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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for systemic infection of this pathogen in mice. Cloning and sequencing of a central region of SPI-2 revealed the presence of genes encoding putative chaperones and effector proteins of the secretion system. The predicted products of the sseB, sseC and sseD genes display weak but significant similarity to amino acid sequences of EspA, EspD and EspB, which are secreted by the type III secretion system encoded by the locus of enterocyte effacement of enteropathogenic Escherichia coli. The transcriptional activity of an sseA::luc fusion gene was shown to be dependent on ssrA, which is required for the expression of genes encoding components of the secretion system apparatus. Strains carrying non-polar mutations in sseA, sseB or sseC were severely attenuated in virulence, strains carrying mutations in sseF or sseG were weakly attenuated, and a strain with a mutation in sseE had no detectable virulence defect. These phenotypes were reflected in the ability of mutant strains to grow within a variety of macrophage cell types: strains carrying mutations in sseA, sseB or sseC failed to accumulate, whereas the growth rates of strains carrying mutations in sseE, sseF or sseG were only modestly reduced. These data suggest that, in vivo, one of the functions of the SPI-2 secretion system is to enable intracellular bacterial proliferation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Several Gram-negative bacterial pathogens secrete virulence proteins via specialized type III secretion systems (Mecsas and Strauss, 1996). These secretion systems comprise a large number of proteins required to transfer specific effector proteins into eukaryotic host cells in a contact-dependent manner (Rosqvist et al., 1994; Zierler and Galan, 1995; Collazo and Galan, 1997). Although several components of the secretion system apparatus show evolutionary and functional conservation across bacterial species (Salmond and Reeves, 1993), the effector proteins are less well conserved and have different functions. The Yersinia effectors YpkA and YopH have threonine/serine kinase and tyrosine phosphatase activities respectively (Galyov et al., 1993; Persson et al., 1995). The actions of these and other Yops inhibit bacterial phagocytosis by host cells, which is thought to enable extracellular bacterial proliferation (for review see Cornelis and Wolf-Watz, 1997). The Shigella Ipa proteins, secreted by the mxi/spa type III secretion system, promote entry of this bacterium into epithelial cells (Menard et al., 1996). The proteins EspA, EspB and EspD, encoded by the locus of enterocyte effacement (LEE) of enteropathogenic Escherichia coli (EPEC) are secreted by a type III secretion system and cause cytoskeletal rearrangements of host epithelial cells resulting in the formation of pedestal-like structures on the host cell surface (for a review see Donnenberg et al., 1997).

Salmonella typhimurium is unusual in that it contains two type III secretion systems for virulence determinants. The first controls bacterial invasion of epithelial cells (Galan and Curtiss, 1989; Galan, 1996) and is encoded by genes within a 40 kb pathogenicity island (SPI-1) located at 63 centisomes on the chromosome (Mills et al., 1995). Evidence for the second type III secretion system is based on a set of corresponding genes within a second pathogenicity island (SPI-2) at 30 centisomes, which are required for systemic growth of this pathogen in its host (Hensel et al., 1995; Ochman et al., 1996; Shea et al., 1996). We proposed that the SPI-2 secretion system genes should be designated as follows: ssa for genes encoding the secretion system apparatus, ssr for genes encoding secretion system regulators, ssc for genes encoding secretion system chaperones and sse for genes encoding secretion system effectors (Hensel et al., 1997a).

Many of the genes encoding components of the SPI-2 secretion system are located in a 25 kb segment beginning at the 31 centisomes boundary of SPI-2 (Hensel et al., 1997b). On the basis of similarities with genes of other bacterial pathogens, the first 13 genes from this boundary (the ssaK/U operon and ssaJ ) encode components of the secretion system apparatus (Hensel et al., 1997a). A number of additional genes including ssaC (orf 11 in Shea et al., 1996; spiA in Ochman et al., 1996) and ssrA (orf 12 in Shea et al., 1996; spiR in Ochman et al., 1996), which encode a secretion system apparatus protein and a two component regulatory protein, respectively, are found in a region ≈8 kb upstream of ssaJ (Shea et al., 1996).

In this paper we describe an analysis of genes that encode other components of the secretion system, located in the region between ssaJ and ssaC of SPI-2. DNA and protein database searches with these genes identified three whose products display weak but significant similarity to proteins secreted by the type III secretion systems of EPEC and Yersinia, and two that are potential chaperones for these proteins. We show that transcriptional activity of sseA is dependent on ssrAB, which encodes a two-component regulatory system required for the expression of ssa genes of SPI-2 (Valdivia and Falkow, 1997). Virulence tests and in vitro assays with strains containing non-polar mutations show that several sse genes are critical for Salmonella virulence in mice and are required for bacterial accumulation within macrophages.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Organization of sse and ssc genes

As part of our effort to characterize SPI-2 genetically and functionally, we cloned and sequenced a central region of the pathogenicity island (Fig. 1A). DNA fragments covering the region between ssaC and ssaJ were subcloned in plasmids p5-2 and p5-4 as indicated in 1Fig. 1C. The arrangement and designation of genes in the 8 kb region between ssaC and ssaK is shown in 1Fig. 1B. This sequence is available from the EMBL database under accession number AJ224892. The sequenced region extends the open reading frame (ORF) of a gene encoding a putative subunit of the type III secretion apparatus referred to as spiB (Ochman et al., 1996). For consistency with the universal nomenclature for type III secretion system subunits (Bogdanove et al., 1996) and the nomenclature of other SPI-2 genes (Hensel et al., 1997a), we propose that this gene be designated ssaD. The deduced amino acid sequence of ssaD is 24% identical to YscD of Y. enterocolitica. This is followed by an ORF with coding capacity for a 9.3 kDa protein, 34% identical to YscE of Y. enterocolitica. Therefore, this gene is designated ssaE. A sequence of 263 bp separates ssaE and a set of nine genes, several of which encode proteins with sequence similarity to secreted proteins or their chaperones from other pathogens. These genes are separated by short intergenic regions or have overlapping reading frames and it is likely that some are co-transcribed and translationally coupled. Therefore, the genes with similarity to those encoding chaperones were designated sscA and sscB, and the others sseA-G. The amino acid sequence deduced from sscA shows 26% identity/49% similarity over 158 amino acid residues to SycD, the product of lcrH of Y. pseudotuberculosis that acts as a secretion-specific chaperone for YopB and YopD (Wattiau et al., 1994). The amino acid sequence deduced from sscB shows 23% identity/36% similarity over 98 amino acid residues to IppI of Shigella flexneri. IppI is a chaperone for S. flexneri invasion proteins (Ipas) (Baundry et al., 1988). As is the case for the secretion chaperones SycD, IppI and SicA (Kaniga et al., 1995), SscB has an acidic pI (Table 1), whereas SscA has an unusually high pI of 8.8.

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Figure 1. . Genetic organization of the effector gene region of SPI-2 and position of mutations. A. The position of this region in relation to other genes of the secretion system is shown. B. Genes encoding proteins with sequence similarity to effector proteins of other type III secretion systems (shaded arrows), putative chaperones of SPI-2 (hatched arrows), components of the type III secretion system (filled arrows) and without sequence similarity (open arrows) are indicated. Positions of a mTn5 insertion in mutant strain P10E11 and the aphT gene cassette in mutant strains HH100, HH102, HH104, HH107 and HH108 are marked. C. A restriction map is shown for BamHI (B), EcoRI (E), HindIII (H), EcoRV(V), SmaI (S) and ClaI (C). The positions of subclones p5-2, p5-4, p5-5, p5-7 and p5-8 are also indicated.

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Table 1. . Features of predicted proteins.Thumbnail image of

SseA, SseE, SseF and SseG showed no significant similarities to DNA or protein database entries. SseB is 25% identical/47% similar to EspA of EPEC over the entire length of the 192-amino-acid-residue protein. SseD is 27% identical/51% similar to EspB of EPEC over 166 amino acid residues. SseC has sequence similarity to a class of effector proteins involved in the translocation of other effectors into the target host cell. These include YopB of Y. enterocolitica, EspD of EPEC and PepB of Pseudomonas aerugunosa. SseC is ≈24% identical/48% similar to both EspD of EPEC and YopB of Y. enterocolitica (Fig. 2). EspD and YopB have two hydrophobic domains that are predicted to insert into target cell membranes (Pallen et al., 1997). SseC contains three hydrophobic regions that could represent membrane-spanning domains. Other features of these predicted effector proteins are shown in Table 1. Using the TMPREDICT program (Hofmann and Stoffel, 1993), transmembrane helices are predicted for all the effector proteins apart from SseA, which is very hydrophilic. Alignments of SseC to homologues in other pathogens are shown in Fig. 2. Conserved amino acids are mainly clustered in the central, more hydrophobic portion of the protein, but unlike YopB, there is no significant similarity to the RTX family of toxins. The conserved residues in SseD are present mainly in the N-terminal half of the protein. Comparison of the deduced amino acid sequences of sseABCDEF with entries in the PROSITE database did not reveal the presence of any characteristic protein motifs. We subjected the predicted amino acid sequences of the sse genes to searches using the programs COIL and MULTICOIL (Lupas, 1997) as described by Pallen et al. (1997). SseA and SseD are predicted to have one trimeric coil each, and SseC is predicted to have two trimeric coils (Table 1). As EspB and EspD are predicted to have one and two trimeric coils, respectively (Pallen et al., 1997), this provides further evidence that these proteins are functionally related.

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Figure 2. . Alignment of the deduced SseC amino acid sequence to EspD of EPEC, YopB of Yersinia enterocolitica (Hakansson et al., 1993) and PepB of Pseudomonas aeroginosa (Hauser et al., 1998). The CLUSTALW algorithm of the MACVECTOR 6.0 program was used to construct the alignments. Similar amino acid residues are boxed, identical residues are boxed and shaded.

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Between sseG and ssaJ [a gene encoding a component of the type III secretion apparatus (Hensel et al., 1997a)], three short ORFs were identified, designated ssaG, ssaH and ssaI, encoding proteins of 7.9 kDa, 8.1 kDa and 9.0 kDa respectively. SsaG has sequence similarity to YscF of Y. enterocolitica (25% identity over 71-amino-acid residues) and MxiH of S. flexneri (34% identity over 46 amino acid residues). No significant sequence similarities were obtained with SsaH and SsaI.

Expression of sseA is dependent on ssrAB

To establish if the sse genes are part of the SPI-2 secretion system, we investigated the expression of an sseA::luc reporter gene fusion, integrated by homologous recombination into the chromosome of different SPI-2 mutant strains. In accordance with results from a previous study (Valdivia and Falkow, 1997), no significant transcription was detected during growth in rich medium. However, transcriptional activity of sseA in a wild-type background was detected during growth in minimal medium (Fig. 3). Transposon insertions in ssrA and ssrB, encoding the sensor component and the transcriptional activator, respectively, resulted in 250- to 300-fold reduced expression of sseA. Inactivation of hilA, the transcriptional activator of SPI-1 (Bajaj et al., 1996), had no effect on sseA gene expression. Transposon insertions in two genes encoding components of the SPI-2 type III secretion apparatus (ssaJ::mTn5; ssaT::mTn5; Shea et al., 1996) also had no significant effect on the expression of sseA. These data show that ssrAB is required for the expression of sseA, but that hilA is not.

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Figure 3. . Expression of an sseA::luc fusion in wild-type and mTn5 mutant strains of S. typhimurium. Expression in each strain was determined in triplicate and the standard errors from the means are shown.

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Virulence tests with strains carrying non-polar mutations

DNA sequence analysis suggested that the sse genes might encode effector proteins of the secretion system, but apart from a possible polar effect from a transposon insertion in sscA (strain P10E11, Fig. 1) no strains carrying mutations in these genes were recovered in the original STM screen for S. typhimurium virulence genes using mTn5 mutagenesis (Hensel et al., 1995), and their role in virulence was unclear. To address this question, we constructed strains carrying non-polar mutations in sseA, sseB, sseC, sseE, sseF and sseG (Fig. 1) and subjected these strains to virulence tests by inoculating groups of BALB/c mice intraperitoneally with each strain, as this was the method by which the original SPI-2 mutants were identified. Table 2 shows that all mice inoculated with strains carrying mutations in sseA, sseB and sseC survived a dose of 1 × 104 cfu, three orders of magnitude greater than the LD50 of the wild-type strain, which is less than 10 cfu when the inoculum is administered by the i.p. route (Buchmeier et al., 1993; Shea et al., 1996). The same strains containing a plasmid carrying the corresponding wild-type allele were also inoculated into mice at a dose of 1 × 104 cfu. No mice survived these infections, which shows that each mutation can be complemented at least partially by the presence of a functional copy of each gene, and that each of these genes plays an important role in Salmonella virulence. Strains carrying non-polar mutations in sseE, sseF and sseG caused lethal infections when ≈1 × 104 cells of each strain were inoculated into mice by the i.p. route (Table 2) and were analysed in more detail by a competition assay with the wild-type strain in mixed infections (three mice per test) to determine whether they were attenuated in virulence. The competitive index, defined as the output ratio of mutant to wild-type bacteria, divided by the input ratio of mutant to wild-type bacteria, shows that sseF and sseG do contribute to virulence, but their absence in mutant strains is not detectable by the relatively insensitive LD50 test (Table 2). By comparison, very low competitive indices were obtained using strains carrying mutations in either ssrA or sseB. The competitive index for the sseE mutant was not significantly different to that of a fully virulent strain carrying an antibiotic resistance marker, which implies that this gene does not play a significant role in systemic Salmonella infection of the mouse.

Table 2. . Virulence of S. typhimurium strains in mice. a. Mice were inoculated intraperitoneally with 1 × 104 cells of each strain and survival was determined after 14 days.b. Result of competition between wild-type strain 12023 and a virulent mTn5 mutant identified in the STM screen.Thumbnail image of

Intramacrophage replication of mutant strains

We tested several mutant strains for their ability to grow inside macrophages and macrophage-like cell lines, as macrophage survival and replication are thought to represent an important aspect of Salmonella pathogenesis in vivo (Fields et al., 1986), and because Ochman et al. (1996) reported that an S. typhimurium strain carrying a mutation in a SPI-2 gene was unable to survive in macrophages. We have reported previously that a number of SPI-2 mutant strains were not defective for survival or replication within RAW macrophages (Hensel et al., 1997a), but subsequent experiments have revealed that some SPI-2 mutants can be shown to have a defect if aerated stationary-phase bacterial cultures opsonized with normal mouse serum are used (see also accompanying paper: Cirillo et al., 1998). The increase in cfu for different strains in RAW macrophages over a 16 h period is shown in Fig. 4. Growth defects were observed for strains carrying mutations in ssaV (encoding a component of the secretion apparatus), sseA, sseB, sseC and to a lesser extent for strains carrying mutations in sseE, sseF and sseG. Partial complementation of this defect was achieved with strains harbouring plasmids carrying functional copies of sseC and sseB, and very slight complementation was observed for sseA. We also investigated the ability of SPI-2 mutant strains to accumulate inside the J774.1 macrophage cell line (Fig. 5A) and in periodate-elicited peritoneal macrophages from C3H/HeN mice (Fig. 5B). Similar defects of S. typhimurium carrying transposon or non-polar mutations in SPI-2 genes were observed, regardless of the phagocyte cell-type examined, although the peritoneal elicited cells had superior antimicrobial activity than either cell line.

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Figure 4. . Intracellular survival and replication of SPI-2 mutant S. typhimurium in RAW 264.7 macrophages. After opsonization and infection, macrophages were lysed and cultured for enumeration of intracellular bacteria (gentamicin protected) at 2 h and 16 h after infection. The values shown represent the fold increase calculated as a ratio of the intracellular bacteria between 2 h and 16 h after infection. Each strain was infected in triplicate and standard error from the mean is shown.

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Figure 5. . Intracellular survival and replication of SPI-2 mutant S. typhimurium in (A) J774.1 cells and (B) periodate elicited peritoneal macrophages from C3H/HeN mice. After opsonization and internalization, phagocytes were lysed and cultured for enumeration of viable intracellular bacteria at time 0 h. The values shown represent the proportion of this intracellular inoculum viable at 20 h ± the standard error of the mean. Samples were processed in triplicate, and each experiment was performed at least twice.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We have identified a group of genes encoding putative effector proteins and chaperones of the SPI-2 type III secretion system. These genes are located between two clusters of genes encoding components of the secretion system apparatus. In terms of their overall arrangement and deduced amino acid sequences, the SPI-2 ssa and sse genes appear to share greatest similarity to the esc and esp genes of the EPEC LEE (Elliot et al., 1998). Although sseBCD are in the same order as the genes to which they show similarity (espADB), there is no gene between espA and espD corresponding to sscA, which encodes a putative chaperone. The corresponding gene products of sseCD and espDB are also predicted to have transmembrane helices and to form trimeric coiled coils which suggests that the two sets of genes have similar functions.

EspA, EspB and EspD are necessary to activate epithelial signal transduction pathways leading to host cell cytoskeletal rearrangements and pedestal-like structures on intestinal epithelial cell surfaces, to which the bacteria adhere (Donnenberg et al., 1997). Recent work has shown that both EspB (Wolff et al., 1998) and EspA (Knutton et al., 1998) are involved in the translocation of EspB into both the plasma membrane and cytoplasm of epithelial cells. EspA is a component of a filamentous surface appendage that forms a contact between the bacterium and the eukaryotic cell surface (Knutton et al., 1998). These structures resemble specific appendages (invasomes) that can be detected on the surface of S. typhimurium during epithelial cell invasion, although their relationship to the SPI-1 type III secretion system is uncertain (Ginocchio et al., 1994; Reed et al., 1998). The SPI-1 secretion system forms a needle-like structure that spans the bacterial cell envelope (Kubori et al., 1998). Both SseC and EspD are similar to YopB, which is a pore-forming protein required for translocation of effector proteins of the Yersinia type III secretion system across the eukaryotic cell plasma membrane (Hakansson et al., 1996). These similarities suggest that at least some of the sse genes encode a SPI-2 translocon that integrates into a target cell membrane and mediates the translocation of other SPI-2 effector proteins. Although intracellular Sse proteins can be detected in bacterial lysates after growth in laboratory media using polyclonal antibodies and epitope tags (our unpublished results), we have not been able to establish in vitro conditions that induce the secretion of these proteins. We are currently investigating their secretion in vivo and within cultured host cells. Evidence that the sse genes encode secreted proteins of the SPI-2 secretion system is therefore based on their chromosomal location and order, similarities of predicted sequence and hydrophobicity to Esps of EPEC and Yersinia YopB, and on the fact that the expression of sseA requires SsrAB, a two-component regulatory system required for the expression of other genes of the secretion system (Valdivia and Falkow, 1997; and accompanying paper Cirillo et al., 1998).

The importance of the sse genes was originally unclear because all the STM-derived transposon insertions in SPI-2 are in genes for structural and regulatory components (Shea et al., 1996) and sscA, which is predicted to encode a chaperone. The failure to recover transposon insertions in the sse genes may have resulted from a low frequency of mTn5 insertions in this region, because strains carrying targeted mutations in sseA, sseB and sseC are strongly attenuated in virulence. These strains also exhibited deficient replication or survival in two different macrophage-like cell lines and elicited mouse peritoneal macrophages. This phenotype was observed using opsonized bacteria but does not require exogenous cellular activation by cytokines. This finding prompted us to re-examine the intramacrophage accumulation of strains carrying transposon insertions in ssrA and ssaV because in an earlier study using non-opsonized bacterial cells grown under conditions that make them highly invasive, these strains were not found to have a replication defect (Hensel et al., 1997a). When ssrA and ssaV mutant strains were opsonized and grown under non-invasive conditions, their intramacrophage numbers were as low as the strains carrying mutations in sseA and sseB. These differences may reflect the different intracellular fate of organisms entering macrophages by pathogen-directed invasion processes (non-opsonized) or by phagocyte-directed uptake (opsonized). Furthermore, as high invasion leads to a greater degree of host cell cytotoxicity through SPI-1-mediated apoptosis (Monack et al., 1996; Chen et al., 1996), cytotoxic effects of replicating wild-type cells may have released bacteria from the intracellular environment and resulted in killing by gentamicin, and therefore an under-estimation of the number of wild-type cells that had undergone intracellular replication in our previous studies.

As the phenotypic behaviour of opsonized bacterial strains carrying transposon or non-polar mutations in various SPI-2 genes inside cultured macrophages reflects their virulence phenotype in vivo, we conclude that the SPI-2 secretion system is required for bacterial proliferation inside macrophages in vivo. However, this does not exclude the possibility that the SPI-2 genes have other functions not apparent from the macrophage assays. These results confirm and extend the earlier findings of Ochman et al. (1996), who found that the secretion system is required for bacterial survival in J774 macrophages. SPI-2 mutant strains were originally found to be attenuated in virulence by mixed infections of mice with pools of signature-tagged mutant strains, the majority of which are virulent (Hensel et al., 1995). This means the SPI-2 mutant strains cannot be rescued in trans by the presence of virulent strains. The demonstration of SPI-2 gene expression within macrophages (Valdivia and Falkow, 1997), along with the reduced numbers of SPI-2 mutant cells in cultured macrophages, suggests that the failure of virulent cells to rescue the SPI-2 defect may be due to physical separation of bacteria within phagocytic cells. Their reduced accumulation could be due to either reduced survival (greater susceptibility to macrophage killing) or reduced replication, or a combination of the two. Ochman et al. (1996) concluded that the function of the secretion system might be to modify host factors required for phagosome–lysosome fusion or phagosome acidification. More recently Valdivia and Falkow (1997) showed that at least one of the SPI-2 secretion system genes is induced in a variety of host cells including macrophage-like RAW cells, and speculated that the function of the secretion system might be to translocate bacterial proteins across the vacuolar membrane. Therefore, the putative translocon encoded by the sse genes reported here might be inserted in the vacuolar membrane and influence the intracellular fate of the Salmonella-containing vacuole. Further investigation of Salmonella–macrophage interactions and the functions of the sse gene products are likely to provide important insights into the mechanism of SPI-2-mediated virulence.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Bacterial strains, phages and plasmids

The bacterial strains, phages and plasmids used in this study are listed in Table 3. Unless otherwise indicated, bacteria were grown in LB broth or on LB agar plates with the addition, when appropriate, of ampicillin (50 μg ml−1), kanamycin (50 μg ml−1) or chloramphenicol (50 μg ml−1).

Table 3. . Phages, plasmids and bacterial strains used in this study (Miller and Mekalanos (1988); Galan et al. (1992); Herrero et al. (1990); Miller et al. (1989)).Thumbnail image of

DNA cloning and sequencing

Clones harbouring fragments of SPI-2 were identified from a library of genomic DNA of S. typhimurium in λ 1059, which has been described previously (Shea et al., 1996). The sse and ssc genes were subcloned from clone λ 5 on a 5.7 kb EcoRI fragment and a 5.8 kb HindIII fragment in pKS+ as indicated in Fig. 1 and Table 3. DNA sequencing was performed using a primer walking strategy. The dideoxy method (Sanger et al., 1977) was applied using the Pharmacia T7 sequencing system for manual sequencing and the dye terminator chemistry for automatic analysis on a ABI377 sequencing instrument. Assembly of contigs from DNA sequences was performed by means of ASSEMBLYLIGN and MACVECTOR software (Oxford Molecular). For further sequence analyses, programs of the GCG package version 8 (Devereux et al., 1984) were used on the HGMP network.

Construction of non-polar mutations

The construction of non-polar mutations in sseA, sseB, sseC, sseE and sseF are described below. All chromosomal modifications were confirmed by PCR and Southern hybridization analysis.

SseA

Plasmid p5-5 was cut at a unique HpaI site within sseA. The linear product was then used as template for a PCR with primers sseA1 (5′-GAAGGCCTTTTTCTTTATCATCATTCCCC-3′) and sseA2 (5′-GAAGGCCTGAAACAACTTAATGCTCAAGCC-3′) to delete an internal region of 246 bp from sseA, corresponding to amino acids 8–90. The linear product contains StuI sites at each terminus and was circularized by ligation after StuI digestion and transferred into E. coli DH5α. A 900 bp HincII fragment of pSB315 containing an aminoglycoside 3′-phosphotransferase gene (aphT ) from which the transcriptional terminator had been removed (Galan et al., 1992) was ligated in the same orientation into the unique StuI site created in the sseA deletion plasmid. An NheI fragment containing the ΔsseA::aphT gene was inserted into the XbaI site of the plasmid pCVD442 (Donnenberg and Kaper, 1991) and transferred by conjugation from E. coli S17-1 λpir to S. typhimurium 12023. Exconjugants were selected for kanamycin resistance and screened for ampicillin sensitivity.

SseB

Plasmid p5-5 was cut at the unique ClaI site within sseB. This linear DNA was used as the template for PCR using primers sseB1 (5′-ATTGGATCCGGTGGAGATACCGTC-3′) and sseB2 (5′-TATGGATCCTGTTGTTAGGGTCGGG-3′). The product, with terminal StuI sites, was digested with StuI and self-ligated to generate an sseB gene containing an internal deletion of 333 bp corresponding to amino acids 55–166. The blunt-ended aphT cassette (see above) was ligated into the StuI site in the sseB deletion. An NheI fragment containing the sseB deletion was ligated into the unique XbaI site of plasmid pCVD442 and transferred to the S. typhimurium chromosome as described above.

SseC

A 2.6 kb fragment was recovered after BamHI and ClaI digestion of p5-2 and subcloned in BamHI/ClaI-digested pKS+. The resulting construct was digested by HindIII, blunt ended using the Klenow fragment of DNA polymerase and ligated to the aphT cassette as indicated above. The resulting plasmid was digested with SalI and XbaI and the insert was ligated to SalI/XbaI-digested pGP704. This plasmid was electroporated into E. coli S17-1 λpir and transferred into S. typhimurium 12023 by conjugation. Exconjugants in which the sseC gene had been replaced by the cloned gene disrupted by insertion of the aphT cassette were selected by resistance to kanamycin and screened for sensitivity to carbenicillin.

SseE

Plasmid p5-8 was used as template for a PCR with primers sseE1 (5′-ATTATGCATGCATGGGAGCGACCTTTACACAGCTT-3′) and sseE2 (5′-ATTTAGCATGCGGCGGTCTCCCCTAAATATGCAGG-3′). The PCR product, containing terminal SphI sites, was digested with SphI and self-ligated to create an internal deletion of 261 bp in sseE corresponding to amino acids 26–112. The plasmid was digested with SalI and SstI and a 2.0 kb fragment containing the deleted sseE gene was ligated into pCVD442. The resulting plasmid was used to transfer the mutated gene onto the Salmonella chromosome as described above, using sucrose selection to obtain cells from which the suicide vector had been lost (Donnenberg and Kaper, 1991).

SseF

The N-terminal and C-terminal regions of sseF were isolated from plasmid p5–2 on a 2.3 kb PstI/HindIII fragment and a 2.7 kb EcoRI/SstII fragment respectively. The PstI/HindIII fragment was subcloned in pBluescript SK, the resulting construct linearized at the polylinker XbaI site and the cohesive ends made flush by treatment with T4 DNA polymerase. The EcoRI/SstII fragment was treated with T4 DNA polymerase and ligated to the linearized vector to form plasmid psseF2, which contains sseF with a central deletion corresponding to amino acids 81–178. Plasmid psseF2 was linearized at a BamHI site adjacent to the destroyed XbaI site and ligated with the aphT gene (see above). A plasmid was identified carrying the aphT gene in the required orientation. SseF::aphT was isolated on a EcoRI/SstII fragment, blunt-ended as before, ligated into the SmaI site of pCVD422 and the mutation transferred to the Salmonella chromosome as described above.

SseG

A 1.0 kb SauIIIA fragment containing sseG was subcloned from p5-7 into pUC18. The resulting construct was digested with AscI, blunt-ended using Klenow fragment of DNA polymerase and ligated to the aphT cassette as indicated above. The resulting plasmid was digested with EcoRI and XbaI and the insert was ligated into EcoRI/XbaI-digested pGP704. This plasmid was introduced into S. typhimurium 12023 and exconjugants carrying the aphT cassette in sseG were isolated as described above.

For complementation of non-polar mutations in sseA, sseB and sseC, the corresponding genes were amplified by PCR from genomic DNA using a series of primers corresponding to the region 5′ of the putative start codons and to the 3′ ends of the genes. These primers introduced BamHI restriction sites at the termini of the amplified genes. After digestion by BamHI, the genes were ligated to BamHI-digested pACYC184 (Chang and Cohen, 1978) and transferred into E. coli DH5α. The orientation of the inserts was determined by PCR, and, in addition, DNA sequencing was performed to confirm the orientation and the correct DNA sequence of the inserts. Plasmids with inserts in the same transcriptional orientation as the Tetr gene of pACYC184 were selected for complementation studies and electroporated into the S. typhimurium strains harbouring corresponding non-polar mutations.

Virulence tests and macrophage survival assays

Groups of female BALB/c mice (20–25 g) were inoculated intraperitoneally with either single or mixed S. typhimurium strains. The inoculum consisted of either 1 × 105 or 1 × 104 cfu in 0.2 ml of physiological saline. Bacterial strains were cultured as described by Shea et al. (1996).

For mixed infections, wild-type and mutant strains were grown separately and mixed before injection. The cfu of both strains was checked by plating a dilution series of the inoculum onto LB and LB supplemented with kanamicin. For mixed infections involving HH106, strain 12023 was first transformed with pACYC184 (which does not affect its virulence, unpublished results) and cfu were checked by plating onto LB and LB supplemented with chloramphenicol. Mice were killed 48 h after inoculation and bacterial cfu were counted after plating dilution series of spleen homogenates onto LB and LB supplemented with the appropriate antibiotic.

RAW 264.7 cells (ECACC 91062702), a murine macrophage-like cell line, were grown in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS) and 2 mM glutamine at 37°C in 5% CO2. S. typhimurium strains were grown in LB to stationary phase and diluted to an OD600 of 0.1 and opsonized for 20 min in DMEM containing 10% normal mouse serum. Bacteria were then centrifuged onto macrophages seeded in 24-well tissue culture plates at a multiplicity of infection of ≈1:10 and incubated for 30 min. After infection, the macrophages were washed twice with PBS to remove extracellular bacteria and incubated for 90 min (2 h after infection) or 16 h in medium containing gentamicin (12 μg ml−1). Infected macrophages were washed twice with PBS and lysed with 1% Triton X-100 for 10 min and appropriate aliquots and dilutions were plated onto LB agar to enumerate cfu.

Survival of opsonized S. typhimurium strains in J774.1 cells (Ralph et al., 1975) or C3H/HeN murine peritoneal exudate cells (from Charles River Laboratories) was determined essentially as described by DeGroote et al. (1997), but without the addition of interferon γ. Briefly, peritoneal cells harvested in PBS with heat-inactivated 10% fetal calf serum 4 days after intraperitoneal injection of 5 mM sodium periodate (Sigma) were plated in 96-well flat-bottomed microtitre plates (Becton-Dickinson) and allowed to adhere for 2 h. Non-adherent cells were flushed out with prewarmed medium containing 10% heat-inactivated fetal calf serum. In previous studies, we have established that >95% of the cells remaining after this procedure are macrophages. S. typhimurium from aerated overnight cultures was opsonized with normal mouse serum and centrifuged onto adherent cells at an effector to target ratio of 1:10. The bacteria were allowed to internalize for 15 min and washed with medium containing 6 μg ml−1 gentamicin to kill extracellular bacteria. At 0 h and 20 h, cells were lysed with PBS containing 0.5% deoxycholate (Sigma), with plating of serial dilutions to enumerate cfu.

Macrophages were examined microscopically over the course of these experiments and did not show significant levels of cytotoxicity.

Construction and analysis of sseA reporter gene fusion

A 1.1 kb SmaI/HincII fragment of p5-4 was subcloned into pGPL01, a suicide vector for the generation of luc fusions (Gunn and Miller, 1996). The resulting construct, in which 1.0 kb upstream and 112 bp of sseA is translationally fused to luc was used to transform E. coli S-17 λpir, and conjugational transfer to S. typhimurium performed as described previously (Gunn and Miller, 1996). Strains that had integrated the reporter gene fusion into the chromosome by homologous recombination were confirmed by PCR and Southern hybridization analysis. Subsequently, the fusion was moved by P22 transduction into the wild-type and various mutant strain backgrounds with mTn5 insertions in SPI-1 or SPI-2 genes (Maloy et al., 1996). As a control, a strain was constructed harbouring a chromosomal integration of pLB02, a suicide plasmid without a promoter fusion to the luc gene (Gunn and Miller, 1996). For the analysis of gene expression, strains were grown for 16 h in minimal medium with aeration. Aliquots of the bacterial cultures were lysed and luciferase activity was determined using a luciferase assay kit according to the manufacturer's protocol (Boehringer Mannheim). Photon detection was performed on a Microplate scintillation/luminescence counter (Wallac). All assay were carried out in triplicate and replicated on independent occasions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

This project was supported by grants from the MRC (UK) to David Holden, and DFG (Germany) (grant no. 1964/2–1) to Michael Hensel. A.V.T. and F.C.F. were supported in part by a grant from the National Institutes of Health (AI39557). We gratefully acknowledge the use of the network service at HGMP Resource Centre, Hinxton, UK. We are grateful to Dr J. S. Gunn for providing pGPL01 and pLB02 and to Dr Carmen Beuzon for help with the sequence analysis.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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