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Journal of Bacteriology, September 1998, p. 4775-4780, Vol. 180, No. 18
Lehrstuhl für Bakteriologie, Max von
Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie
der Ludwig-Maximillians-Universität München, Munich,
Germany,1 and
Department of Infectious
Diseases, Imperial College of Medicine, Hammersmith Hospital,
London, United Kingdom2
Received 20 January 1998/Accepted 11 July 1998
The Salmonella typhimurium genome contains two
pathogenicity islands (SPI) with genes encoding type III secretion
systems for virulence proteins. SPI1 is required for the penetration of the epithelial layer of the intestine. SPI2 is important for the subsequent proliferation of bacteria in the spleens of infected hosts.
Although most mutations in SPI2 lead to a strong reduction of
virulence, they have different effects in vitro, with some mutants
having significantly increased sensitivity to gentamicin and the
antibacterial peptide polymyxin B. Previously we showed that certain
mutations in SPI2 affect the ability of S. typhimurium to
secrete SPI1 effector proteins and to invade cultured eukaryotic cells.
In this study, we show that these SPI2 mutations affect the expression
of the SPI1 invasion genes. Analysis of reporter fusions to various
SPI1 genes reveals highly reduced expression of sipC,
prgK, and hilA, the transcriptional activator
of SPI1 genes. These observations indicate that the expression of one type III secretion system can be influenced dramatically by mutations in genes encoding a second type III secretion system in the same cell.
A large number of virulence factors
are important for the systemic pathogenesis of mice by Salmonella
typhimurium. One group of genes important for the invasion of host
epithelial cells by S. typhimurium is clustered at 63 centisomes on the chromosome (26). This virulence locus was
termed Salmonella pathogenicity island 1 (SPI1), and
molecular analysis of SPI1 has revealed that more than 28 genes
encoding a complex type III secretion system (spa,
inv, prg, and org), as well as
secreted effector proteins (sip or ssp;
spt) and regulatory components (invF and
hilA), are involved in the invasion phenotype and induction
of morphological changes of the host cell (for a review, see reference
9). Many of these invasion genes were identified by
using cell culture-based screens (10, 22). By screening
mutants directly in the animal host via signature-tagged mutagenesis
(STM), we identified a second cluster of genes encoding a separate type
III secretion system at 30 centisomes of the chromosome. This locus was
termed Salmonella pathogenicity island 2 (SPI2) (16,
30). Inactivation of SPI2 genes resulted in a dramatic
attenuation of virulence and the inability of mutants to colonize the
spleens of infected animals. SPI2 contains genes for a type III
secretion system apparatus (ssa) (17, 28, 30) and
a two-component regulatory system (ssr) (28, 30),
as well as candidate genes for a set of secreted effectors
(sse) and their specific chaperones (ssc) (our
unpublished observations). By comparison of the genetic organizations
and sequences of SPI1 and SPI2 genes, it seems clear that the two systems were acquired independently from an unknown source rather than
by duplication of one of the loci (15, 17).
Several phenotypic effects of SPI2 mutations have been identified. Some
SPI2 mutants show reduced levels of serum resistance, whereas others
appear to be more resistant (17). Some mutant strains show
reduced survival inside J774 macrophages (28). Surprisingly,
several SPI2 mutants are less able to invade cultured epithelial cells
or macrophages, and reduced amounts of the SPI1 secreted protein SipC
were detected in the culture medium (17).
These observations prompted us to undertake a more thorough analysis of
SPI2 mutant phenotypes in vitro and to investigate the basis of the
effect of SPI2 mutations on SPI1 secretion in more detail. In this
study, we demonstrate that mutations in SPI2 affect the expression of
SPI1 genes. Furthermore, mutations in SPI2 result in an altered
resistance of S. typhimurium to various antibacterial
agents.
Bacterial strains, plasmids, and construction of reporter fusion
strains.
S. typhimurium NCTC 12023 (identical to ATCC
14028s) was used as the wild-type strain throughout this study. All
mutants and reporter fusions described were constructed in the S. typhimurium 12023 background. S. typhimurium mutations
in SPI1 and SPI2 were identified after STM with a derivative of
mTn5 as described previously (16, 27, 28a).
mTn5lacZY insertions in various SPI1 genes resulting in
lacZ reporter gene fusions have been described previously (2, 3, 18) and were kindly provided by C. A. Lee
(Harvard University). lacZ fusions were transferred into
various SPI1 and SPI2 backgrounds by P22 transduction according to
standard procedures (23). Lack of lysogeny of transductants
was routinely confirmed by streaking on green plates (23),
and correct integration of the reporter fusions was checked by Southern
hybridization analysis. Details of strains used in this study are
presented in Table 1.
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Mutations in Salmonella Pathogenicity
Island 2 (SPI2) Genes Affecting Transcription of SPI1 Genes and
Resistance to Antimicrobial Agents
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
Bacterial growth conditions.
For detection of SipC in
culture supernatants and total cells, as well as for reporter gene
assays, bacterial cultures were grown in LB containing 1% (wt/vol)
NaCl without addition of antibiotics. Cultures were grown overnight at
37°C with a culture volume of 3.5 ml in glass test tubes with
agitation of 50 rpm in a roller drum. Proteins secreted by S. typhimurium were precipitated as described previously
(18). Briefly, bacteria were pelleted by ultracentrifugation
for 20 min at 125,000 × g at 4°C. Residual cellular
debris was removed by filtration through 0.45-µM-pore-size nitrocellulose filters, and supernatant protein was precipitated by
addition of trichloroacetic acid to a final concentration of 10% and
incubation for 1 h at 0°C. Precipitates were collected by
centrifugation for 20 min at 125,000 × g at 4°C,
washed with ice-cold acetone, and air dried. For analysis of total
bacterial protein, cells from overnight cultures were recovered by
centrifugation for 5 min at 4,000 × g. Supernatant
proteins and total bacteria were resuspended in sample buffer (12.5%
glycerol, 4% sodium dodecyl sulfate [SDS], 2% 
-mercaptoethanol,
0.01% bromophenol blue, 50 mM Tris-HCl [pH 6.8]), boiled for 5 min,
and electrophoretically separated on SDS-12% polyacrylamide gels
(21).
DNA biochemistry. The fine mapping of transposon (Tn) insertions in ssaV was performed by DNA sequencing using primers corresponding to the I and O termini of mTn5 (16) as well as specific primers. Sequencing was performed by using dye terminator chemistry on a PE 377XL DNA sequencer.
For the generation of a nonpolar mutation in the ssaV gene, a 7.5-kb PstI fragment from
clone 7 (30)
harboring ssaV was isolated and ligated into the
PstI site of pUC18. This construct was linearized at the
EcoRV site in the ssaV gene and ligated with a
0.8-kb HincII fragment containing the aphT
cassette (lacking transcriptional terminators) from plasmid pSB315
(12). The direction of transcription of the aphT
gene within the disrupted ssaV gene was confirmed by a
HindIII/ClaI double digest and a plasmid
selected which contains the aphT gene transcribed in the
same direction as the ssaV gene. The disrupted
ssaV gene was then isolated on an EcoRI fragment
and ligated into the suicide vector pGP704 (25) at the
EcoRI site to form plasmid pNPssaV.
pNPssaV was transformed into S17-1 pir and
transferred by conjugation to S. typhimurium 12023 Nalr, using selection for kanamycin resistance. The correct
integration of the ssaV::aphT mutation
onto the chromosome was verified in individual exconjugants by
restriction of genomic DNA with ClaI and
HindIII in single digests and Southern hybridization.
This mutation was transduced in the wild-type S. typhimurium
12023, and the resulting strain, NPssaV, was used for
further studies. For the complementation of P9B7, ssaT was
amplified by using primers SSAT-FOR
(5'-CTAGGATCCGGCAGATAATGTTACG-3'), and SSAT-REV
(5'-GCTGGATCCGCTCATACAGATGGAAAC-3'), and
ssaTU was amplified by using primers SSAT-FOR and SSAU-REV (5'-GCTGGATCCATTTATGGTGTTTCGGTAG-3'),
introducing BamHI restriction sites (underlined).
Plasmids pssaT and pssaTU were generated by ligation of the respective BamHI-digested PCR products to
BamHI-digested pACYC184.
Expression of recombinant SipC; biochemical and serological procedures. For the expression of recombinant SipC, the sipC gene of S. typhimurium 12023 was amplified by using primers SIPC-FOR 5'-CTCGGATCCCGCCGCTTATTTA-3' and SIPC-REV 5'-TGAAAGCTTAAGCGCGAATATTGCC-3', introducing restriction sites for BamHI and HindIII, respectively (underlined). A single PCR product of about 1.2 kb was obtained, digested with BamHI and HindIII, and purified by electrophoresis on an agarose gel. The purified fragment was ligated into BamHI- and HindIII-digested His tag fusion vector pQE30 (Qiagen, Hilden, Germany) to generate pJD10. E. coli M15[pREP] was transformed to ampicillin resistance by electroporation with pJD10. Expression of the His-tagged SipC fusion protein (recombinant SipC [rSipC]) from pJD10-harboring strains was detected with a Ni-alkaline phosphatase conjugate according to the instructions of the supplier (Qiagen). A highly expressing strain was selected and used for large-scale expression of rSipC. rSipC was purified by affinity chromatography on HiTrap chelating columns as instructed by the manufacturer (Pharmacia, Freiburg, Germany). The eluate was further purified by preparative SDS-polyacrylamide gel electrophoresis (PAGE), and a 42-kDa protein corresponding to rSipC was excised. rSipC was recovered from gel slices by electroelution.
For immunization, about 1 mg of rSipC was emulsified with complete and incomplete Freund's adjuvants for primary and booster immunizations, respectively. A rabbit was immunized subcutaneously according to standard procedures (14), and antiserum was used without further modification. SipC was detected with the antiserum raised against rSipC after electrophoretic separation of proteins from culture supernatants or total cell fractions and transfer onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany), using a semidry blotting device (Bio-Rad). Bound antibody was visualized by using a secondary antibody-alkaline phosphatase conjugate according to standard procedures (14).Reporter gene assays. Expression levels of lacZ reporter gene fusions to SPI1 genes were assayed as described by Miller (24). Cultures of various S. typhimurium strains were grown overnight under conditions identical to those described for the analysis of secreted and cellular SipC. All reporter assays were performed at least in triplicate on different occasions.
Sensitivity assays. Bacteria were grown to mid-logarithmic phase in LB broth, washed twice with RPMI 1640 (RPMI), and resuspended in RPMI at a concentration of approximately 108 CFU/ml. Gentamicin sensitivity assays were performed by adding an equal volume of RPMI containing twice the final concentration of gentamicin (0.62 µg/ml; Sigma) to bacterial suspensions and incubating the cells at 37°C for 1 h. To enumerate the number of viable bacteria present, an equal volume of RPMI without gentamicin was added to the bacterial suspensions, which were then incubated as described above. After incubation, the bacteria were harvested by centrifugation at 18,000 × g for 4 min, washed once with RPMI, and resuspended in RPMI. A dilution series was prepared in RPMI and plated onto LB or LB containing kanamycin (50 µg/ml) to enumerate CFU. Sensitivity to polymyxin B was assayed essentially as described by Roland et al. (29). Briefly, bacteria were grown to mid-logarithmic phase in LB broth, washed and resuspended in 0.5% tryptone saline at 104 CFU/ml, and incubated at 37°C for 1 h with or without polymyxin B (Sigma) at a final concentration of 500 ng/ml. After incubation, the test samples were diluted in saline and plated onto LB to determine the number of CFU, and the percent survival was calculated. Serum sensitivity assays were performed with pooled normal human serum as previously described (17).
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RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Effects of mutations in SPI2 on a secreted effector protein of SPI1. The positions and orientations of Tn insertions of mutant strains used in this study are indicated in Fig. 1. Analysis of proteins secreted into the growth medium by the S. typhimurium SPI2 mutant strains P9B7 (ssaT::mTn5) and P11C3 (ssaV::mTn5) revealed the absence or strong reduction in the amounts of the secreted SPI1 effector protein SipC (17). These SPI2 mutants are also reduced in the ability to invade cultured epithelial cells or cultured macrophages (17). To examine this phenomenon in greater detail, we expressed rSipC and raised antibodies against rSipC in rabbits. On Western blots, antiserum against rSipC reacted with a 42-kDa protein from precipitates of culture supernatants of S. typhimurium wild-type strain 12023. No reaction was observed with supernatants from cultures of EE638, a strain deficient in SipC (18) (data not shown) and a mutant (P7B12) from our STM collection harboring a mTn5 insertion in sipC (Fig. 2B). Furthermore, on Western blots SipC was not detected in culture supernatants of the SPI2 mutants P8G12 (ssrB::mTn5), P9B7 (ssaT::mTn5), P11C3 (ssaV::mTn5), and P11D10 (ssaJ::mTn5) and SPI1 mutants P4H2 (hilA::mTn5) and P6E11 (spaRS::mTn5). However, SipC was detected in culture supernatants of SPI2 mutants P2D6 (ssaV::mTn5), P9B6 (ssaV::mTn5), and NPssaV (ssaV::aphT). The detection by antiserum of SipC in culture supernatants of various strains was in accord with the presence or absence of SipC as detected by SDS-PAGE (Fig. 2A).
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Effects of SPI2 mutations on the expression of SPI1 genes.
To
assay the effect of SPI2 mutations on the expression of SPI1 genes,
previously characterized fusions of lacZ to various SPI1
genes (2, 3) were transduced into various SPI2 and SPI1
mutants to generate a set of reporter fusion strains. The expression of
the reporter -galactosidase in cultures grown under conditions
inducing for SPI1 expression (see above) was assayed. A Tn insertion in
hilA (P4H2) reduced the expression of both prgK and sipC, while an insertion in spaRS (P6E11)
affected the expression of only sipC (Fig. 3). Some mutant
strains with Tn insertions in SPI2 genes encoding components of the
type III secretion apparatus (P11C3, P11D10, and P9B7) or the
two-component regulatory system (P8G12) showed reduced expression of
reporter fusions to prgK and sipC (Fig.
3). The effects on the expression of both
genes were similar. Other mutant strains with Tn insertions in
ssaV (P2D6 and P9B6), as well as mutant NPssaV
harboring a nonpolar insertions in ssaV, had levels of
expression of prgK and sipC comparable to those
of corresponding reporter fusions in a wild-type genetic background.
Analysis of lacZ fusions to prgH and
invF revealed an effect on expression similar to that shown
for prgK and sipC (data not shown).
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SPI2 mutations affect expression of the SPI1 regulator hilA. Analysis of reporter fusions to sipC and prgK indicated that expression of genes in two different operons of SPI1 can be affected by SPI2 mutations, suggesting that these mutations affect other SPI1 genes involved in regulation of sipC and prgK. It has been demonstrated previously that the expression of SPI1 genes is under the control of the transcriptional activator HilA (2, 3). The expression of hilA was therefore analyzed in the presence of various SPI2 mutations. The SPI2 mutant strains P8G12, P9B7, P11C3, and P11D10 had largely diminished levels of hilA expression (Fig. 4). Again, very low levels of hilA expression were observed in mutants that had reduced levels of prgK and sipC expression. To analyze whether the effect of SPI2 mutations on SipC expression resulted from the reduced expression of hilA, we next performed complementation experiments in various mutant strains harboring pVV135 (constitutive expression of hilA) (3) or pVV214 (expression of hilA from the native promoter) (2). In accordance with a previous study (2), the hilA mutation of strain P4H2 was complemented by pVV214. However, SipC expression was not restored in mutant strain P11C3, P9B7, or P8G12 harboring either pV135 or pVV214 (data not shown).
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Effects of mutations in ssaV and ssaT. Different mutations in ssaV produced different phenotypic effects. While the Tn insertion of mutant P11C3 resulted in a strong reduction of SPI1 gene expression, strains P2D6 and P9B6 with insertions at other positions of the same gene exhibited wild-type levels of SPI1 gene expression. To investigate the role of ssaV in more detail, a nonpolar mutation in ssaV was constructed. We analyzed the effect of the inactivation of ssaV on the virulence of S. typhimurium by determining the 50% lethal dose (LD50) in susceptible BALB/c mice. After intraperitoneal inoculation, an LD50 of 4.5 × 106 CFU was obtained for NPssaV. This level of attenuation is comparable to SPI2 mutants with mTn5 insertions in other parts of the ssaK-U operon (30). However, synthesis of SipC and expression of SPI1 genes were not significantly affected in NPssaV, showing that ssaV is not required for SPI1 secretion system function. We also observed that a mTn5 insertion in ssaT (strain P9B7) (Fig. 1) resulted in reduction of SPI1 gene expression. Since ssaT is the penultimate gene of the ssaK-U operon, this observation suggests that ssaT and/or ssaU are important for SPI1 gene expression. SsaT and SsaU are homologs of YscT and YscU of the virulence plasmid-encoded type III secretion system of Yersinia spp. (17). Both proteins contain several putative transmembrane helices and may form structural components of the type III secretion system (1, 5). To further analyze the effect of SsaT and/or SsaU on expression of SPI1 genes, complementation experiments were performed with plasmids pssaT and pssaTU, which constitutively express ssaT and ssaTU, respectively, from the Tetr promoter of pACYC184. The virulence of strain P9B7[pssaTU] in BALB/c mice after intraperitoneal infection with 104 CFU was restored, indicating that the function of SPI2 was complemented by pssaTU. The presence of pssaT or pssaTU had no effect on SipC expression in the wild-type strain; however, SipC expression was not restored in mutant strains P9B7 and P11C3 harboring either pssaT or pssaTU (data not shown).
Sensitivity of SPI2 mutants to serum, gentamicin, and polymyxin B. In a previous study (17), we found that some mutations in the ssaV, ssaT, and ssaJ genes resulted in altered resistance to killing by complement, whereas other resulted in wild-type levels of resistance. These data suggested that mutations in these genes caused a perturbation of the cell wall. Accordingly, we investigated whether mutations in these and other SPI2 genes could affect sensitivity to serum, gentamicin, and polymyxin B, since these compounds affect or need to cross the cell wall for activity. Mutations in the ssaT and ssr genes cause increased sensitivity to killing by complement and gentamicin, while some mutations in ssaV and ssaJ show enhanced resistance to killing by complement and wild-type levels or enhanced resistance to gentamicin. All of the SPI2 mutants analyzed were more sensitive to polymyxin B than the wild-type strain, with ssaT mutant P9B7 being the most sensitive to polymyxin B (Table 2).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We previously showed that certain mutations in SPI2 affect the ability of S. typhimurium to invade cultured epithelial cells and cultured macrophage-like cells, and it has been suggested that this is a secondary effect due to a decreased secretion of SPI1 effector proteins (17). Here we have confirmed and extended these findings by showing that the effect on SPI1 functions is correlated to reduced expression of SPI1 genes in certain SPI2 mutant strains. Collazo and Galan (7) have demonstrated that a functional type III secretion system of SPI1 is required for the secretion of SPI1 effector proteins. According to the model proposed by Bajaj et al. (2), HilA is the key local regulatory protein for the expression of SPI1 genes, and it has been demonstrated that the expression of SPI1 operons encoding type III secretion system components (such as PrgK) and secreted effector proteins (such as SipC) is under the control of HilA (2, 3). SirA and PhoPQ, encoded by genes outside the SPI1 and SPI2 loci, also regulate the expression of hilA (19). A possible explanation for the reduced expression of sipC may be the reduced expression of hilA resulting from mutations in SPI2. However, our results indicate that this is unlikely since regulated or constitutive expression of hilA in trans did not complement the effects of mutations in SPI2 on sipC expression. If the expression of SPI1 genes is not directly dependent on the amount of HilA present in the cell, SPI2 mutations may affect other proteins also regulating SPI1 gene expression. Alternatively, mutations in SPI2 genes may affect the activity of promoters of SPI1 genes more directly. It has been demonstrated that the activity of HilA is modulated by environmental factors (3), but the molecular nature of this modulation has not been elucidated. The recognition of such environmental signals affecting HilA activity may be disturbed in strains carrying certain SPI2 mutations. This hypothesis is supported by the observation that certain mutations in SPI2 have pleiotropic effects resulting in increased sensitivity to complement, gentamicin, and polymyxin. As all three of these antimicrobial agents require an interaction with the bacterial cell surface or membrane components to be effective (for review, see reference 20), this finding suggests that the cell membrane or surface is perturbed in these mutants. This may be due to mutant SPI2 components present in or spanning the cell membrane or to the lack of SPI2 components. However, it should be noted that not all SPI2 mutants with reduced expression of SPI1 genes show increased sensitivity to antimicrobial agents. The global regulation by DNA supercoiling is another regulatory factor for SPI1 gene expression (11). If such regulation by DNA topology is disturbed in the background of SPI2 mutations, this effect could act in parallel on several promoters within SPI1. Under such conditions, SPI1 promoters may not be active even in the presence of HilA. Alternatively, some products of SPI2 genes may directly affect the activity of HilA. The expression of SPI2 genes is largely reduced in an ssrB mutant strain (P8G12) (14a, 31). This mutation also led to reduced expression of SPI1 genes, indicating that the presence of one or more SPI2 proteins is required for expression of SPI1 genes.
The effect of a mutation in ssaT on S. typhimurium virulence was complemented by constitutive expression of ssaTU in trans. Presence of pssaTU in the wild-type background did not influence SipC expression; however, the effect of the ssaT mutation in strain P9B7 on SPI1 gene expression was not complemented by pssaTU. This result indicates that the mutation in ssaT may have a dominant effect on the expression of SPI1 genes that cannot be complemented by the addition of the wild-type allele. Alternatively, the levels of SsaTU required for the complementation of the SPI2 virulence function may be different from the amounts of SsaTU required for SPI1 function. There is no obvious explanation for the observation that only certain SPI2 mutations in the ssaK-U operon affect expression of SPI1 genes. It has been observed that the transcriptional terminator of the aph gene of Tn5 can cause polar effects (13). The Tn insertion may also introduce promoters activating the transcription of genes downstream of the Tn insertion site (4). Either effect of a Tn insertion could result in a disturbance of the ratio between SPI2 subunits encoded by the ssaK-U operon. Further work is needed to determine if the lack of components of the SPI2 secretion system or the presence of components of the SPI2 secretion system in unbalanced amounts affects the expression of SPI1 genes.
The similar intraperitoneal LD50s obtained for the ssrA::mTn5 strain P3F4, ssaT::mTn5 strain P9B7 (30), and mutant NPssaV (this study) demonstrate that the effect of antimicrobial agents such as complement, gentamicin, and polymyxin are very likely to be a secondary consequence of mutations in SPI2 genes, and their physiological relevance is uncertain. However, even if these effects are secondary, they have important implications for the study of SPI2 mutants, particularly in cell culture invasion or replication assays that use gentamicin over long periods of time to kill extracellular bacteria.
In conclusion, our observation of the interaction between virulence loci in S. typhimurium suggests that an intact SPI2 secretion system may be required for normal expression of genes in SPI1. Present understanding of the regulation of gene expression of SPI1 and SPI2 is not sufficient to explain the molecular basis of effects of SPI2 mutations on SPI1 function. However, the potential regulatory interaction described here should be considered with respect to experiments designed to understand the function of the type III secretion system of SPI1 and the role of SPI1 in pathogenesis.
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ACKNOWLEDGMENTS |
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This project was supported by the Deutsche Forschungsgemeinschaft (grant He 1964/2-2 to M.H.) and by a grant from the Medical Research Council (United Kingdom) to D.W.H.
We are indebted to C. A. Lee (Harvard University) for providing strains and plasmids and to J. E. Galan (Yale University) for plasmid pSB315. We especially thank J. Heesemann (Munich, Germany) for support and discussion.
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FOOTNOTES |
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* Corresponding author. Mailing address: Lehrstuhl für Bakteriologie, Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Pettenkofer Str. 9a, D-80336 Munich, Germany. Phone: 49 (0)89 5160 5248. Fax: 49 (0)89 5160 5223. E-mail: hensel{at}m3401.mpk.med.uni-muenchen.de.
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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