proteins for their ability to induce iNOS in RAW264.7 cells. The
results of such an experiment are shown in the Western blot of Fig.
2A, which indicates that the strains
SB136, which lacks InvA (a structural component of the type III
apparatus), and EE638, which lacks the effectors SipC, SipD, and SipA,
are impaired in iNOS induction, while strain GG5, which lacks the
effector protein SopE, induces iNOS to levels comparable to the
wild-type SL1344 strain (lanes 1 to 5). Similarly, the serovar Dublin
strain B1, which lacks Sips B, C, D, and A, is impaired in iNOS
induction compared to the wild-type 2229 strain (lanes 6 to 8).
Quantitation of iNOS band intensities from several such experiments is
shown in Fig. 2B, which indicates that the mutant strains induce iNOS
protein to levels which are about 50% of that induced by the wild-type
strain. Plating of serial dilutions of the bacterial suspensions used
to infect the cells confirmed that the numbers of input bacteria were
comparable for all of the strains.
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FIG. 2.
(A) Western blot of iNOS and EF1 expression in RAW264.7
cells infected with different strains of Salmonella. Strain
designations are indicated above the lanes. (B) Quantitation of
mean iNOS band intensities from five separate experiments similar to
that shown in panel A. Strain designations are indicated below the
columns. Error bars indicate the standard deviations.
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Similar experiments were carried out with a large number of mutant
strains of both the Typhimurium and the Dublin serovars.
The results of
these experiments are summarized in Table
1. Inspection
of the results shown in the
table indicates that strains that
lack a functional SPI1 type III
secretion system (VV341, SB201,
and SB136) are unable to induce iNOS to
wild-type levels. EE638,
which lacks Sips C, D, and A, is also impaired
in iNOS induction,
but EE633, which lacks SipA, behaves like the
wild-type strain.
The results from the latter two strains indicate that
Sips C and
D, but not SipA, are required for inducing iNOS expression.
The
same conclusion can be drawn from the phenotypes of the Dublin
strains A1 and B1, i.e., that SipA is not required for induction
of
iNOS. The results also indicate that SipB by itself is not
sufficient
to induce iNOS expression since strain EE638 (which
expresses SipB, but
not Sips C, D, and A) is deficient in this
function.
Induction of iNOS by Salmonella does not require
invasion of cells.
Studies in epithelial cell lines have indicated
that structural components of the SPI1-encoded type III secretion
system such as InvG and InvA, as well as Sips C and D, are essential
for invasion (21). SipB is also involved in this process
since Sips B, C, and D are mutually interdependent for translocation
into host cells (4). Since macrophages are professional
phagocytic cells and may not behave like the nonphagocytic epithelial
cells, we addressed the invasiveness of the various
Salmonella strains in RAW264.7 by means of a standard
gentamicin protection assay. The results of such an experiment are
shown in Fig. 3. It is clear that the
strains SB136 (deficient in the InvA structural component of the type
III secretion system), EE638 (deficient in Sips C, D, and A), and B1
(deficient in Sips B, C, D, and A) are significantly defective in
invasion compared to the two wild-type strains SL1344 and 2229. The
strain EE633 (deficient in SipA) had wild-type levels of invasion (data
not shown). Similar, although less dramatic, differences in
internalization between wild-type and mutant bacteria were observed
when bacteria were opsonized with 20% normal mouse serum (ca. 15%
internalized for strain SL1344 versus ca. 3% internalized for strain
EE638). These results indicate that even in the phagocytic RAW264.7
cells, internalization of Salmonella is facilitated by an
intact type III secretion system and by Sips B, C, and D.
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FIG. 3.
Invasiveness of different Salmonella strains
based on the mean numbers of internalized bacteria (percent input)
2 h after infection of RAW264.7 cells. Error bars indicate the
standard deviations.
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Based on these studies of invasiveness, it seemed possible that the
effects of Sips B, C, and D on iNOS induction described
in the previous
section could be secondary to decreased entry
of the corresponding
mutant bacteria into the cells. To address
this possibility, we
inhibited the internalization of the wild-type
Salmonella
strain SL1344 by treating the RAW264.7 cells with the
actin
depolymerizing agent cytochalasin D for 1 h before and during
the
course of infection (
12). The number of internalized
bacteria
was determined, and representative results are displayed in
Fig.
4A. The results indicate clearly
that cytochalasin D treatment
produces a dose-dependent decrease in the
number of internalized
wild-type
Salmonella. At 15 µg of
cytochalasin D per ml, the number
of internalized wild-type bacteria is
comparable to the number
of internalized mutant strains SB136 and
EE638.
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FIG. 4.
(A) Effect of cytochalasin D (at 5 or 15 µg/ml) on
internalization of wild-type Salmonella (SL1344) into
RAW264.7 cells compared to the number of internalized SB136 and EE638
mutant strains. Error bars indicate the standard deviations. (B)
Western blot indicating the effect of cytochalasin D treatment on iNOS
and EF1 expression in RAW264.7 cells. Lane 1, uninfected cells; lane 2, untreated cells infected with SL1344; lane 3, cytochalasin D-treated (5 µg/ml) cells infected with SL1344; lane 4, cytochalasin D-treated (15 µg/ml) cells infected with SL1344.
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We then tested the effect of cytochalasin D on the ability of wild-type
Salmonella to induce expression of iNOS. As shown
in Fig.
4B, in contrast to the effects on internalization, cytochalasin
D had
little or no effect on iNOS expression, even at a dose of
15 µg/ml.
Cytochalasin D treatment alone did not alter iNOS protein
expression
from control levels (data not shown). These observations
indicate that
cell invasion is not required for iNOS induction
by
Salmonella, making it unlikely that the requirement for Sips
B, C, and D in this process is secondary to their role in
invasiveness.
There were no significant differences between wild-type and mutant
bacterial strains in their attachment to RAW264.7 cells
(data not
shown), indicating that the differences in iNOS induction
could not be
attributed to variations in adherence of the bacteria
to the
cells.
Effect of Salmonella on iNOS mRNA.
To examine the
effect of Salmonella infection on iNOS mRNA, Northern blots
were carried out on total RNA extracted from infected RAW264.7 cells.
In initial experiments, induction of iNOS mRNA was found to be maximal
at about 18 h after infection and required bacterial viability, as
observed for the iNOS protein (data not shown). We then went on to
examine the mutant strains of Salmonella for their ability
to induce iNOS mRNA. As shown in Fig. 5,
the wild-type strain SL1344 (lane 2) induced a dramatic increase in iNOS mRNA level over control (lane 1), but the mutants SB201 (deficient in InvG; lane 3), SB136 (deficient in InvA; lane 4), and EE638 (deficient in the effectors Sips C, D, and A; lane 5) did not. A strain
deficient in the effector SopE (GG5; lane 6) showed wild-type levels of
iNOS induction, as did the strain EE633 (deficient in the effector
SipA; data not shown). Similar results were obtained with the Dublin
serovar; the wild-type strain 2229 showed a marked increase in iNOS
mRNA, but the mutant B1 (deficient in the effectors Sips B, C, D, and
A) showed very little increase over uninfected cells (data not shown
and also see the Northern blot of Fig. 7). Confirming the results
obtained with iNOS protein, treatment of the cells with 15 µg of
cytochalasin D per ml did not inhibit iNOS mRNA induction by wild-type
Salmonella (Fig. 5, lane 7), while cytochalasin D by itself
had no effect (lane 8). Thus, the results of the Northern blot analysis
substantiate the iNOS protein data and suggest that the regulation of
iNOS expression by Salmonella occurs at the level of mRNA.
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FIG. 5.
Northern blot of iNOS mRNA expression in RAW264.7 cells
18 h after infection with different strains of
Salmonella. Lane 1, control; lane 2, SL1344; lane 3, SB201;
lane 4, SB136; lane 5, EE638; lane 6, GG5; lane 7, cytochalasin
D-treated (15 µg/ml) cells infected with SL1344; lane 8, cytochalasin
D-treated (15 µg/ml) cells. The ethidium bromide-stained 28S rRNA
band (EtBr) is shown to indicate the loading of lanes.
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Complementation of mutant Salmonella strains with Sip
expression plasmids.
The data presented above implicate SipB,
SipC, and SipD in the regulation of iNOS expression by
Salmonella. To confirm this idea and to determine which of
these proteins is actually required for iNOS induction, we created
expression plasmids expressing SipC and SipD, either individually or in
combination, and transformed them into strains EE638 (which expresses
SipB) and B1 (which does not express SipB). Transformants were selected
and grown overnight in standing cultures, and expression of the
plasmid-encoded Sips was examined by analyzing the proteins present in
the bacterial supernatants. The results of such an experiment are shown
in Fig. 6A, which indicates that proteins
of the appropriate size (SipC, ca. 42 kDa; SipD, ca. 36 kDa) are
present at similar levels in the supernatants of the relevant
transformed strains. The plasmid-encoded proteins were expressed even
when the bacteria were not induced with IPTG (compare lanes 5 and 6 in
Fig. 6A).
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|
FIG. 6.
(A) Expression of Sips C and D in wild-type, mutant, and
transformed strains of Salmonella. Lane 1, SL1344; lane 2, EE638; lane 3, EE638/C; lane 4, EE638/D; lane 5, EE638/CD; lane 6, EE638/CD, no IPTG; lane 7, B1; lane 8, B1/CD. The numbers to the left
of the gel indicate molecular mass in kilodaltons. (B) Invasiveness of
wild-type, mutant, and transformed strains of Salmonella.
The column numbers refer to the same strains and conditions as the lane
numbers in panel A. Error bars indicate the standard deviations.
|
|
To confirm the functionality of the introduced proteins, we carried out
invasion assays on the wild-type, mutant, and transformed
strains. The
results are shown in Fig.
6B and indicate that strain
EE638/CD (the
transformant expressing Sips B, C, and D) displays
invasiveness
comparable to wild-type
Salmonella, with or without
IPTG
induction, while EE638/C (the transformant expressing Sips
B and C but
not SipD), EE638/D (the transformant expressing Sips
B and D but not
SipC), and B1/CD (the transformant expressing
Sips C and D but not
SipB) are significantly impaired in invasiveness.
These findings are
consistent with previously reported observations
that Sips B, C, and D
are all required for invasion (
4,
21)
and confirm that the
plasmid-encoded SipC and SipD are capable
of carrying out at least one
of the functions ascribed to
them.
We then went on to examine the ability of the transformed strains to
induce iNOS expression 18 h after infection of RAW264.7
cells. As
shown in the Northern blot of Fig.
7A,
strain EE638/CD
(the transformant expressing Sips B, C, and D, lane 6)
induced
iNOS mRNA expression comparable to wild-type
Salmonella (lane
2). In contrast, strains EE638/C (the
transformant expressing
Sips B and C but not SipD; lane 4), EE638/D
(the transformant
expressing Sips B and D but not SipC; lane 5), and
B1/CD (the
transformant expressing Sips C and D but not SipB; lane 11)
are
as defective as the parental mutant strains (EE638, lane 3; B1,
lane 10) in the induction of iNOS mRNA. Treating the cells with
cytochalasin D did not significantly reduce the ability of EE638/CD
to
induce iNOS (lane 7), a result consistent with our observations
on the
wild-type strain. In keeping with the observation that
IPTG induction
was not required for expression of plasmid-encoded
Sips C and D (Fig.
6), iNOS induction occurred in response to
EE638/CD even without IPTG
treatment of the bacteria (lane 8).
Similar results were obtained when
Western blotting was used to
analyze iNOS protein expression (Fig.
7B).
These observations
confirm the involvement of Sips B, C, and D in iNOS
induction
by
Salmonella and further indicate that all three
of these proteins
are required for this function.
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|
FIG. 7.
(A) Northern blot of iNOS expression in RAW264.7 cells
infected with wild-type, mutant, and transformed strains of
Salmonella. Lane 1, uninfected cells; lane 2, SL1344; lane
3, EE638; lane 4, EE638/C; lane 5, EE638/D; lane 6, EE638/CD; lane 7, EE638/CD, cells treated with 15 µg of cytochalsin D per ml; lane 8, EE638/CD, no IPTG; lane 9, 2229; lane 10, B1; lane 11, B1/CD. EtBr,
ethidium bromide-stained 28S rRNA band. (B) Western blot of iNOS and
EF1 expression in RAW264.7 cells infected with wild-type, mutant, and
transformed strains of Salmonella. Lane 1, uninfected cells;
lane 2, SL1344; lane 3, EE638; lane 4, EE638/C; lane 5, EE638/D; lane
6, EE638/CD.
|
|
Involvement of Sip-dependent effectors in iNOS induction.
The
analysis of the mutant strains is consistent with the idea that an
effector protein or proteins whose translocation is dependent on both
the type III secretion system and the Sips B, C, and D might be
involved in inducing iNOS expression. Four such effectors, SopB, SopE,
SptP, and AvrA (9, 11, 15, 16, 40) have been considered, and
the results shown in Table 1 indicate that they are not required for
iNOS induction. Recently, a structural homolog of SopE, SopE2, has been
identified (2). SopE2 is expressed in all
Salmonella strains tested, in contrast to SopE, which is
encoded in a temperate bacteriophage found only in some strains
(16, 29). Like SopE, SopE2 is found in protein aggregates in
culture supernatants of SipB-deficient strains, suggesting that the
translocation of the latter protein is also likely to be dependent on
Sips B, C, and D (2).
We tested a SopE-deficient, wild-type serovar Typhimurium strain, F98,
as well as its isogenic, SopE2-deficient derivative,
SE2.1, for the
ability to induce iNOS expression in RAW264.7 cells.
The results
shown in Fig.
8 indicate that F98 induced
iNOS expression
as well as the wild-type strain SL1344, a finding
consistent with
the results obtained with two other
SopE-deficient strains that
we have tested (GG5 and SE1). However, iNOS
induction by strain
SE2.1 was reduced, suggesting that the absence of
SopE2 (in a
SopE-deficient background) impaired this function. These
results
indicate that SopE2 is involved in inducing iNOS expression.
View larger version (38K):
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[in a new window]
|
FIG. 8.
(A) Western blot of iNOS and EF1 expression in RAW264.7
cells infected with wild-type and mutant strains of
Salmonella. (B) Northern blot of iNOS expression in RAW264.7
cells infected with wild-type and mutant strains of
Salmonella. EtBr, ethidium bromide-stained 28S rRNA band.
Strain designations are indicated above the lanes.
|
|
| |
DISCUSSION |
The production of NO is an important antimicrobial defense
mechanism, but when uncontrolled it can lead to pathological
inflammatory states (28). Indeed, elevated NO levels and
increased expression of iNOS have been implicated in clinical problems
such as rheumatoid arthritis and inflammatory bowel disease (1, 7,
19, 27). Elucidating the mechanisms that regulate iNOS expression
is thus relevant to understanding both innate immunity, and the
pathogenesis and treatment of chronic inflammatory conditions.
Like several other microorganisms, Salmonella has been noted
to increase expression of iNOS in mammalian cells, although the mechanism of induction has not been worked out in detail. Two recent
studies have shown that infection of intestinal epithelial cell lines
by Salmonella is associated with increased iNOS expression, and a correlation between invasiveness and iNOS induction was observed
(33, 39). Our findings extend these studies to macrophages and also demonstrate the involvement of SipB, SipC, and SipD in the
regulation of iNOS expression. These effectors have been previously shown to play an important role in invasion, thereby providing an
explanation for the link between invasiveness and iNOS induction noted
in the earlier studies (33, 39). However, our results show
clearly that the two processes can be dissociated based on the effects
of cytochalasin D treatment (Fig. 4 and 5). This uncoupling from
invasion has been noted for other effects induced by
Salmonella, i.e., the activation of NF-
B, as well as
transepithelial signaling for neutrophil recruitment (8,
12), and is consistent with the observation that Sips can be
translocated into mammalian cells by extracellularly situated bacteria
(4).
One of the functions of Sips B, C, and D is the formation of a
translocation apparatus for the delivery of certain effector proteins
(AvrA, SopB, SopE, and SptP) into the mammalian cytosol (9, 11,
15, 16, 40). The involvement of one of these effectors in
iNOS induction seemed likely based on our observation that Sips B, C,
and D were all required for the function. However, our initial series
of experiments ruled out a role for AvrA, SopB, SopE, and SptP (Table
1), suggesting that yet another Sip-dependent effector might be
the ultimate mediator of iNOS induction. Indeed, we found that a strain
deficient in SopE2, a recently identified homolog of SopE that is also
likely to be dependent on Sips B, C, and D for translocation
(2), was clearly impaired in its ability to induce
iNOS expression (Fig. 8).
Our findings thus suggest a simple model in which SopE2, translocated
into the mammalian cell via the SPI1 type III secretion system and the
SipB, -C, and -D complex, initiates signals leading to increased iNOS
expression. SopE2 is likely to have the same function as SopE in
relation to the activation of Rho GTPases (14), thus
providing an obvious mechanism for the initiation of signaling. The
close structural, and presumed functional, homology between the two
molecules raises the possibility that SopE may also have the ability to
induce iNOS expression. Although strains GG5 and SE1 (both deficient in
SopE) behaved like the corresponding wild-type strains with respect to
iNOS expression (Table 1), the presence of SopE2 in these strains
probably compensated for the lack of SopE. A strain expressing SopE but
deficient in SopE2 will be required to test the involvement of the
former effector in iNOS induction. Finally, it is worth mentioning here
that since Rho GTPases have been implicated in the
Salmonella-activated signaling pathways leading to the
expression of proinflammatory cytokines in epithelial cells
(20), SopE and SopE2 are likely to have a broad role in
inducing inflammation that extends beyond the production of NO in macrophages.
Complementation of the SopE2-deficient strain SE2.1 with a
plasmid-encoded SopE2 gene (2) did not restore iNOS
induction to wild-type levels (data not shown). Although this failure
of rescue raises the possibility that some other gene might be involved in the regulation of iNOS expression, we think it unlikely since (i) the sopE2 mutation does not cause obvious effects on the
expression or secretion of any other Salmonella secretory
protein (2) and (ii) our analysis of the SipB-, SipC-, and
SipD-deficient mutants and their transcomplemented derivatives clearly
indicates a role for a Sip-dependent effector in iNOS induction. The
failure of complementation may reflect a requirement for specifically regulated expression of SopE2 (expression at a specific time during invasion, or at a specific site within the macrophage) that cannot be
achieved by the plasmid-encoded effector.
Although our findings have highlighted the involvement of Sips B, C,
and D and SopE2 in iNOS induction, they do not exclude a role for LPS
in this process. LPS is an important inducer of iNOS expression in
macrophages, especially in conjunction with cytokines such as
interferon gamma (42). Furthermore, a mutant of
Salmonella with altered LPS structure has been shown to be impaired in inducing NO production, both in vitro and in vivo (26). Sip-deficient, as well as SopE2-deficient, mutants of Salmonella have residual iNOS inducing ability (Fig. 2 and
8, for example), which could be attributable to the effects of LPS acting through the recently described Toll-like receptors
(30). It is likely that the final level of iNOS expression
is determined by signals activated by both LPS and SopE2.
We have not directly addressed the in vivo significance of our
observations. However, evidence from the published literature suggests
that our findings are likely to be relevant to Salmonella pathogenicity. B1, the mutant strain of serovar Dublin deficient in
Sips A, B, C, and D, has been found to be markedly impaired in inducing
intestinal fluid secretion and neutrophil transmigration in bovine
ligated ileal loops (11). Similar findings were obtained when serovar Typhimurium mutant strains deficient in SipB, SipC, or
SipD were fed to calves (37). Although the decreased
pathogenicity of the Sip-deficient strains in these studies could be
related to their poor invasiveness, at least some aspects of virulence (neutrophil transmigration, NF-B activation) have been shown to be
independent of cell invasion (8, 12). An alternative possibility, therefore, is that one of the effectors dependent on Sips
B, C, and D for translocation might be involved in inducing intestinal
pathology. The involvement of several such effectors has been examined
in bovine models. Strains deficient in SopB, SipA, or SptP all behave
like wild-type Salmonella after being fed to calves
(36, 37). In the bovine ligated ileal loop model, strains
deficient in SptP or AvrA are unimpaired in enteropathogenesis, and
strains deficient in SopB or SopE are only modestly impaired (11,
31, 35, 38). Thus, these in vivo observations have an excellent
correlation with our in vitro findings: Sips B, C, and D are
required for inducing both intestinal pathology and iNOS expression;
conversely, SipA, SopB, SopE, AvrA, and SptP are not required for
either of these functions. Together with the known involvement of NO in
inflammatory conditions of the bowel (7, 19, 27), our
results suggest that the SopE2-dependent induction of iNOS in
epithelial cells and tissue macrophages of the gut will have an
important role in the intestinal pathology associated with
Salmonella infection. Experiments to address this possibility will be a fruitful avenue of further research.
| |
ACKNOWLEDGMENTS |
This work was supported by National Institutes of Health grants
T-32-DK-07477 (B.J.C.) and DK-50989 (B.A.M.).
We thank Jorgé Galan, Edouard Galyov, and Catherine Lee for
kindly providing bacterial strains; Isabel Fernandez and Milton Silva
for assisting with some of the experiments; Catherine Lee, Cathryn
Nagler-Anderson, and Shiv Pillai for helpful comments on the
manuscript; and W. Allan Walker for his continued support.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Mucosal
Immunology Laboratory, Combined Program in Pediatric Gastroenterology
and Nutrition, Massachusetts General Hospital, Rm. 3303, MGH East,
Bldg. 149, 13th St., Charlestown, MA 02129. Phone: (617) 726-4170. Fax:
(617) 726-4172. E-mail:
cherayil{at}helix.mgh.harvard.edu.
Editor:
J. T. Barbieri
| |
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Infection and Immunity, October 2000, p. 5567-5574, Vol. 68, No. 10
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
