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1 Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK
2 Molecular Microbiology Group, Institute of Food Research, Norwich NR4 7UA, UK
3 School of Clinical Veterinary Sciences, University of Bristol, Langford House, Langford, Bristol BS40 5DU, UK
Correspondence
Clare E. Bryant
ceb27{at}cam.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work.
Present address: Division of Microbial Diseases, UCL Eastman Dental Institute, University College London, 256 Gray's Inn Road, London WC1X 8LD, UK.
Present address: School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
||Present address: School of Genetics and Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland.
The ArrayExpress accession numbers for the complete gp91–/–, TLR4–/– and hydrogen peroxide datasets associated with this paper are E-MEXP-1089 and E-MEXP-1067.
Five supplementary tables are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). A crucial aspect of the pathogenicity of invasive Salmonella infections is the ability of the pathogen to invade and survive within phagocytes (Fields et al., 1986). Following phagocytosis of a pathogen by a macrophage, and its compartmentalization into a phagosome, a sequence of antibacterial processes is initiated in the host cell. These include superoxide production, catalysed by NADPH oxidase, and nitric oxide production, catalysed by inducible nitric oxide synthase, acidification of the phagosome through the action of the vacuolar ATPase, and fusion with lysosomes to deliver hydrolases and cationic antimicrobial peptides. These processes generate a highly bactericidal environment within the phagosome (Steele-Mortimer, 2008; Haas, 2007). Following internalization S. Typhimurium becomes compartmentalized within a phagosome which fails to completely mature, known as the Salmonella-containing vacuole (SCV). S. Typhimurium subverts the phagosomal maturation pathway through prevention or delay of fusion of selected endosomes harbouring antimicrobial components with the SCV (Steele-Mortimer, 2008; McCollister & Vazquez-Torres, 2006). Effectors delivered by type III secretion systems encoded by Salmonella pathogenicity islands (SPI) 1 and 2 are involved in regulating SCV biogenesis, with SPI2 effectors being required for intra-macrophage replication and systemic infection of mice (Waterman & Holden, 2003). However, knowledge of the molecular details of the exact actions of SPI effectors, and the extent to which individual effector proteins subvert the phagosomal pathway, is currently limited (Steele-Mortimer, 2008).
During infection, the cellular components of bacteria are recognized by the innate immune system via specific ligand–receptor binding. This involves host cell pattern recognition receptors including Toll-like receptors (TLRs) (Akira et al., 2006). There are 12 known TLRs across different species, which are capable of binding to specific pathogen-associated molecular patterns (Akira et al., 2006). One of these is TLR4, which is a key component of the innate immune response to Gram-negative bacteria (Takeuchi et al., 1999; Lembo et al., 2003) and is critical in controlling S. Typhimurium infections in mice (Weiss et al., 2004). Mice lacking active TLR4 are unable to suppress bacterial growth in the spleen and liver, and exhibit substantially reduced survival when experimentally infected with S. Typhimurium (O'Brien et al., 1980; Vazquez-Torres et al., 2004; Eisenstein et al., 1982). TLR4 recognizes the lipid A portion of bacterial LPS through cooperation with three murine accessory proteins: LPS-binding protein (LBP), CD14 and MD-2 (Teghanemt et al., 2007; Kim et al., 2007).
Infection of macrophages with S. Typhimurium induces a similar pattern of changes in host inflammatory response and antimicrobial gene expression to that seen when macrophages are stimulated by LPS (Rosenberger et al., 2000). Thus it is likely that during Salmonella infection it is predominantly LPS that activates the macrophage inflammatory response, establishing production of inflammatory cytokines such as TNF-
and antibacterial mediators such as nitric oxide (Rosenberger et al., 2000; Royle et al., 2003). In addition, TLR4 and the phagosomal NADPH oxidase are functionally linked: the oxidative burst is elicited in monocytes in response to LPS in a TLR4-dependent manner (Ryan et al., 2004) and LPS stimulation activates NADPH oxidase via IRAK-4 (Pacquelet et al., 2007).
The host cell response to S. Typhimurium infection is increasingly well understood, but the responses of the infecting salmonellae to the changing macrophage environment remain poorly characterized. Changes in bacterial gene expression may occur as a consequence of detecting the hostile intracellular environment generated by the host response to infection. It is important therefore to understand how the bacteria respond to changes in the host cell environment. Transcriptome profiling of S. Typhimurium during infection of J774-A.1 macrophage-like cells, and of S. Typhi in THP-1 macrophages, has revealed large changes in gene expression compared to in vitro growth, giving an indication of how S. enterica responds to the intracellular environment (Eriksson et al., 2003; Faucher et al., 2006).
In this study we used primary bone marrow-derived macrophages (BMDMs) from knockout mice deficient in either TLR4 or gp91 NADPH oxidase to determine whether the alteration of the intracellular environment by these key components of the innate immune system in turn leads to gene expression changes in S. Typhimurium living inside these cells. We discovered that there were changes in the expression of many S. Typhimurium genes involved in resistance to the oxidative burst following TLR4 signalling. The fact that the deletion of any one of these genes individually does not cause attenuation in murine infection models suggests that S. Typhimurium possesses redundant mechanisms that facilitate resistance to the initial innate immune response and allow subsequent persistence needed to establish systemic infection.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and gp91–/– knockout mice (lacking NADPH oxidase activity: Pollock et al., 1995) were bred at the University of Cambridge under Specific Pathogen Free conditions.
Bacterial strains, media and reagents.
The S. Typhimurium strain SL1344 (Hoiseth & Stocker, 1981) and its parental strain 4/74 (Jones et al., 1991; Rankin & Taylor, 1966) were used in this study. These two strains only differ in the hisG mutation carried by SL1344. Both strains are virulent in a variety of animals (Villarreal-Ramos et al., 2000; Paulin et al., 2007). S. Typhimurium was grown in Luria–Bertani (LB) broth or on LB agar at 37 °C. The bacterial strains used in this study are summarized in Table 1.
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). For infection experiments leading to RNA isolation, BMDMs were plated onto 6-well plates at a density of 2x106 cells per well and infected with S. Typhimurium SL1344 (opsonized for 30 min at 37 °C with 10 %, v/v, normal mouse serum) at an m.o.i. of 200 : 1. To minimize SPI1 expression, which could lead to macrophage apoptosis (Monack et al., 1996), bacteria were grown overnight on LB plates at 37 °C and suspended in PBS before opsonization (Eriksson et al., 2003). After 1 h of phagocytosis, extracellular bacteria were killed with 30 µg gentamicin ml–1 for 1 h followed by further incubation with 5 µg gentamicin ml–1.
RNA extraction.
At 2 h or 4 h post-infection, infected macrophages were lysed on ice for 30 min in 0.1 % SDS, 1 % acidic phenol and 19 % ethanol in water as described previously (Eriksson et al., 2003; Hinton et al., 2004). S. Typhimurium cells were pelleted after centrifugation and RNA was prepared using the Promega SV and Ambion RiboPure-Bacteria total RNA purification kits. Bacterial RNA was further purified by phenol/chloroform extraction. Each sample yielded 5–10 µg of total bacterial RNA.
Peroxide stress conditions for transcriptomic analysis.
A 25 ml overnight culture of S. Typhimurium 4/74 was diluted 1000-fold in flasks containing 25 ml LB broth and grown to OD600 0.1. Hydrogen peroxide was added to half of the flasks at a final concentration of 1 mM and the other half were used as controls. The cultures were allowed to grow for a further 12 min before samples were taken for transcriptomic analysis (Nagy et al., 2006). Triplicate biological replicates were performed (Porwollik et al., 2003).
Microarray procedures.
Transcriptomic techniques involved S. Typhimurium microarrays constructed at the Institute of Food Research, Norwich, UK, as described previously (Nagy et al., 2006; Lucchini et al., 2005). All transcriptomic analyses were defined using microarrays covering 4193 genes, 92 % of which are common between S. Typhimurium strains LT2 and SL1344 (Kelly et al., 2004).
Probe preparation and scanning.
The yield of bacterial RNA extracted from infected BMDMs was relatively low; therefore RNA was first reverse-transcribed into cDNA and subsequently labelled by random priming with the Klenow fragment of DNA polymerase I according to the ‘Labelling protocol for reduced amounts of RNA’ described at http://www.ifr.ac.uk/Safety/MolMicro/protocols.html. Fluorescently labelled genomic DNA was used as a reference channel for each experiment. Slides were scanned on an Axon 4000B scanner (Axon Instruments) using GenePix version 1.4 software (Axon Instruments). Each hybridization was performed twice. Two biological replicates were performed for the wild-type and TLR4–/– BMDM, and three for the gp91–/– BMDM.
Data analysis.
Data analysis was performed as described by Eriksson-Ygberg et al. (2006). Spots showing a reference signal lower than background plus two standard deviations, or obvious blemishes, were excluded from subsequent analyses. Local background was subtracted from spot signals, and fluorescence ratios were calculated. To compensate for unequal dye incorporation or any effect of the amount of template, data centring was performed by bringing the median natural logarithm of the ratios for each group of spots printed by the same pin to zero. In a few cases, when comparing results from different hybridizations, we observed slight deviations which were dependent on gene expression levels. These were corrected using the Loess function in R (Limma package). Differentially expressed genes were identified by a Perl implementation of the rank products (RP) method (Breitling et al., 2004), which performs well with limited numbers of samples or datasets that exhibit some biological noise (Jeffery et al., 2006). The log fold-changes for all pair-wise comparisons between the two sample classes (BMDM and gp91–/– BMDM, or BMDM and TLR4–/– BMDM) were used to calculate the RP values. The false discovery rate (FDR) for each gene was determined by using 1000 random permutations of the data to assign significance to those RP values. This was only used to analyse the TLR4 and NADPH oxidase data. Forty-nine and 36 genes were up- and downregulated, respectively, in wild-type BMDM compared with gp91–/– BMDM samples, and 44 and 28 genes up- and downregulated, respectively, in wild-type BMDM compared with TLR4–/– BMDM samples, with significance defined as an FDR of 
1 %. The complete dataset is available as supplementary material with the online version of this paper (Supplementary Tables S1 and S2). For the hydrogen peroxide data, an ANOVA test with a Benjamini and Hochberg FDR of 0.05 was used to identify genes that were significantly differentially expressed by strain 4/74 between LB and LB plus peroxide. Data that passed the quality controls were analysed using Genespring version GX7.3 software (Agilent). The complete gp91–/–, TLR4–/– and hydrogen peroxide datasets have been submitted to ArrayExpress with the accession numbers E-MEXP-1089 and E-MEXP-1067.
Construction of S. Typhimurium mutants.
Chromosomal deletion mutants of S. Typhimurium SL1344 were constructed using the one-step gene-disruption technique (Datsenko & Wanner, 2000; Mo et al., 2006). Primers listed in Supplementary Table S3 were used to amplify gene cassettes encoding resistance to chloramphenicol (from pKD3 or pACYC184) or kanamycin (from pKD4 or pACYC177) with the addition of 5' and 3' homology arms complementary to the flanking regions of the chromosomal gene to be deleted at either end of the appropriate resistance cassette. SL1344 was transformed with pBAD
red carrying the phage lambda genes exo, bet and gam, induced with 0.2 % (w/v, final concentration) arabinose, followed by electroporation of the mutagenic PCR products and selection with the appropriate antibiotic. Allelic replacement was verified by a test PCR using primers listed in Supplementary Table S4. After DNA sequencing to confirm the sequence at the junction of the antibiotic resistance cassette and the disrupted gene, the mutations were moved by P22 bacteriophage transduction (Schmieger, 1972) into a clean SL1344 background and verified by colony PCR, or alternatively cured of pBADred by serial passage, and loss of plasmid confirmed by testing for ampicillin resistance and by attempted plasmid purification.
Infection of mice.
Bacteria were grown overnight at 37 °C as a static culture in LB medium, then washed and diluted in PBS to obtain 5x103 c.f.u. ml–1. Aliquots (200 µl, 103 c.f.u.) of this bacterial suspension were injected into the tail veins of six- to eight-week-old mice. Appropriate dilutions of the inoculum were plated on LB agar for enumeration of the number of viable bacteria given to the mice. At each time point post-infection, four mice were killed by cervical dislocation and spleens and livers were removed. The organs were placed into 10 ml sterile distilled water and homogenized in a Colworth stomacher for subsequent determination of viable bacterial counts by plating out appropriate dilutions of the homogenates on LB agar.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). We used a transcriptomic approach to determine the impact of TLR4 activation upon salmonellae infecting macrophages in culture. We compared the gene expression profiles of intracellular S. Typhimurium infecting BMDMs from either wild-type or TLR4–/– mice. Expression levels of 72 S. Typhimurium genes were significantly different when infections in wild-type BMDMs were compared to those in TLR4–/– BMDMs at a false discovery rate (FDR) of 0.01, suggesting that these genes are specifically differentially regulated in response to TLR4 signalling (Supplementary Table S1). Forty-four of these genes were expressed at higher levels and 28 of them were expressed at lower levels in wild-type macrophages when compared to the TLR4-deficient cells. The list of differentially expressed genes was limited further by the application of a stringent fold-change cut-off of 3, in order to focus solely on genes with substantially different expression levels during infection of the wild-type and TLR4–/– BMDMs. The expression of 17 genes was altered more than 3-fold, and all of these genes were more highly expressed in the wild-type compared to the TLR4–/– BMDMs (Fig. 1a, Supplementary Table S1). For convenience we designate these as ‘TLR4-inducible’ S. Typhimurium genes. A number of these protect the bacterium from oxidative damage in vitro, including katG (catalase), ahpCF (alkyl hydroperoxide reductase), dps (DNA-binding protein in stationary phase) (Janssen et al., 2003) and sufABCDSE (repair of oxidatively damaged [Fe–S] clusters) (Nachin et al., 2003). Infection studies have shown that dps, for example, is required for bacterial survival inside macrophages and for full virulence of S. Typhimurium in mice (Halsey et al., 2004).
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). Many of the S. Typhimurium genes that respond to both TLR4 and NADPH oxidase protect against oxidative damage, including ahpCF, dps and sufABC (Zheng et al., 2001; Storz et al., 1989; Porwollik et al., 2003; Nachin et al., 2003; Lee et al., 2004; Janssen et al., 2003; Halsey et al., 2004). These data link the antibacterial effect of TLR4 in the phagosome with NADPH oxidase and are consistent with the literature suggesting a role for TLR4 in the regulation of this enzyme (Ryan et al., 2004; Pacquelet et al., 2007).
Identification of eight TLR4-inducible, NADPH oxidase-inducible genes that respond to peroxide in vitro
We hypothesized that the ‘TLR4-inducible’ and ‘NADPH oxidase-inducible’ genes would contribute to resisting environmental stress. Many of the bactericidal effects of NADPH oxidase are mediated by hydrogen peroxide, making it a useful in vitro mimic of NADPH oxidase, albeit with limitations. We therefore determined whether the 21 NADPH oxidase-inducible genes were also induced by hydrogen peroxide in vitro. S. Typhimurium was grown to mid-exponential phase and subjected to a short treatment with hydrogen peroxide. Expression of 309 and 428 genes was up- or downregulated more than 3-fold, respectively, in response to hydrogen peroxide (Supplementary Table S5). Twelve genes were ‘NADPH oxidase-inducible’, but not peroxide-inducible and eight genes were ‘TLR4-inducible’ but not peroxide-inducible (Fig. 1d and Supplementary Tables S1 and S2). In contrast, nine genes (ahpC, ahpF, katG, dps, sufA, sufB, sufC, trxC and mntH) were induced by both peroxide in vitro and intracellular NADPH oxidase during macrophage infection, and all but one (mntH) of these were also upregulated in the presence of TLR4 (Fig. 1b, c, d; Supplementary Table S1).
Deletion of individual TLR4- and NADPH oxidase-inducible genes results in sensitivity of S. Typhimurium to oxidative stress in vitro
We made deletion mutants of genes that were both NADPH oxidase-inducible and TLR4-inducible and characterized the resistance of the resulting mutants to hydrogen peroxide stress in vitro. We focused initially on the katG, dps, sufA and STM0561 genes, which were all induced more than 3-fold in the presence of NADPH oxidase. The katG, dps and sufA genes were also most strongly induced by hydrogen peroxide in vitro (673-, 104- and 88-fold, respectively; Supplementary Table S5). The addition of peroxide to the S. Typhimurium parent strain SL1344 caused an initial approximately 10-fold reduction of bacterial numbers from an initial inoculum of approximately 5x104 c.f.u. ml–1 and prevented bacterial growth for approximately 6 h (Fig. 2a). The four mutants showed substantial reductions in viable counts upon addition of hydrogen peroxide (Fig. 2b–e). The strains lacking katG or STM0561 were killed by exposure to hydrogen peroxide for 45 min (Fig. 2b, e). S. Typhimurium lacking dps was killed by 200 min of hydrogen peroxide challenge. In contrast, bacteria lacking sufA showed a rapid decrease in bacterial numbers over the initial 3 h of the experiment, followed by a stabilization to around 5x102 c.f.u. ml–1 over the next 5 h of the experiment (Fig. 2d). At 20 h post-challenge, this strain had managed to grow and had apparently entered stationary phase (data not shown).
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). In total 12 mutant strains, carrying deletions in 14 of the 17 TLR4-inducible genes, and 9 of the 10 genes that were both TLR4- and NADPH oxidase-inducible, were constructed. All the S. Typhimurium mutants grew similarly to the SL1344 wild-type in LB broth and on LB agar (data not shown). We infected C57BL/6 mice intravenously with each of the S. Typhimurium mutants. Only the S. Typhimurium dps mutant had reduced growth in spleen and liver (Fig. 3a–d). Lower spleen and liver viable counts compared with those seen using SL1344 were also observed after infection of gp91–/– mice with the dps mutant, suggesting that Dps contributes to resistance to other control mechanisms in addition to oxidative stress (Fig. 3e, f). None of the other deletion mutants exhibited a significantly reduced bacterial load in either spleen or liver.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Through the comparison of S. Typhimurium gene expression in TLR4–/– and wild-type BMDMs, we identified 17 genes that were clearly induced in response to the effects of TLR4 signalling during macrophage infection. Several of these genes have been ascribed functions related to resistance to oxidative damage. These include dps, katG, ahpC and ahpF, and four components of the suf operon (Buchmeier et al., 1995; Halsey et al., 2004; Lee et al., 2008; Poole & Ellis, 1996; Taylor et al., 1998). The induction of multiple genes known to protect S. Typhimurium against oxidative stress could reflect an intracellular environment rich in oxygen radicals, and suggests that TLR4 contributes to this environment.
There is a substantial degree of overlap between the S. Typhimurium genes induced in response to TLR4 and those induced in response to active NADPH oxidase (10 out of 17 TLR4-inducible genes). This suggests a functional link between these two components of the innate immune system. Our identification of eight genes induced in response to TLR4 and NADPH oxidase that also respond to hydrogen peroxide in vitro suggests that TLR4 contributes to oxygen radical generation in macrophages. The involvement of TLR4 in production of reactive oxygen species remains contentious. Previous experiments have shown that TLR4-deficient macrophages are able to elicit the same level of early killing of S. Typhimurium as wild-type peritoneal macrophages (Cook et al., 2007). This period corresponds to the ‘NADPH oxidase-mediated’ phase, and at 1 h after infection no measurable difference in hydrogen peroxide or superoxide was detected in TLR4-deficient cells (O'Brien et al., 1980; Vazquez-Torres et al., 2004; Eisenstein et al., 1982). Peritoneal macrophages lacking the TLR adaptor protein, MyD88, exhibit impaired killing of S. Typhimurium and a deficiency in NADPH oxidase-mediated oxygen radical generation. This defect in production of reactive oxygen species was not observed in TLR4-deficient macrophages (Laroux et al., 2005). However, LPS is known to elicit a TLR4-dependent oxidative burst in THP-1 monocyte-like cells and BMDM (Ryan et al., 2004). More recent data have shown that IRAK-4, a regulatory kinase that operates downstream of TLR4, phosphorylates the p47phox subunit to activate NADPH oxidase in response to LPS in human neutrophils (Pacquelet et al., 2007). The data that we present here support the idea that there is a link between TLR4 signalling and NADPH oxidase activity in murine BMDMs.
We constructed defined deletion mutants of a number of NADPH oxidase-inducible genes and tested their hydrogen peroxide sensitivity in vitro, revealing that under these conditions katG, dps, sufA and STM0561 are apparently required for full resistance to peroxide-induced stress. For three of these four mutant strains (katG, dps and STM0561), hydrogen peroxide was completely bactericidal, with no viable bacteria recovered after 200 min of challenge. These mutants were not complemented, and thus it cannot be definitively stated that the observed hydrogen peroxide sensitivity is due to deletion of these genes, and not some polar effect or secondary site mutation. However, in the case of the Dps- and catalase-deficient mutants, these results are consistent with published data (Buchmeier et al., 1995; Halsey et al., 2004). Deletion of the suf operon in Escherichia coli results in increased sensitivity to various superoxide generators but not to hydrogen peroxide, whereas hydrogen peroxide sensitivity was observed in S. Typhimurium suf mutants (Lee et al., 2008). STM0561 is a gene of unknown function; the inferred amino acid sequence has 74 % identity to a 104 residue stretch of E. coli CusS, a putative two-component sensor histidine kinase involved in copper-inducible expression of a potential copper ion anti-porter (Munson et al., 2000). However, this identity only covers the histidine kinase domain of CusS, so while it is probably safe to assume that this protein is involved in bacterial signal transduction nothing can be assumed about what it is responding to in the environment.
The sensitivity of S. Typhimurium to hydrogen peroxide in vitro only mimics part of the oxidative stress that bacteria are exposed to in vivo. The ability of pathogens to resist oxidative stress involves multiple genes with redundant or complementary functions, and their role in Salmonella virulence has proven to be difficult to dissect. However, the role of three oxidative stress-responsive genes has been defined. The heat-shock protein HtrA is involved in resistance to oxidative stress in vitro, and htrA mutants are attenuated in mice but cause a lethal infection in animals lacking a functional NADPH oxidase (Mutunga et al., 2004). Thioredoxin 1, encoded by trxA, promotes intracellular replication and virulence of S. Typhimurium, but thioredoxin 2, encoded by trxC, does not play a significant role during S. Typhimurium infection (Bjur et al., 2006). SodCI mutants lacking a Cu/Zn-superoxide dismutase are slightly attenuated in mice, as measured by reduced mortality after intraperitoneal infection in Nramp-resistant mice. It should be noted however that the mutants were still able to kill approximately 50 % of the genetically resistant mice infected at the dose used, indicating that there is no absolute requirement for SodC1 for murine infection with S. Typhimurium (Ammendola et al., 2008).
In this study, deletion of many of the TLR4- and NADPH oxidase-inducible genes, alone or in combination, did not cause significant attenuation in intravenous murine infections. Only deficiency in dps resulted in reduced bacterial loads in the spleens and livers of infected mice when compared to wild-type SL1344 infection. This lack of attenuation of mutants lacking oxidative stress response genes has also been observed elsewhere, for example with katG, ahpC and grxA (Boyer et al., 2002; Buchmeier et al., 1995; Taylor et al., 1998). No global mutagenesis approach has yet identified a role for individual oxidative stress response genes in murine infection by S. Typhimurium (Hensel et al., 1995; Lawley et al., 2006).
Our findings illustrate the strength of global gene expression profiling as a means to investigate the response of an intracellular pathogen to specific innate immune responses. This approach has revealed a complete and subtle set of information that illustrates the complexity of the bacterial response to host innate immunity. The response of S. Typhimurium to the innate immune mechanisms encountered within the phagosome is complex, and involves induction of numerous genes with seemingly redundant functions. This dataset would not have been arrived at if a simple single gene mutagenesis method had been used to investigate these complex pathways. Moreover, some of the genes identified clearly play a role in resisting other host resistance mechanisms. For instance, the mutants in the NADPH oxidase-responsive genes exhibit strong peroxide sensitivity in vitro, yet only one, dps, is significantly attenuated in vivo. Interestingly infection of gp91–/– mice with the S. Typhimurium dps mutant also resulted in recovery of reduced numbers of bacteria compared to the numbers recovered from infection with the wild-type S. Typhimurium strain, especially in the spleen, suggesting that Dps does not simply protect against NADPH oxidase. Dps also binds to and coats DNA (Almiron et al., 1992) and it is thought thus to protect DNA from damage in a non-specific fashion. This clearly illustrates that there are many complex elements to the host–pathogen interaction.
We conclude that S. Typhimurium induces a variety of genes in response to the activity of the potent TLR4- and NADPH oxidase-mediated innate immune mechanisms. Most of the induced genes do not contribute to virulence in isolation, but are likely to represent multiple redundant mechanisms involved in resisting these host resistance factors, suggesting that these mechanisms are important for survival and proliferation of S. Typhimurium within the host cell.
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ACKNOWLEDGEMENTS |
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Edited by: D. L. Gally
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Received 28 April 2009;
accepted 12 June 2009.
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) and absence (
) of 5 mM hydrogen peroxide. (a) SL1344 (b) 
katG, (c) 