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Journal of Bacteriology, August 1999, p. 4949-4954, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Department of Microbiology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095,1 and Departments of Molecular Microbiology2 and Pediatrics,3 Washington University School of Medicine, St. Louis, Missouri 63110
Received 17 March 1999/Accepted 10 June 1999
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ABSTRACT |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The expression of genes encoding proteins secreted by the SPI1
(Salmonella pathogenicity island) type III secretion
apparatus is known to require the transcriptional activators SirA and
HilA. However, neither SirA nor HilA is believed to directly activate the promoters of these genes. invF, the first gene of the
inv-spa gene cluster, is predicted to encode an AraC-type
transcriptional activator and is required for invasion into cultured
epithelial cells. However, the genes which are regulated by InvF have
not been identified. In this work, an in-frame deletion in
invF was constructed and tested for the expression of
(sigD-lacZYA),
sipC::Tn5lacZY, and a plasmid-encoded
(sicA-lacZYA). SigD (Salmonella invasion gene) is a secreted protein required for the efficient invasion of
Salmonella typhimurium into cultured eucaryotic cells.
sicA (Salmonella invasion chaperone) is the
first gene of a putative operon encoding the Sip/Ssp
(Salmonella invasion/Salmonella secreted proteins) invasion proteins secreted by the SPI1 type III export apparatus. invF was required for the expression of the
sigD, sicA, and sipC fusions. This
is the first demonstration that there is a functional promoter in the
intergenic sequence between spaS and sicA. In
addition, several proteins were either absent from or found in reduced
amounts in the culture supernatants of the invF mutant.
Therefore, invF is required for the optimal expression of
several genes encoding SPI1-secreted proteins. Genetic evidence is also
presented suggesting there is HilA-dependent readthrough transcription
from the invF promoter at least through sipC.
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INTRODUCTION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The expression of genes required for the invasion of eucaryotic cells is stimulated by environmental cues including osmolarity (12), pH (4), and growth state and oxygen tension (10, 27, 38). Several regulators of SPI1 (Salmonella pathogenicity island 1) gene expression have been identified, but it is not known how they recognize environmental signals which affect gene expression. A pho-24 mutant produces constitutively active PhoP, a response regulator which directly or indirectly represses the expression of SPI1 genes (4, 6, 15, 21). One of these genes, hilA, encodes an activator of SPI1 gene expression (3, 28). HilA is believed to directly activate expression from the invF and prgH promoters (3), although this has not been established biochemically. invF is predicted to encode an AraC-type transcriptional activator (for a review, see reference 13), but it is not known which genes it activates (23). invF is not autoregulated, nor does it activate the expression of invH, a gene which encodes an outer membrane lipoprotein component of the SPI1 type III secretion apparatus and which is divergently transcribed from invF (2, 8, 23). Nonetheless, invF is required for efficient invasion of cultured epithelial cells (23), suggesting that it is important for the expression of other genes required for invasion. This hypothesis was tested by examining the effect of an invF mutation on the expression of genes known to be required for invasion.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bacterial strains and plasmids.
Bacterial strains and
plasmids used in this study are described in Table
1. Electroporation of plasmids into
bacteria was carried out as previously described (36).
Plasmids that were manipulated in Escherichia coli were
passaged through a restriction-minus (hsd) Salmonella
typhimurium LT2 strain (LB5000) (37) prior to
electroporation into S. typhimurium SL1344 (19).
P22 HT int lysates were harvested and used for transductions
as previously described (30).
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-D-thiogalactopyranoside
(IPTG)-inducible sigDE clone pHH37, a 3.2-kb
EcoRI-BamHI fragment from pHH20 (20)
was cloned into pVLT33 (9). Induction of sigDE expression was done by the addition of IPTG to a final concentration of
100 µM in overnight cultures.
pHD3 (invF-lacZYA) and pHD11 (sicA-lacZYA) were
made by PCR amplification (Pfu polymerase; Stratagene) of
putative promoter sequences and cloning into pRW50 (29). For
pHD3, an invH primer with an EcoRI linker
(invH-1
[5'-GGAATTCCGGCGCCATGTTTTTACACAACCGTCAGAAC-3']) and an
invF primer with a BamHI linker
(invF-1
[5'-CGGGATCCCGGCAGCTTTTGCGCGGAACACGTCTGTATAAACC-3']) (Gibco BRL) were used to amplify ~460 bp between
invF and invH from pDL7-2. The resulting fragment
was cloned into the BamHI and EcoRI sites of
pRW50. For pHD11, the primers spaS-EcoRI-3 (5'-GGAATTCCGCGGAGAAGGTTGGCGTACCTG-3') and
sicA-BamHI-1
(5'-CGGGATCCCGGCGTGGCGCCTTCACTAACGGCATCC-3') were used to
amplify the intergenic sequence (137 bp) between spaS and
sicA along with 192 bp of the 3' end of spaS and
76 bp of sicA. This amplified product was cloned into pRW50
as described above, and both strands were sequenced with the same
primers used for amplification to confirm the sequence.
To construct the sigD chromosomal lacZYA
reporter, plasmid pFUSE was used (5). pFUSE contains an R6K
origin of replication and cannot replicate in SL1344. A 1-kb
XbaI fragment from pHH15 (20) containing 0.4 kb
of sequence upstream of sigD and 0.6 kb of sigD
coding sequence was cloned into the unique XbaI site of
pFUSE. Ligations were transformed into E. coli
S17-1
pir, and clones were screened by restriction
digestion with PstI and EcoRI for inserts in the
correct orientation relative to lacZYA. One clone was
selected and named pHD5. pHD5 was transferred by conjugation into
SVM252 (14028s sirA::Tn10dTc) (20,
22), where it integrated into sigD, leaving an intact
sigDE+ copy in addition to a
sigD-lacZYA operon fusion. The
sirA::Tn10dTc strain was used as a
recipient in order to provide a selectable marker (tetracycline
resistance) for S. typhimurium. Correct integration of pHD5
was confirmed in several exconjugants by transduction linkage analysis
using SVM167 (sigE::Tn10dTc) (86%
linkage). The (sigD-lacZYA) fusion was then transduced
into the appropriate strains.
Growth conditions.
S. typhimurium and E. coli strains were grown in Luria-Bertani (LB) broth (Difco) at
37°C with aeration on a roller drum or without aeration in standing
cultures, as indicated. Antibiotics were used at the following final
concentrations: chloramphenicol, 25 µg/ml; kanamycin, 100 µg/ml;
streptomycin, 100 µg/ml; spectinomycin, 100 µg/ml; and
tetracycline, 15 or 30 µg/ml for single-copy or multicopy
tetracycline resistance, respectively. For the detection of
-galactosidase activity, solid medium (LB agar) was supplemented with 5-bromo-4-chloro-3-indolyl--D-galactoside (X-Gal)
at 40 µg/ml. IPTG was used at a final concentration of 100 µM.
Tissue culture invasion assays. HEp-2 cells were maintained and passaged as recommended by the American Type Culture Collection. For invasion assays, 2 × 105 cells/ml were seeded into Falcon 24-well tissue culture plates (Becton Dickinson, Lincoln Park, N.J.) to obtain about 90% confluent monolayers on the following day. For bacterial cultures, single colonies were inoculated into 2 ml of LB broth and grown for 18 h without shaking at 37°C. Aliquots of 5 µl (107 to 108 CFU) were used per well of tissue culture cells. The invasion assay was performed as previously described (20).
Construction of spaS disruption mutants.
To
construct the spaS::
Str/Sp mutant SMV514, a
2.5-kb SalI-BamHI fragment containing
spaQRS was cloned into pMAK705, creating pHD12. A
BamHI streptomycin-spectinomycin resistance cassette from
pSmUC was cloned into the unique BglII site within the 5' region of spaS. This plasmid, pHD14, was used to exchange
the disrupted spaS allele with the wild-type allele on the
chromosome as previously described (16).
Str/Sp mutant was chosen and called SVM514.
Construction of the invF in-frame deletion mutant. To make an in-frame deletion in invF, pHD10-1 was digested with ClaI and SacII, the 5' and 3' single-stranded overhangs were removed with mung bean nuclease (New England Biolabs), and the blunt ends were ligated with T4 DNA ligase (New England Biolabs). Mung bean nuclease can sometimes remove double-stranded DNA in addition to single-stranded DNA; therefore, several clones were sequenced to identify clones with an in-frame deletion in invF. One clone, pHD13, contained an in frame deletion of 465 bp. A 1.25-kb PstI fragment from pHD13 was subcloned into pMAK705, producing pHD15, which was then used to exchange the deletion onto the chromosome as previously described (16).
To screen for the invF deletion on the wild-type chromosome, bacteria with resolved plasmids were pooled in phosphate-buffered saline, subcultured into LB broth, and infected with P22 HT int containing pHH21 (sigD-lacZYA reporter plasmid, medium copy number) (20). If invF were required for the expression of sigD as predicted, the expression of(sigD-lacZYA) would be reduced in an
invF strain. Transductants were grown on MacConkey
lactose agar supplemented with ampicillin. Four of several hundred
transductants that formed less red or white colonies were purified and
subsequently cured of the reporter plasmid by growing bacteria in LB
broth for 5 days, subculturing 1:500 on each day. Individual colonies
were screened for ampicillin sensitivity on LB agar containing
ampicillin. One mutant was chosen and called SVM579. The
invF mutation in this strain was complemented by
invF in a low-copy-number vector (pHD9-1), suggesting that
the phenotype of this mutant (see Results) was due to the deletion in
invF and not another mutation elsewhere on the chromosome.
The same technique was used to introduce the invF deletion
onto the chromosome of BJ68
(sipC::Tn5lacZY) (34), creating SVM725. The invF resulted in reduced
lacZY expression from the
sipC::Tn5lacZY fusion when tested on LB
agar plates supplemented with X-Gal.
To confirm the presence of the deletion on the chromosome and the
absence of any gross chromosomal rearrangements, Southern analysis was
performed (36). Chromosomal DNA was digested with PstI and probed with the 1.7-kb PstI fragment
from pHD10-1. The probe hybridized to a 1.7-kb PstI fragment
of chromosomal DNA from SL1344 and a 1.2- to 1.3-kb fragment of SVM579
and SVM725 as predicted (data not shown).
To construct the invF spaS::Str/Sp
sipC::Tn5lacZY triple mutant, SVM754,
the spaS::Str/Sp mutation in SVM514 was
transduced into SVM579 (invF). Nine purified
transductants were screened by Southern hybridization to confirm that
the invF deletion had not been lost upon transduction due to
its spaS-linked location on the chromosome (data not shown).
One mutant, SVM733 (invF spaS::Str/Sp), was
used to make a P22 HT int lysate. The
spaS::Str/Sp mutation from SVM733 was
transduced into SVM725 (invF
sipC::Tn5lacZY). Tetracycline-,
spectinomycin-, and streptomycin-resistant transductants were purified
and checked for P22 sensitivity.
Analysis of culture supernatants. Cultures were grown in 5 to 10 ml of LB broth with antibiotics for 18 h without aeration, and equivalent units of optical density at 600 nm were harvested as previously described (20). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (26) on 7.5% gels for silver stain analysis (7) and 5% gels for immunoblot (Western) analysis (ECL Western blotting detection system; Amersham Pharmacia Biotech). It is notable that in these gels (30% acrylamide to 1.6% bisacrylamide), some proteins migrated more slowly than through gels poured with 0.8% acrylamide (data not shown). For immunoblots, proteins were transferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore).
Antibodies.
To make antibodies to SigD, the first 575 bp of
sigD were cloned into the expression vector pMAL-c1 (New
England Biolabs). Expression and affinity purification of the maltose
binding protein (MBP)-SigD' fusion protein expressed from this plasmid
were performed as described by the manufacturer. Protein eluted from
the amylose column was separated on a 7.5% SDS-polyacrylamide gel, and
the fusion protein was excised from the gel. Lyophilized gel slices containing MBP-SigD' were used to raise rabbit antibodies to SigD (Covance Research Services, Denver, Pa.). The anti-SigD antibodies were
preadsorbed with an acetone powder of E. coli DH5
expressing MBP (17) to reduce cross-reactivity of the
antibody to proteins other than SigD. The preadsorbed antiserum was
used at a 1:1,000 dilution. Anti-rabbit horseradish
peroxidase-conjugated secondary antibodies were obtained from Sigma
(St. Louis, Mo.).
Enzyme assays.
-Galactosidase assays were performed and
values were calculated as previously described (31).
Cultures were grown in 5 ml of LB with appropriate antibiotics for
18 h at 37°C in 13- by 100-mm screw-capped tubes. Samples of 1.5 ml were harvested, washed once in 0.88% NaCl, and resuspended in 1 ml
of working buffer; 100 µl of each cell suspension was used per assay.
Sequence analysis. Sequencing was performed by using the BigDye Terminator Cycle Sequencing Ready Reaction system (PE Applied Biosystems, Foster City, Calif.). Reactions were analyzed by the Washington University Nucleic Acid Chemistry Laboratory (St. Louis, Mo.). Sequence analyses (homologies, mapping, etc.) were performed using the Wisconsin Sequence Analysis Package (Genetics Computer Group, Inc.).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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InvF is essential for the expression of effector genes.
invF is the first open reading frame of a large gene cluster
encoding components of the SPI1 type III secretion apparatus (23). It has been proposed that InvF activates the
expression of genes required for invasion (23), but this has
not been tested. Therefore, a strain with an in-frame deletion of 465 bp in invF, SVM579, was constructed and tested for
expression of the (sigD-lacZYA) (chromosomal),
sipC::Tn5lacZY, and
(sicA-lacZYA) (episomal) reporters. sigD,
which is unlinked to SPI1, encodes a protein secreted by the SPI1 type
III secretion apparatus and is required for efficient invasion into
cultured epithelial cells (20). The SigD homologue in
S. dublin, SopB (Salmonella outer protein), has
been implicated as a factor important for causing enteritis in a calf
model of infection (14). Contrary to our previous report
(20), sigD expression was found to be
hilA dependent (1). Therefore, strain VV302
(hilA-523) was resent to us and retested for the
expression of (sigD-lacZYA) in pHH21. In addition to the
episomal sigD-lacZYA fusion, a chromosomal reporter fusion,
(sigD-lacZYA)/sigDE+, was
constructed and tested in the wild-type and VV302 strains. The
expression of both (sigD-lacZYA) fusions was
significantly reduced in the hilA background (data not
shown). Thus, although the hilA deletion in VV302 in our
previous work was confirmed by Southern analysis and by tissue culture
invasion assays (20), it appeared that the original strain
of VV302 we received had acquired a suppressing mutation which allowed
for hilA-independent expression of
(sigD-lacZYA).
(sigD-lacZYA) and
(sicA-lacZYA) in SVM579 was found to be much lower than
in the wild-type strain SL1344 (Table 2).
The regulation defect due to the invF deletion was complemented by pHD9-1 (invF+), demonstrating
that the regulation phenotype was due to invF. Expression
of the (sicA-lacZYA) and (sigD-lacZYA)
reporters was not complemented by philA
(hilA+) (Table 2), suggesting that the
regulatory effect of InvF on the sigD and the
sicA promoters is downstream of HilA.
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invF background. The expression of
sipC::Tn5lacZY was significantly
reduced in the invF mutant (Table 2). This regulatory
defect could be complemented by pHD9-1 (invF+).
Interestingly, hilA provided in multicopy could also
increase the expression of
sipC::Tn5lacZY, suggesting that
sipC expression could be activated from either a HilA- or an
InvF-dependent promoter (Table 2). The HilA-dependent expression of
sipC::Tn5lacZY in the
invF mutant could be virtually eliminated by a polar
disruption in spaS, a gene upstream of sicA and
sipC. In contrast, the InvF-dependent expression of
sipC::Tn5lacZY was not affected by the
polar disruption in spaS. These results suggest the
production of a readthrough transcript beginning upstream of
spaS that is HilA dependent and a second transcript
beginning downstream of spaS (probably the sicA
promoter) that is InvF dependent (Table 2).
Expression from the invF promoter was measured from pHD3
(invF-lacZYA) in SVM579 to determine if invF was
autoregulated. As previously reported for a different invF
mutant (23), the in-frame deletion in invF did
not significantly affect expression of the invF promoter (64 (wild type) versus 53 [invF] U of -galactosidase activity).
InvF is not sufficient to activate the expression of
(sicA-lacZYA) or (sigD-lacZYA) in
E. coli.
Previous work demonstrated that an
(invF-lacZYA) reporter (pVV562) could be activated by
hilA (in pVV214) expressed in E. coli
(3). To determine if HilA and/or InvF directly activates sigD or sicA expression, the effect of providing
hilA or invF in trans on the
expression of (sigD-lacZYA) in pHH21 (20) and of (sicA-lacZYA) in pHD11 was tested in E. coli. Although philA and pVV214 (data not shown) were
able to activate (invF-lacZYA) (pHD3) in E. coli, neither pVV214 nor philA could activate
(sicA-lacZYA) or (sigD-lacZYA),
respectively, in E. coli (Table
3). Expression of these fusions was also
tested in the presence of invF. Like hilA,
invF, with or without hilA, was unable to
activate the expression of either fusion in E. coli (Table
3). Therefore, invF may require additional factors or
signals that are absent from E. coli for the activation of
the sigD and the sicA promoters.
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InvF is required for efficient invasion into cultured epithelial
cells.
Invasion into cultured epithelial cells was also
quantitated for the SVM579 mutant and wild-type S. typhimurium. Invasion of HEp-2 cells by the invF
deletion strain was reduced significantly (Table
4), but not to the extent previously
reported (23). It is possible that the invF
mutation tested by Kaniga et al. (23) was having a polar
effect on the expression of the inv genes and thus had a
stronger phenotype. Moreover, invasion by the invF mutant
was not as reduced as invasion by a secretion-defective invA
(SB154) or spaS (SVM514) mutant (Table 4). Invasion by
SVM579 into HEp-2 cells was fully complemented to wild-type levels by pHD9-1. Interestingly, invasion was partially restored by
philA (Table 4).
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Analysis of secreted proteins from the invF mutant. Proteins that are secreted by the SPI1 type III machinery are believed to be the effectors which stimulate the uptake of bacteria by eucaryotic host cells (18, 20, 21, 24, 25). Because the expression of genes encoding several of these proteins was reduced in the invF mutant, the secreted protein profile of SVM579 was analyzed by SDS-PAGE and silver staining. Compared to the wild-type strain, several proteins were missing from the culture supernatants of the invF mutant, including SigD (confirmed by immunoblotting with anti-SigD antibodies [data not shown]) and a smaller protein migrating at about 36 kDa (Fig. 1, lanes 2 and 5) (it is notable that most of the known proteins, including SipA, SipB, and SigD, did not migrate according to their predicted molecular weights in these gels [see Materials and Methods]). Unexpectedly, Sip/SspA was clearly observed in culture supernatants of the invF mutant, although in slightly lower amounts, compared to the wild-type supernatant proteins. Sip/SspA and the other SPI1 secreted proteins were clearly absent from the supernatants of the spaS mutant SVM514 (Fig. 1, lane 1). pHD9-1 restored the secretion of SigD and the ~36-kDa protein into culture supernatants of the invF mutant and increased Sip/Ssp secretion to wild-type levels (Fig. 1, lane 6).
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invF mutant.
However, philA did increase the amounts of several other
proteins including the Sip/Ssps to culture supernatants of the
invF mutant (Fig. 1, lane 7). Because philA
could increase the expression of
sipC::Tn5lacZY in the
invF mutant, it is not surprising that the Sip/Ssp
proteins in culture supernatants are increased accordingly (Fig. 1,
lane 7). This result may explain why philA partially
restores invasion of an invF mutant into tissue culture cells.
To determine if InvF was required for the secretion of SigD in addition
to the regulation of sigDE expression, an inducible clone of
sigDE, pHH37 (Ptac-sigDE), was
transformed into SVM579. If invF were required for
secretion, SigD would not appear in culture supernatants even after the
induction of sigDE expression with IPTG. The secretion of
SigD was clearly restored to the supernatants of the invF
mutant containing pHH37, demonstrating that InvF is not required for
the secretion of proteins by the SPI1 type III secretion system (Fig.
1, lane 9).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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|---|
invF was identified by sequence analysis of the SPI1
region and was predicted to encode an AraC-type transcriptional
activator (23). However, InvF-dependent genes were not
identified. In this study, invF was shown to be required for
the expression of (sigD-lacZYA) and
(sicA-lacZYA) fusions. An in-frame invF
deletion mutant was fully complemented for the expression of these
reporters by invF cloned into a low-copy-number vector but
was not complemented by hilA, a central activator of SPI1
gene expression (3). In contrast, although a
sipC::Tn5lacZY reporter also required
invF for optimal expression, hilA provided in
multicopy was also able to increase sipC expression in the
invF mutant.
Several of the genes encoding secreted proteins, specifically the sip/ssp genes, are immediately downstream of the inv-spa gene cluster (18, 21, 24, 25). Because the sip/ssp locus is only 137 bp downstream of the spa genes, it was possible that sip/ssp expression could be activated from the invF promoter ~12 kb upstream of sicA; no transcriptional terminator is evident in this intergenic region. This observation is supported by the analysis of secreted proteins from the invF mutant which revealed the presence, arguably in lesser amounts, of several of the Sip/Ssp proteins. Moreover, these proteins could be restored to wild-type levels in the invF mutant by providing hilA on a low-copy-number plasmid. hilA could also partially complement the invasion defect of the invF mutant. Most importantly, the expression of sipC::Tn5lacZY could be activated in an invF mutant with the addition of hilA in multicopy, and this expression could be eliminated by a polar disruption in spaS. These results suggest that readthrough expression of the sip/ssp genes could be activated from the invF promoter by HilA.
To determine if a promoter was present immediately upstream of the
sip/ssp gene cluster, an episomal,
(sicA-lacZYA) reporter fusion in a low-copy-number vector
was made. Expression of this fusion was dramatically reduced in the
invF mutant. The regulation defect was complemented by
invF but not hilA. This result demonstrates for
the first time the presence of an InvF-dependent promoter immediately
upstream of sicA. From these results taken together with the
invasion assays, the -galactosidase assays, and the SDS-PAGE data,
it appears that expression of the sip/ssp genes can be
driven in part from the HilA-dependent invF promoter in addition to an InvF-dependent promoter immediately upstream of sicA (Fig. 2).
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The expression of a (sigD-lacZYA) fusion in the
invF deletion mutant was also dramatically reduced. SigD was
absent from culture supernatants of the invF mutant and
restored by pHD9-1 (invF+). In contrast to the
Sip/Ssp proteins, SigD was not restored to culture supernatants when
philA was placed in the invF mutant. In addition
to SigD, a ~36-kDa protein was absent from invF mutant culture supernatants. This protein was also restored by the presence of
invF but not hilA on a low-copy-number plasmid.
Therefore, unlike the sip/ssp genes, sigD (and
possibly the ~36-kDa protein) is not likely to be directly dependent
on hilA for expression. It is notable that unlike the case
for hilA, providing invF in multicopy does not
result in the hypersecretion of proteins found in the culture
supernatants of the wild-type strain (Fig. 1, lane 3 versus lane 4).
Although InvF activates the expression of genes encoding secreted
proteins, it probably does not increase the transcription of the
apparatus genes required for the secretion of these proteins. This may
explain why the hypersecretion of proteins does not occur despite the
hyperexpression of the effector genes observed when invF is
present in multicopy.
It is clear that the regulation of SPI1 gene expression is complicated
and multifactorial (Fig. 2). hilA expression is dependent, directly or indirectly, on SirA, a protein which is known to be conserved in several of the Enterobacteriaceae (22, 33,
35). HilA, a member of the OmpR/ToxR family of regulators, in
turn activates the expression of genes encoding the type III secretion apparatus (3). This effect is predicted to be direct because hilA expressed in E. coli can activate the
expression of either (invF-lacZYA) or
(prgH-lacZYA) (3). This work provides the first demonstration that the AraC-type transcriptional activator InvF
is required for the expression of genes encoding proteins secreted by
the type III secretion system. Further analysis will be necessary to
determine if InvF itself binds to sequences upstream of sicA
and sigD or if it activates the expression of another gene
required for their expression. Because invF and
hilA cannot activate the expression of
(sicA-lacZYA) or (sigD-lacZYA) in E. coli, it seems unlikely that InvF alone is sufficient for
activation. Perhaps InvF, like AraC, requires a cofactor (in the case
of AraC, arabinose) (13) which induces a conformational
change in InvF allowing it to bind to the appropriate promoters. Future
studies will elucidate if and how InvF directly interacts with the
promoters of genes encoding secreted effectors.
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ACKNOWLEDGMENTS |
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We thank Andrew Darwin for critically reviewing the manuscript. We also thank Brad Jones and Catherine Lee for strains indispensable for this work. We especially thank C. Lee for helpful and open discussions about this project.
This work was supported by National Institutes of Health grant AI01230 to V.L.M.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid, Campus Box 8230, St. Louis, MO 63110. Phone: (314) 747-2132. Fax: (314) 747-2135. E-mail: virginia{at}borcim.wustl.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| 1. | Ahmer, B. M. M., J. van Reeuwijk, P. R. Watson, T. S. Wallis, and F. Heffron. 1999. Salmonella SirA is a global regulator of genes mediating enteropathogenesis. Mol. Microbiol. 31:971-982[Medline]. |
| 2. | Altmeyer, R. M., J. K. McNern, J. C. Bossio, I. Rosenshine, B. B. Finlay, and J. E. Galán. 1993. Cloning and molecular characterization of a gene involved in Salmonella adherence and invasion of cultured epithelial cells. Mol. Microbiol. 7:89-98[Medline]. |
| 3. | Bajaj, V., C. Hwang, and C. A. Lee. 1995. hilA is a novel ompR/toxR family member that activates the expression of Salmonella typhimurium invasion genes. Mol. Microbiol. 18:715-727[Medline]. |
| 4. | Bajaj, V., R. L. Lucas, C. Hwang, and C. A. Lee. 1996. Co-ordinate regulation of Salmonella typhimurium invasion genes by environmental and regulatory factors is mediated by control of hilA expression. Mol. Microbiol. 22:703-714[Medline]. |
| 5. | Baumler, A. J., R. M. Tsolis, A. W. M. van der Velden, I. Stojiljkovik, A. Anic, and F. Heffron. 1996. Identification of a new iron regulated locus of Salmonella typhi. Gene 183:207-213[Medline]. |
| 6. |
Behlau, I., and S. I. Miller.
1993.
A PhoP-repressed gene promotes Salmonella typhimurium invasion of epithelial cells.
J. Bacteriol.
175:4475-4484 |
| 7. | Blum, H., H. Beier, and H. J. Gross. 1987. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93-99. |
| 8. | Daefler, S., and M. Russel. 1998. The Salmonella typhimurium InvH protein is an outer membrane lipoprotein required for the proper localization of InvG. Mol. Microbiol. 28:1367-1380[Medline]. |
| 9. | de Lorenzo, V., L. Eltis, B. Kessler, and K. N. Timmis. 1993. Analysis of Pseudomonas gene products using lacIq/Ptrp-lac plasmids and transposons that confer conditional phenotypes. Gene 123:17-24[Medline]. |
| 10. |
Ernst, R. K.,
D. M. Dombroski, and J. M. Merrick.
1990.
Anaerobiosis, type 1 fimbriae, and growth phase are factors that affect invasion of HEp-2 cells by Salmonella typhimurium.
Infect. Immun.
58:2014-2016 |
| 11. | Friedman, A. M., S. R. Long, S. E. Brown, W. J. Buikema, and F. M. Ausubel. 1982. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene 18:289-296[Medline]. |
| 12. |
Galán, J. E., and R. Curtiss, III.
1990.
Expression of Salmonella typhimurium genes required for invasion is regulated by changes in DNA supercoiling.
Infect. Immun.
58:1879-1885 |
| 13. | Gallegos, M.-T., R. Schleif, A. Bairoch, K. Hofman, and J. L. Ramos. 1997. AraC/XylS family of transcriptional regulators. Microbiol. Mol. Biol. Rev. 61:393-410[Abstract]. |
| 14. | Galyov, E. E., M. W. Wood, R. Rosqvust, P. B. Mullan, P. R. Watson, S. Hedges, and T. S. Wallis. 1997. A secreted effector protein of Salmonella dublin is translocated into eucaryotic cells and mediates inflammation and fluid secretion in infected ileal mucosa. Mol. Microbiol. 25:903-912[Medline]. |
| 15. |
Gunn, J. S.,
E. L. Hohmann, and S. I. Miller.
1996.
Transcriptional regulation of Salmonella virulence: a PhoQ periplasmic mutation results in increased net phosphotransfer in phoP.
J. Bacteriol.
178:6369-6373 |
| 16. |
Hamilton, C. M.,
M. Aldea,
B. K. Washburn,
P. Babitzke, and S. R. Kushner.
1989.
New method for generating deletions and gene replacements in Escherichia coli.
J. Bacteriol.
171:4617-4622 |
| 17. | Harlow, E., and D. Lane. 1988. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 18. | Hermant, D., R. Ménard, N. Arricau, C. Parsot, and M. Y. Popoff. 1995. Functional conservation of the Salmonella and Shigella effectors of entry into epithelial cells. Mol. Microbiol. 17:781-789[Medline]. |
| 19. | Hoiseth, S. K., and B. A. D. Stocker. 1981. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291:238-239[Medline]. |
| 20. |
Hong, K. H., and V. L. Miller.
1998.
Identification of a novel Salmonella invasion locus homologous to Shigella ipgDE.
J. Bacteriol.
180:1793-1802 |
| 21. | Hueck, C. J., M. J. Hantman, V. Bajaj, C. Johnston, C. A. Lee, and S. I. Miller. 1995. Salmonella typhimurium secreted invasion determinants are homologous to Shigella Ipa proteins. Mol. Microbiol. 18:479-490[Medline]. |
| 22. | Johnston, C., D. A. Pegues, C. J. Hueck, C. A. Lee, and S. I. Miller. 1996. Transcriptional activation of Salmonella typhimurium invasion genes by a member of the phosphorylated response-regulator superfamily. Mol. Microbiol. 22:715-727[Medline]. |
| 23. | Kaniga, K., J. C. Bossio, and J. E. Galán. 1994. The Salmonella typhimurium invasion genes invF and invG encode homologues of the AraC and PulD family of proteins. Mol. Microbiol. 13:555-568[Medline]. |
| 24. |
Kaniga, K.,
D. Trollinger, and J. E. Galán.
1995.
Identification of two targets of the type III protein secretion system encoded by the inv and spa loci of Salmonella typhimurium that have homology to the Shigella IpaD and IpaA proteins.
J. Bacteriol.
177:7078-7085 |
| 25. |
Kaniga, K.,
S. Tucker,
D. Trollinger, and J. E. Galán.
1995.
Homologs of the Shigella IpaB and IpaC invasins are required for Salmonella typhimurium entry into cultured epithelial cells.
J. Bacteriol.
177:3965-3971 |
| 26. | Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685[Medline]. |
| 27. |
Lee, C. A., and S. Falkow.
1990.
The ability of Salmonella to enter mammalian cells is affected by bacterial growth state.
Proc. Natl. Acad. Sci. USA
87:4304-4308 |
| 28. |
Lee, C. A.,
B. D. Jones, and S. Falkow.
1992.
Identification of a Salmonella typhimurium invasion locus by selection for hyperinvasive mutants.
Proc. Natl. Acad. Sci. USA
89:1847-1851 |
| 29. | Lodge, J., J. Fear, S. Busby, P. Gunasekaran, and N. R. Kamini. 1992. Broad host range plasmids carrying the Escherichia coli lactose and galactose operons. FEMS Microbiol. Lett. 95:271-276. |
| 30. | Maloy, S. R., V. J. Stewart, and R. K. Taylor. 1996. Genetic analysis of pathogenic bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 31. | Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 32. | Mills, D. M., V. Bajaj, and C. A. Lee. 1995. A 40 kb chromosomal fragment encoding Salmonella typhimurium invasion genes is absent from the corresponding region of the Escherichia coli K-12 chromosome. Mol. Microbiol. 15:749-759[Medline]. |
| 33. |
Moolenar, G. F.,
C. A. van Sluis,
C. Backendorf, and P. van de Putte.
1987.
Regulation of the Escherichia coli excision repair gene uvrC. Overlap between the uvrC structural gene and the region coding for a 24 kD protein.
Nucleic Acids Res.
15:4273-4289 |
| 34. | Penheiter, K. L., N. Mathur, D. Giles, T. Fahlen, and B. D. Jones. 1997. Non-invasive Salmonella typhimurium mutants are avirulent because of an inability to enter and destroy M cells of ileal Peyer's patches. Mol. Microbiol. 24:697-709[Medline]. |
| 35. |
Rich, J. J.,
T. G. Kinscherf,
T. Kitten, and D. K. Willis.
1994.
Genetic evidence that the gacA gene encodes the cognate response regulator for the lemA sensor in Pseudomoas syringae.
J. Bacteriol.
176:7468-7475 |
| 36. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 37. | Sanderson, K. E., and B. A. D. Stocker. 1987. Salmonella typhimurium strains used in genetic analysis, p. 1220-1224. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C. |
| 38. |
Schiemann, D. A., and S. R. Shope.
1991.
Anaerobic growth of Salmonella typhimurium results in increased uptake by Henle 407 epithelial and mouse peritoneal cells in vitro and repression of a major outer membrane protein.
Infect. Immun.
59:437-440 |
| 39. | Stewart, G. S. A. B., S. Lubinsky-Mink, C. G. Jackson, A. Cassel, and J. Kuhn. 1986. pHG165: a pBR322 copy number derivative of pUC8 for cloning and expression. Plasmid 15:172-181[Medline]. |
| 40. | Wang, R. F., and S. R. Kushner. 1991. Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene 100:195-199[Medline]. |
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