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1 Zentralinstitut für Ernährungs- und Lebensmittelforschung (ZIEL), Abteilung Mikrobiologie, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany
2 Institute of Food Science and Nutrition, ETH Zürich, Schmelzbergstr. 7, 8092 Zürich, Switzerland
Correspondence
Thilo M. Fuchs
thilo.fuchs{at}wzw.tum.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The interaction between S. typhimurium and professional phagocytes results in a membrane-bound compartment known as the Salmonella-containing vacuole (Garcia-del Portillo, 2001; Mills & Finlay, 1998). This interference with the host vacuolar trafficking machinery ends at a stage at which accumulation of bacterial cells leads to destruction of the host cell, probably by a delayed apoptotic-like process that depends on ompR, Salmonella pathogenicity island 2 (SPI-2) and spvB (Browne et al., 2002; Richter-Dahlfors et al., 1997; van der Velden et al., 2000).
Among the most successful strategies used so far to identify S. typhimurium virulence-associated genes has been a screen of approximately 10 000 independent transposon insertions for mutants with growth defects in phagocytic cells (Fields et al., 1986). Attenuated mutants with auxotrophic requirements, altered lipopolysaccharide structure or hypersensitivity to host bactericidal activities were found to be required for full virulence in vivo, demonstrating that survival within macrophages is essential for Salmonella pathogenesis. Analysis of other attenuated mutants led to the identification of metabolic genes such as aroA (Hoiseth & Stocker, 1981) or purD (Bäumler et al., 1994), of the spv operon located on the virulence plasmid pSLT (Gulig & Doyle, 1993), and of the two-component regulatory system encoded by phoP/phoQ (Miller, 1991). PhoP/Q controls the expression of numerous genes such as pagC, which was shown to be induced up to 100-fold in macrophage phagosomes and to be required for full in vivo virulence (Alpuche-Aranda et al., 1992; Pulkkinen & Miller, 1991). Further examples of virulence genes are the recA and recBC genes involved in the repair of DNA damage (Buchmeier et al., 1993; Cano et al., 2002), several stress-induced genes such as htrA, dnaK and groEL (Bäumler et al., 1994; Buchmeier & Heffron, 1990), and rpoS, which encodes an alternative sigma factor (Fang et al., 1992). In several more recent attempts to dissect the biology of intracellularly replicating salmonellae, numerous bacterial genes have been identified that strongly affect bacterial proliferation in macrophages. These include the SPI-2 genes encoding a type III secretion apparatus (Hensel et al., 1995), and virK, rcsC, somA and mig-14, whose products are suggested to confer Salmonella resistance against specific microbiocidal mechanisms of the macrophage (Brodsky et al., 2005; Detweiler et al., 2003). A functional periplasmic superoxide dismutase, SodC, that is encoded by the inducible prophage Gifsy-2, has been demonstrated to be required for survival in phagocytes, probably by conferring protection against reactive oxygen species (De Groote et al., 1997; Figueroa-Bossi & Bossi, 1999). Many Salmonella genes required for intracellular proliferation have been identified by non-systematic in vivo screens of transposon mutants, thus favouring the isolation of strongly attenuated bacteria. Alternatively, in vivo expression technologies (IVET) and differential fluorescence induction (DFI) have successfully been applied to quantify gene expression in infected hosts (Bumann et al., 2000; Mahan et al., 1993; Valdivia & Falkow, 1997), resulting in the finding that genes such as mgtBC, responsible for magnesium uptake, or entF, involved in iron uptake, are induced within macrophages (Heithoff et al., 1999). Impaired growth in macrophages was also observed for gaiA, which is up-regulated upon Salmonella typhi invasion of eukaryotic cells (Basso et al., 2002). A more comprehensive microarray-based negative screen led to the identification of at least 62 genes contributing to long-term systemic infection (Lawley et al., 2006). A pioneering attempt in the field of microarray analysis determined the complete transcriptional profile of intracellular S. typhimurium (Eriksson et al., 2003). The impact of intracellularly up-regulated genes on virulence, however, has still to be experimentally proven. Interestingly, almost 50 % of in vivo-regulated genes are of as yet unknown function, suggesting that novel macrophage-associated determinants remain to be discovered. This assumption is further supported by the estimation that 4 % of the Salmonella genome is required for fatal infection in mice (Bowe et al., 1998); some of those as yet unknown virulence factors are expected to be identified by in vitro cell culture assays.
To complete the picture of Salmonella factors required for intracellular replication, an insertion mutant library of S. typhimurium was established and analysed for growth-deficient mutants identified in the macrophage model. Following localization of gene disruption, a striking coincidence was found between our data and ten of the 29 larger islands interspersing the collinear regions of the chromosome of salmonellae. The approach led to the identification of 33 genes located on genomic islands, most of them as yet not known to contribute to S. typhimurium replication in macrophages. The results presented here provide experimental evidence of putative roles in macrophage-associated functions for a novel group of genes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Escherichia coli and S. typhimurium were grown in liquid Luria–Bertani (LB) medium or on LB agar plates at 30 °C or 37 °C. Minimal medium (MM) was composed of 1x M9 medium (Sambrook & Russell, 2001), 2 mM MgSO4, 0.1 µM CaCl2 and 20 mM glucose. For growth studies, overnight cultures were diluted 1 : 1000 in 200 ml flasks with 50 ml medium (MM) or in 50 ml Falcon tubes with 28 ml medium (LB) and incubated at 37 °C under vigorous shaking until the cultures reached stationary phase. If appropriate, tetracycline (17.5 µg ml–1), ampicillin (100 µg ml–1) or kanamycin (50 µg ml–1) was added to the media.
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), and following the manufacturer's instructions. GeneRuler DNA Ladder Mix from MBI Fermentas was used as a marker for DNA analysis. Plasmid DNA was transformed via electroporation using a Bio-Rad Gene Pulser II as recommended by the manufacturer. The Gene Pulser settings included a voltage of 2.5 kV, a capacitance of 25 µF and a resistance of 200 
. After electroporation, cells were immediately placed into 1 ml SOC medium, incubated with shaking for 45 or 90 min at 30 or 37 °C, respectively, and plated on LB agar with the appropriate antibiotic. Products of ligation reactions with the temperature-sensitive vector pIDM1 were transformed into helper strain EC101, and cells were incubated at 30 °C for 48 h. PCRs were carried out with Taq polymerase under the following conditions: 96 °C for 4 min; 35 cycles at 96 °C for 30 s, 53 °C for 30 s, and 72 °C for 30–120 s depending on the expected fragment length; 72 °C for 8 min. As template for PCR, we used 100–400 ng chromosomal DNA, 2–40 ng plasmid DNA, or an aliquot of a single colony resuspended in 100 µl H2O.
Sequence analysis.
Fragments cloned into pIDM1 were amplified by PCR using the oligonucleotides lacZ1 (5'-CATGCCATGGAAGAGCGCCCAATAC-3') and IDM2 (5'-ATACCGTCGACCTCGAG-3') that hybridize to plasmid sequences flanking the cloned DNA. Sequencing was performed by Biolux GmbH, using oligonucleotide IDM2. The genomic location of fragments resulting in attenuated mutants was determined by BLASTN analysis (http://genome.wustl.edu) against the S. typhimurium LT2 sequence (McClelland et al., 2001). S. typhimurium (STM) gene numbers were taken from the above-indicated Salmonella genome homepage of the Washington University. The homepages of the BDGP Neural Networks (http://www.fruitfly.org/seq_tools/promoter.html) and of the HUSAR Bioinformatics Lab (http://genius.embnet.dkfz-heidelberg.de) were used for promoter and terminator predictions, and the enteric server of Washington University (http://globin.cse.psu.edu/enterix/) was used to determinate the distribution of S. typhimurium ORFs in other enterobacterial genomes.
Construction of an insertion mutant library.
We established the conditionally replicating plasmid pIDM1 (Fuchs et al., 2006), which carries a multiple cloning site, a tetracycline-resistance gene and a gene encoding the thermosensitive RepAts protein allowing replication in S. typhimurium at 30 °C, but not at 37 °C (see Fig. 1). As described recently (Knuth et al., 2004), randomly generated chromosomal fragments of S. typhimurium with lengths of 350–450 bp were ligated into pIDM1. The resulting mutagenic fragment library was amplified in helper strain EC101 at 30 °C and then transformed into S. typhimurium. Clones grown at 30 °C were individually isolated in 96-well microtitre plates containing 200 µl LB medium per well and amplified by shaking at 30 °C for 24 h. The redundancy of the fragment library was determined to be <10 % (Knuth et al., 2004). Insertion mutants were then isolated upon growth of a single colony at 37 °C in the presence of tetracycline. In a high-throughput approach, 20 µl aliquots from each well were dropped at the edge of square agar plates using a 12-channel multipipette, and the plates were tilted to allow the liquid to run down the plate. The plates were then incubated at 37 °C for 16 h. Cells from colonies representing viable insertion mutants were transferred into 150 µl LB medium with tetracycline in 96-well microtitre plates. After amplification for 16 h at 37 °C, 50 µl 50 % glycerol was added to each well, and the plates were stored at –80 °C.
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To transfer an insertion mutation into a deletion mutant, resulting in double mutants, insertion mutants were streaked out on LB agar plates in turn with and without tetracycline and grown at permissive temperature. Following several passages, the excised and intracellularly recovered plasmid was isolated and transformed into a deletion mutant to allow homologous recombination of the chimeric plasmid (Fuchs et al., 2006).
In-frame deletion mutants were constructed by the one-step method based on the phage 
Red recombinase (Datsenko & Wanner, 2000). PCR products comprising the kanamycin-resistance cassette of plasmid pKD4 including the flanking FRT sites, were generated using pairs of 70 nucleotide primers that included 20 nucleotide priming sequences for pKD4 as template DNA. Homology extensions of 50 bp overlapped 18 nucleotides of the 5' end and 36 nucleotides of the 3' end of the target gene (Link et al., 1997). For transformations into Salmonella cells harbouring plasmid pKD46, 500–1000 ng of fragment DNA was used. The deletion of each gene was verified by PCR using oligonucleotides located upstream and downstream of the deleted region, as well as by kanamycin sensitivity. Successful deletion was verified by subsequent sequencing of the target site of two mutants, by PCR spanning the deleted region using chromosomal DNA of all mutant strains as template, and by Southern blot analysis of chromosomal DNA of one mutant.
Macrophage infection assays.
Bacteria were grown overnight in LB medium with tetracycline in 96-well plates, and the OD620 was determined using the Tecan ELISA reader, with an OD620 of 0.1 corresponding to approximately 8.0x106 c.f.u. per ml. To test deletion mutants, strains were grown in 20 ml LB medium to mid-exponential phase (OD600 
0.6) and supplemented with glycerol (15 % final concentration). Aliquots of 1 ml were frozen at –80 °C. Prior to infection, samples were thawed and the number of viable bacteria was determined as c.f.u. per ml. J774A.1 macrophages were seeded at 4x105 cells per well in 24-well culture plates on the day prior to the macrophage assay. Cells were washed three times with DMEM without FCS before the experiment. Bacteria diluted in PBS were added to each well at a ratio of approximately 10 bacteria per macrophage (m.o.i.) in 200 µl DMEM, and the plates were centrifuged at 1000 r.p.m. for 5 min at room temperature to enhance and synchronize infection. Macrophages were incubated for 30 min at 37 °C and 5 % CO2 to permit phagocytosis. Extracellular bacteria were removed by three washes with PBS. DMEM supplemented with 10 % FCS and 100 µg gentamicin ml–1 was added. Gentamicin concentration was lowered to 10 µg ml–1 after 1 h of incubation. The cell cultures were microscopically examined for apoptosis at regular intervals in each assay run. At indicated time points after infection, cells were washed three times with PBS and lysed with 0.5 % deoxycholate. Using 96-well plates, dilutions were made in PBS and plated onto LB agar for enumeration of viable bacteria. Duplicate samples for each time point and strain were performed, and the mean and standard deviation for each time point was calculated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The nucleotide sequences obtained were compared to the complete genome sequence of S. typhimurium, and 177 different fragments were identified that had been inserted into the chromosome by insertional-duplication mutagenesis. Comprehensive analysis of the S. typhimurium genome led to the identification of 62 so-called non-collinear islands with respect to the E. coli genome (McClelland et al., 2001), six of them identical to the pathogenicity islands SPI-1 to SPI-6 (Hensel, 2004). Genome mapping of the mutagenic insertions resulting in at least 3.6-fold intracellular attenuation indicated that 33 of the 177 insertions are located in ten of those islands. This accumulation was revealed to be significant, since these ten islands comprise 7.1 % of all S. typhimurium genes (318 of 4489), and the 33 mutants represent approximately 18.6 % of all mutants identified in this screen. Three genes (flgE, cobS and recT) were found to be interrupted by homologous recombination via two independent fragments. The targeted genomic islands (GEIs) are GEI 0266/0307, GEI 0715/0727, GEI 1005/1056 (Gifsy-2 prophage), GEI 1171/1184, GEI 1379/1422 (SPI-2), GEI 1664/1678, GEI 2016/2058 (cob-cbi-pdu cluster), GEI 2584/2636 (Gifsy-1 prophage), GEI 2741/2767 and GEI 2770/2788, designated according to the number of their first and last genes. The proteins encoded by the loci identified in the macrophage screen were further characterized by classification into clusters of orthologous proteins (COG). The majority of vector insertions in these islands led to the identification of as yet uncharacterized genes with putative functions only (Table 2).
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). To predict the genes or operons that functionally contribute to the selected attenuated phenotype, we examined the chromosomal sites targeted by IDM for putative operon structures according to the genome annotation of S. typhimurium and to predicted promoter and terminator sequences. This analysis revealed that 16 of the genes identified by this approach are monocistronically transcribed or are the terminal gene of a putative operon (Table 2
). Nine fragments were found to be part of non-terminal, closely spaced co-directional genes that are possibly organized in operons. In five of these insertion mutants, the mutated gene is followed by an ORF that is involved in the same functional pathway, namely fimbrial biosynthesis, motility, propanediol utilization and cobalamin synthesis. In four cases we were only able to define loci rather than genes as being involved in intracellular replication (Table 2), but polar effects might be excluded for some of these mutants in which the outward-directed lac promoter might rescue the transcription of genes located downstream of the insertion (Merlin et al., 2002). A biased result was obtained for one fragment overlapping the ORFs of two neighbouring genes that are transcribed in opposite directions.
All but one of the mutants show wild-type-like growth behaviour in vitro
To rule out the possibility that the mutants had general growth defects instead of deficiencies in intracellular proliferation, they were grown in LB medium at 37 °C. The derived in vitro growth curves showed that all but one of the tested insertion mutants behaved like the control strain 14028-phoN628 : : pIDM1 (Fig. 1a). One strain with an insertion in gene STM1664 showed severe growth deficiency in LB medium and was therefore not further considered for analysis of intracellular behaviour. Possible auxotrophies could be excluded due to wild-type-like growth of all insertion mutants in minimal medium (data not shown).
Validation of the screen
Five strains with insertions in the SPI-2 genes ssaB, sseE, ssaN, ssaO and ssaS validated the screening method applied (Kuhle & Hensel, 2004) and were not further investigated. Three of the genes listed in Table 2, namely virK, gipA and STM2745, have been described previously as affecting the virulence or intracellular replication properties of S. typhimurium (Brodsky et al., 2005; Detweiler et al., 2003; Lawley et al., 2006; Stanley et al., 2000), again confirming the present results. We have shown recently that pIDM1 inserted into the chromosome is not excised during a macrophage assay at 37 °C and in the absence of tetracycline, indicating that insertion mutants are as stable as transposon mutants (Fuchs et al., 2006). In all cell culture experiments with immortalized J774A.1 murine macrophages, 14028-phoN : : pIDM1 was the reference for wild-type behaviour. We assumed that part of the intracellular growth defect observed in the mutants is due to a non-specific effect derived from the chromosomal insertion of the large thermosensitive vector and its tetracycline-resistance gene (Abromaitis et al., 2005). Therefore, we tested the replication properties of 14 randomly chosen mutants with pIDM1 inserted at unknown chromosomal sites, and the average of the data obtained was used as an appropriate control. As shown in Fig. 2, the replication rate of the control group was only slightly lower with respect to the wild-type (96.6 % versus 100 %). The insertion mutants were then screened in a 24-well plate macrophage assay (Buchmeier & Heffron, 1989) to quantify the relevance of the mutated genes for intracellular replication. To exclude the possibility that intracellular growth defects are a result of attenuated adhesion or invasion properties of the Salmonella mutants, bacteria were also quantified 1 h post-infection. As a control, we used a strain with an insertion in the promoter region of ratA for which only 0.92 % of intracellular bacteria with respect to the wild-type strain were recovered 1 h after infection. The data indicate that this mutant is severely deficient in its invasion properties, possibly due to a polar effect on the transcription of ratB, which is known to be involved in caecum colonization (Kingsley et al., 2003). A remarkable deviation from the wild-type behaviour comparable to that of 14028-ratA : : pIDM1 could not be observed for any of the mutants described here (data not shown).
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and Fig. 2. The degree of intracellular attenuation ranges from 3.5 % to 33.8 % with respect to the wild-type, showing a highly variable effect of single knockout mutations on the intracellular phenotype of S. typhimurium. A fivefold and tenfold degree of intracellular attenuation was observed for the virK and gipA knockout mutants (Table 2). Interestingly, several insertion mutants exhibited an intracellular proliferation rate (
13.1 %) similar to or below that of the control strain 14028-phoP : : pIDM1 in which the virulence gene phoP is disrupted (10.3±2.3 %). Amongst others, those strongly attenuating insertions were identified in three genes of the Gifsy-2 prophage, including STM1033, encoding a putative Clp-like protease, in STM1677, encoding a LysR-like transcriptional regulator, and in STM2633 of the Gifsy-1 prophage, which encodes a protein with similarity to enterohaemolysin 1 of E. coli (Fig. 2). We also compared our data to the expression profile recently derived for intracellular-replicating Salmonella strains (Eriksson et al., 2003) and found that only safC was identified to be significantly up-regulated (4.73-fold) during intracellular replication. In addition, we determined a three- to fivefold diminished intracellular growth for two flagellar mutants (flgE and flgH), thus confirming former results that flagella mutants are more sensitive to intracellular killing mechanisms (Bäumler et al., 1994; Weinstein et al., 1984). Our further analysis of genomic islands involved in intracellular proliferation was then focused on GEI 0266/0307, GEI 1005/1056 (Gifsy-2 prophage), GEI 1664/1678, GEI 2016/2058 and GEI 2584/2636 (Gifsy-1 prophage) because we had identified at least two attenuating insertion mutants in each of these islands (Table 3). A significant deviation from the mean chromosomal G+C content of 53 mol%, characteristic of pathogenicity islands, was found in the case of GEI 1664/1678 exhibiting a G+C content of 47.2 mol%.
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Red recombinase system (Datsenko & Wanner, 2000) to construct seven non-polar deletions affecting nine genes of S. typhimurium identified in our screen (Table 2). The genes chosen are representatives of the five genomic islands of interest, and at least four of the target genes are assumed to be transcribed polycistronically. A phoP deletion mutant, 14028
phoP, was used as a control and showed a tenfold reduction of intracellular replication compared to the wild-type (9.5±8.8 % from 13 independent experiments). In one strain, two identified genes (STM2603 and STM2605), probably located in an operon within the Gifsy-1 prophage, were deleted simultaneously with STM2604, thus generating a triple deletion. No growth deficiencies in comparison with the wild-type strain could be observed (Fig. 1b). In all but one case, the results of the cell culture experiments confirmed that the deleted genes are indeed responsible for the observed attenuated phenotypes (Fig. 3). The replication rate of the safC deletion mutant did not validate the attenuation of the corresponding insertion mutant, possibly due to polar effects on safD. This finding is reminiscent of a similar phenotype of a sefD mutant of S. enteritidis, suggesting a role in intracellular infection (Edwards et al., 2000). Other discrepancies between the results of both assays, e.g. in the case of STM1042, cobS or STM2603–2605, might be attributed to a polar effect of the insertion on downstream-located genes as mentioned in Table 2.
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). The resulting mutant, 14028-pocR385 : : pIDM4, showed an intracellular growth rate 8.3-fold lower than that of the wild-type strain (Fig. 2), suggesting a role for PocR in Salmonella virulence.
Double mutants
Several as yet uncharacterized genes co-localized on genomic islands have been shown in this study to be important for S. typhimurium proliferation in macrophages. The attenuation rate of a few mutants, especially in GEI 0266/0307 and in the cob-cbi-pdu cluster (GEI 2016/2058), fell short of a critical threshold of approximately fivefold that might correlate with detectable in vivo attenuation (Table 2). However, additive effects are expected if more than one cellular function encoded on such a genomic island is eliminated. To test this hypothesis, an additional insertion mutation was introduced into four deletion mutants, resulting in mutants lacking two transcriptionally independent gene products of three selected genomic islands (Table 4). These double mutants were then tested for their proliferation ability in macrophages. Statistical exploration of the data obtained revealed a significant increase of attenuation for all but one double mutant when compared to the respective single mutants, indicating that genes responsible for independent functions have been affected (Beuzón et al., 2001). No such additive effect could be observed in the case of strain 140281677/1672 : : pIDM1, indicating that both genes are involved in the same function, and that the regulatory factor encoded by STM1677 might influence the transcription of STM1672.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Mei et al., 1997; Merrell & Camilli, 2000) and are known to play a role in virulence. Examples from our study demonstrating the important role of nutrient acquisition for intracellular growth are cobS, catalysing the last step of the synthesis of adenosylcobamide, as well as pduK and pocR. The latter two genes are involved in the vitamin B12-dependent degradation of 1,2-propanediol or ethanolamine as an alternative source of carbon, nitrogen and energy (Rondon et al., 1995). IVET techniques have indicated that pdu genes may be important for growth in host tissues (Heithoff et al., 1999), and competitive index studies with mice have shown that pdu mutations, but not cob mutations, are responsible for a virulence defect (Bjorkman et al., 1996; Conner et al., 1998). Ethanolamine-utilization mutants of S. typhimurium were attenuated in macrophages and BALB/c mice when administered per os but not when injected intraperitoneally (Stojiljkovic et al., 1995). Only recently, eutB was shown to be required for Listeria monocytogenes growth in epithelial cells (Joseph et al., 2006). The 10-fold decrease of intracellular replication of a pocR mutant that is affected in both the control of vitamin B12 synthesis and propanediol degradation (Bobik et al., 1992) demonstrates that the cob-cbi-pdu gene cluster increases the intracellular fitness of Salmonella. This finding provides a solution to the B12 paradox that although nearly 1 % of the S. typhimurium genome is involved in cobalamine metabolism, no laboratory growth phenotype of mutants defective in B12 synthesis has been observed (Roth et al., 1996). Interestingly, both the eut and the pdu operons are repressed in a mutant of csrA, which encodes the CsrA regulator of Salmonella pathogenicity (Lawhon et al., 2003), and a fivefold abundance of Pdu proteins of strain 14028 in comparison with the less virulent variant LT2 was detected in acidic minimal medium (Adkins et al., 2006). Propanediol and ethanolamine are abundant compounds in the human intestine, and because their utilization pathways are also present in the genomes of L. monocytogenes and Clostridium perfringens, these genetic determinants were predicted to be associated with food poisoning (Korbel et al., 2005). During intracellular replication, these two substrates might derive from phospholipase activity on fucose glycoconjugates and phosphatidylethanolamine as components of the host cell membrane (Fig. 4).
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). This is in line with the result of a microarray-based negative screen that identified 15 S. typhimurium phage genes as being involved in long-term systemic infection of mice (Lawley et al., 2006). Gifsy-2 functional prophages have already been shown to play a role in bacterial virulence, since curing of Gifsy-2 prophage significantly reduces the ability of Salmonella to establish a systemic infection in mice (Figueroa-Bossi et al., 2001). The sodC gene alone, encoding a periplasmic superoxide dismutase, was not able to restore virulence of the prophage-cured strain. Interestingly, a novel virulence factor gtgE, a synonym of STM1055, was recently shown to be activated by SlyA, which is also involved in the regulation of virulence genes such as mig-14, sopD2 and virK (Brumell et al., 2003; Ho et al., 2002; Navarre et al., 2005). Here, we provide evidence that at least four other Gifsy-2 prophage genes, among them STM1033 encoding a putative Clp-like protease, contribute to wild-type like replication of salmonellae within macrophages and possibly also to in vivo virulence of salmonellae.
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). Our data indicate that despite their lack of relevance for systemic infection, other factors encoded by the Gifsy-1 prophage beside GipA, among them a PagK homologue and a putative enterohaemolysin, play an important role in wild-type-like replication of S. typhimurium in murine macrophages (Fig. 5). Gene STM2633 encoding RecT has a functionally equivalent allele on the Gifsy-2 genome, and the deletion of both alleles from the chromosome might result in an avirulent S. typhimurium mutant as postulated earlier (Figueroa-Bossi & Bossi, 1999).
Genomic islands 0266/0307 (SPI-6) and 1664/1678
pIDM1 insertions into four genes of genomic island 0266/0307 (termed SPI-6 or SCI) led to significant reduction of S. typhimurium replication in J774A.1 cells. Deletion of the entire SPI-6 locus had no effect on systemic Salmonella pathogenesis, but led to a reduced invasion of cultured cells (Folkesson et al., 2002). Several putative virulence-associated factors encoded by genes located on GEI 0266/0307, such as a shiga-like toxin A subunit, a homologue of PagN possibly involved in invasion (Folkesson et al., 1999), or a homologue to Shigella VirG protein, might contribute to this phenotype. According to our data, a putative ClpB-like protease encoded by STM0272 also plays a role in intracellular replication. Protease genes are present in pathogenicity islands (Hacker & Kaper, 2000; Schmidt et al., 2001) and are known to be involved in resistance mechanisms to antimicrobial peptides (Peschel, 2002). While the island is restricted to Salmonella spp., STM0272 is highly conserved among Gram-negative bacteria. Since salmonellae have to modulate the expression of virulence-associated genes in response to their specific environmental niche, regulators play an important role in intracellular growth. Two of the intracellularly attenuated mutants were found to carry vector insertions in genes encoding as yet uncharacterized transcriptional regulators, namely SinR in SPI-6 and the product of STM1677 located on GEI 1664/1678 with homology to LysR-like proteins known to regulate virulence in different pathogens (Finlay & Falkow, 1997). Their cellular function remains to be disclosed.
Concluding remarks
It is well documented that an intracellular phase of S. typhimurium is associated with virulence (Fields et al., 1986; Miller, 1995), and optimal growth of S. typhimurium was observed in the murine macrophage-like cell line J774A.1 (Buchmeier & Heffron, 1989). Although tissue-culture assays do not exactly resemble in vivo conditions (Hurley & McCormick, 2003), the replication of S. typhimurium mutants in macrophages was found to have the clearest correlation with attenuated bacterial virulence in animal models of systemic infection (Bowe et al., 1998; Leung & Finlay, 1991). Bowe et al. (1998) estimated a minimum of 4 % of the S. typhimurium genome to be required for fatal infection in mice, corresponding to a total of 180 virulence genes. Thus, it can be expected that some of the identified IDM mutants are avirulent in the mouse model, especially those that show an intracellular replication rate of less than 13 %, the stringent threshold of attenuation of a phoP mutant in tissue culture of J774A.1 cells. Although several of the genes listed in Table 2 confer more subtle growth advantages on intracellular salmonellae (Moors & Portnoy, 1995), we propose that the additive effect of such small contributions might have a significant impact on fitness within the host, as suggested by the increased attenuation of three double mutants (Table 4).
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ACKNOWLEDGEMENTS |
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Edited by: P. H. Everest
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