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Centre for Infectious Disease Research and Biosafety Laboratories, Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India
Correspondence
Dipshikha Chakravortty
dipa{at}mcbl.iisc.ernet.in
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ABSTRACT |
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The GEO accession number for the microarray data for this study is GSE11820.
Supplementary tables with details of strains, vectors and primers, and a spreadsheet of the complete microarray data are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Hensel et al., 1995). The mechanisms by which phagocytes kill virulent Salmonella are not completely understood; however, the role of the NADPH phagocytic oxidase system has been strongly implicated (Mastroeni et al., 2000). The generation of reactive oxygen species (ROS) occurs via a membrane-bound flavocytochrome b558, consisting of two phagocytic oxidase components (gp91phox and p22phox) and four cytosolic components, p40phox, p47phox, p67phox and a GTP-binding Rac protein. During activation, the cytosolic components translocate to the site of gp91phox/p22phox on the phagosomal membrane, forming a functional enzyme complex, which generates ROS by catalysing electron transfer from NADPH to molecular oxygen (Leusen et al., 1994). NADPH phagocytic oxidase (NADPH phox) is active in murine macrophages infected with S. Typhimurium (De Groote et al., 1997), and greater numbers of Salmonella are recovered from the livers and spleens of gp91phox–/– C57BL/6 mice than from wild-type mice (Mastroeni et al., 2000). Further, in the phagocytes, replication of S. Typhimurium is limited by NADPH oxidase and MEK/ERK kinase (Rosenberger & Finlay, 2002). Patients deficient in the NADPH phagocytic oxidase are susceptible to recurrent microbial infection, including salmonellosis (Mouy et al., 1989). To combat the damaging effects of this active oxygen, virtually all aerobic organisms have evolved complex defence and repair mechanisms (Farr & Kogoma, 1991). For example, the type III secretion system encoded in the SPI2 chromosomal gene cluster of Salmonella has been found to be critical to minimize the contact of Salmonella phagosomes with vesicles harbouring NADPH oxidase or inducible nitric oxide synthase (iNOS) complexes (Vazquez-Torres et al., 2000; Chakravortty et al., 2002).
The LysR family in S. Typhimurium is composed of 44 auto-regulatory LysR-type transcriptional regulators (LTTRs), which apparently evolved from a distant ancestor into subfamilies found in diverse prokaryotic genera (Schell, 1993). In response to different inducers, LTTRs activate transcription of linked target genes or unlinked regulons encoding extremely diverse functions including amino acid biosynthesis, nitrogen fixation, oxidative stress response and bacterial virulence (Ochsner et al., 2000; Pagán-Ramos et al., 1998; Russell et al., 2004; Sun et al., 2002). OxyR, a member of the LysR family of bacterial transcriptional factors, regulates expression of many genes required for combating hydrogen peroxide (H2O2)-mediated bacterial killing, such as those encoding catalase, glutathione reductase, glutaredoxin, alkyl hydroperoxide reductase, OxyS and Fur (Christman et al., 1985). Two-dimensional gel analysis of Salmonella proteins labelled during the 60 min period following treatment with a low dose of H2O2 (a ROS) indicated enhanced expression of 30 proteins when compared to untreated cells (Christman et al., 1989).
Microarray data of Eriksson et al. (2003) revealed 8-fold and 10-fold upregulation of two LTTR genes, STM0952 and STM1625, respectively. In this paper, we report that STM0952, but not STM1625, counteracts H2O2 stress and is implicated in the virulence of S. Typhimurium. We hypothesize that STM0952 (which we have named hrg, for hydrogen peroxide resistance gene) can resist oxidant-based killing mechanisms of the host immune system by interfering with ROS production.
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METHODS |
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Construction of gene deletions in S. Typhimurium.
The STM0952 (hrg) and STM1625 mutants in S. Typhimurium were constructed using a one-step deletion strategy (Datsenko & Wanner, 2000). Briefly, transformants carrying a ‘red’ helper plasmid (pKD46) were grown in LB with ampicillin and 10 mM L-arabinose(Sigma) at 30 °C to an OD600 of 0.35–0.4 and then made electrocompetent by washing three times with ice-cold 10 % glycerol and MilliQ water. PCR products containing the kanamycin-resistance gene (from plasmid pKD4) flanked by sequences upstream and downstream of the hrg and STM1625 gene were obtained with the sets of primers described in Supplementary Table S2. Electroporation was done according to the manufacturer's instructions (Bio-Rad), using 500 ng PCR product. Transformants were selected on LB agar containing kanamycin. The knockouts were confirmed by PCR using confirmatory primers. In the knockout strains (
hrg and 1625) a 1.7 kb band was amplified, whereas in the WT strain a 1.1 kb band for both hrg and STM1625 was amplified (data not shown).
Construction of the hrg-complemented (c-hrg) and the hrg-overexpressing strain.
DNA extracted from WT S. Typhimurium was used as a template to amplify the hrg gene using primers as listed in Table S1. The amplified product was purified using the Eppendorf Gel Cleanup kit and this insert along with vector pQE-60 were digested with restriction enzymes. The vector and the insert were mixed at 1 : 4.5 molar concentrations and ligated at 16 °C for 16 h. The vector-containing insert was then transformed into competent cells of Escherichia coli and plated on LB-carbencillin plates after 1 h incubation in SOC medium (Sambrook et al., 1989). The colonies were screened for the plasmid harbouring the insert, and this purified plasmid was then transformed into hrg competent cells. The overexpressing strain was constructed similarly (primers in Supplementary Table S1) using the pBAD vector; expression was induced by addition of 1 mM L-arabinose to bacterial cultures (OD600 0.5) for 3 h.
Cell culture and bacterial infection.
The murine macrophage cell line RAW264.7 was kindly gifted by Professor Anjali Karande (Department of Biochemistry, Indian Institute of Science, India). RAW264.7 and INT 407 cells (obtained from the National Centre for Cell Science, Pune, India) were maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma) containing 10 % heat-inactivated fetal calf serum (Sigma) at 37 °C in an atmosphere of 5 % CO2. The cells were seeded in 24-well plates at a density of 2.5x105 cells per well for the experiments.
For the infection of RAW264.7 cells, S. Typhimurium strains were grown to stationary phase in LB with the appropriate antibiotic. For infection of epithelial cells, stationary-phase cultures were diluted 1 : 33 in LB and grown for 3 h. The OD600 of the cultures was adjusted to 0.3 and the resulting bacterial suspensions were used to infect the cells at an m.o.i. of about 10. Bacteria were centrifuged onto the cells at 500 g for 5 min. After infection for 25 min, cells were washed three times with PBS and incubated for 1 h in cell culture medium containing 50 µg gentamicin ml–1 (Sigma). The medium was replaced with medium containing 10 µg gentamicin ml–1 for the rest of the experiment. In some experiments, IFN
(Sigma, 100 IU ml–1), phenylarsine oxide (PAO, 5 µM) or Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; Calbiochem, 50 µM, 30 min pretreatment) was added to the cells along with 10 µg gentamicin ml–1.
For the enumeration of intracellular bacteria, macrophages were washed three times with PBS and lysed with 0.1 % Triton X-100 for 10 min at room temperature and serial dilutions were plated onto LB agar.
H2O2 sensitivity assay.
Cells (107 c.f.u.) from early stationary-phase cultures (9 h growth) of the WT, hrg and (L-arabinose-induced) Hrg-overexpressing strains were suspended separately in 1 ml PBS containing 600 µM H2O2. The cultures were grown aerobically with shaking at 225 r.p.m. at 37 °C and after every time point 1000 U catalase (Sigma) was added to quench the remaining H2O2. Serial dilutions of the bacteria were plated every hour to monitor c.f.u.
Measurement of extracellular H2O2 by phenol red assay.
H2O2 production by RAW264.7 cells was determined by a modification of the method of Wautier et al. (2001), which uses the horseradish peroxidase (HRPO)-dependent conversion of phenol red by H2O2 into a compound with increased absorbance at 610 nm. Briefly, RAW264.7 cells after infection with the WT or the hrg strain were incubated in phenol red solution containing NaCl (0.14 M), potassium phosphate (0.01 M; pH 7.0), glucose (0.0055 M), phenol red (0.2 g l–1) and HRPO (19 U ml–1; Sigma). At the end of the incubation period, the supernatant was assayed in a spectrophotometer at 610 nm to check the amount of H2O2. From each infected well total protein was estimated using the Bradford method and the data are represented as amount of H2O2 produced h–1 (mg total protein)–1. A standard curve was generated using H2O2 concentrations ranging from 0.1 to 5 µM in the phenol red solution.
Measurement of intracellular ROS.
Intracellular ROS level was measured by flow cytometry (Von Knethen & Brüne, 2001) by using the redox-sensitive dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes). H2DCFDA is a cell-permeable indicator for ROS that is non-fluorescent until the acetate groups are removed by intracellular esterases and oxidation to the highly fluorescent polar derivative H2DCF occurs within the cell. For this assay, 106 cells were incubated for 30 min with 10 µM H2DCFDA in the dark. Cells were quickly washed and immediately 10 000 events were acquired on a FACSCalibur (BD Biosciences).
Localization of NADPH subunit gp91phox by fluorescence microscopy.
Localization of gp91phox in RAW264.7 cells infected with the WT or hrg S. Typhimurium was determined by standard immunocytochemical methods. The WT- or hrg-infected cells were washed free of medium,were fixed for 10 min in paraformaldehyde (3.5 %) at room temperature and washed with PBS. Fixed cells were then incubated with goat anti-mouse gp91phox immunoglobulin G (BD Transduction) diluted 1 : 100 in PBS containing 2 % BSA, 2 % goat serum and 0.1% saponin. The cells were then washed three times in PBS and incubated in identical conditions but with rabbit anti-goat IgG conjugated to Cy3 (Jackson ImmunoResearch Laboratories). Bacteria were stained with anti-LPS antibody raised in mouse followed by FITC-conjugated anti-mouse IgG. Samples were viewed on a confocal laser-scanning microscope equipped with an argon laser (Zeiss).
Mouse experiments.
Six- to eight-week-old BALB/c mice (Central Animal Facility, Indian Institute of Science, Bangalore, India) were maintained under specific-pathogen-free conditions. All procedures with animals were carried out in accordance with institutionally approved protocols. Bacterial strains were grown with shaking overnight at 37 °C, centrifuged, washed, resuspended to the appropriate concentration in sterile PBS, and administered to mice at the indicated doses. For the survival studies, 10 mice were used for each strain of bacteria and observed daily for survival. For the organ infiltration experiments, liver, spleen and mesenteric lymph nodes were taken aseptically 5 days after infection. The organs were weighed and homogenized in 1 ml PBS. The homogenate was centrifuged and plated at different dilutions to determine bacterial numbers.
Competitive index assay.
The competitive indices (Beuzon & Holden, 2001) were calculated by oral infection with the WT and hrg or WT and c-hrg strains at 1 : 1 ratio, i.e. organisms of two strains, in three independent experiments. After the 5th day of infection, homogenized samples of liver, spleen and mesenteric lymph nodes of infected mice were plated and the competitive index for the knockout bacteria was calculated by dividing the number of the mutant bacteria from the infected animal by the number of theWT bacteria recovered. This value was then corrected by the initial ratio of the mutant to WT bacteria used to infect the mice. For control experiments, the competitive index of a different isogenic WT strain was calculated in the same way.
Microarray analysis.
The microarray analysis of the gene expression profile comparing the WT and the hrg strain was performed according to the manufacturer's protocol (Genotypic Technology, Agilent Technologies). Total RNA was isolated following treatment with 600 µM H2O2 for 4 h. The hybridization procedure for the total RNA and the complete normalized data with ratio and fold change are available as supplementary data with the online version of this paper. In the knockout strain, the genes that were upregulated more than 1.8-fold and those downregulated more than 0.55-fold were considered to be significant. The microarray was repeated for these genes and a P-value <0.05 for any gene was considered significant. The complete dataset has been submitted to the GEO public database (accession no. GSE11820).
Statistical analysis and software.
Each assay was repeated at least three times. In vitro data were analysed by paired t test (two sample, equal variance) and P values below 0.05 were considered significant. FACS data were plotted and analysed using WinMDI software. Results of mouse in vivo challenge studies were evaluated by using the Mann–Whitney U test from the GraphPad Prism 4.0 software. Differences between experimental groups were considered significant at P<0.05.
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RESULTS |
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shows the analysis with five known LTTRs from different bacteria; the homology of Hrg with these proteins is around 65 %, suggesting that Hrg is indeed a member of the LysR family of proteins.
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, the fold increase in the WT strain from 2 to 16 h is 10.7-fold, whereas in the hrg strain, it is only 2.7-fold. This decrease in the fold replication was not seen in the hrg-complemented strain. However, a knockout mutant of another LTTR, STM1625, which was also found to be upregulated in the macrophages, replicated like the WT strain when macrophages were infected with this strain (1625). This defect in the intracellular proliferation of the hrg strain was restricted to macrophage cells and was not observed in INT 407, an intestinal epithelial cell line (Fig. 2b). The growth of the WT and the hrg strains was comparable in LB and in minimal media (data not shown).
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hrg strain showed decreased replication in macrophages compared to the WT strain, we sought to investigate the mechanism behind this attenuation. The WT, hrg, c-hrg and 1625 strains were therefore subjected to various oxidative and nitrosative stresses. No difference in growth was observed among the strains when methylene blue (0.5 mg ml–1), sodium hypochlorite (0.2 mg ml–1) or acidified sodium nitrite (1 mM, pH 5) was used to confer stress (data not shown). However, when the cells were treated with 600 µM H2O2, starting from the fourth hour of treatment the hrg strain exhibited greater sensitivity to H2O2 than the WT and 1625, whereas the Hrg-overproducing strain showed increased survival advantage over the WT (Fig. 3b). Overexpression of the Hrg protein in the overexpressing strain was confirmed by SDS-PAGE (Fig. 3a).
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hrg strain compared to infection with the WT strainhrg strain showed a significant difference in the phenol red assay: after 8 h of infection, the hrg-infected macrophages produced 186.3±4 nmol H2O2 h–1 (mg protein)–1 compared to 107.7±5 nmol H2O2 h–1 (mg protein)–1 in the case of the WT infection.
We then tested ROS production in the macrophages after Salmonella infection using the dye H2DCFDA, which is converted to its fluorescent derivative in the presence of ROS. Only 5.8 % of the macrophages produced ROS upon infection with the WT strain, whereas 15 % of the cells produced ROS when infected with the hrg strain (Fig. 3c
). In INT 407 cells, where no compromise was observed in the intracellular proliferation of the hrg strain, ROS production was similar in both the WT- and the hrg-infected cells (data not shown). In all cases, uninfected cells served as control.
Inhibition of ROS production by MnTMPyP or PAO reverses the growth attenuation phenotype of the hrg strain
We next checked the fate of the hrg strain when ROS production was induced in macrophages with IFN. IFN (100 IU ml–1) induced a high level of ROS as assessed by DCFDA staining (data not shown). As shown in Fig. 4(a), addition of IFN led to a 4-fold decrease in the intracellular proliferation of the WT and a 10-fold decrease in the case of the hrg strain.
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hrg strain contributes to the growth defect. MnTMPyP was used to quench the ROS produced in the cells. As depicted in Fig. 4(b), pretreatment of macrophages with MnTMPyP completely restored the growth of the hrg strain to the WT level. PAO is a pharmacological inhibitor of NADPH oxidase. Incubation of macrophages with PAO gave a similar result to MnTMPyP (Fig. 4c).
Increased co-localization of gp91phox with the hrg strain
As it was evident that ROS production was significantly higher in macrophages infected with the hrg strain than in those infected with the WT, we next examined whether there is any difference in the NADPH phagocytic oxidase localization between the WT and hrg strain. After infecting RAW264.7 macrophages with either the WT or hrg for 20 h, cells were stained for Salmonella LPS and gp91phox subunit of NADPH phagocytic oxidase. Interestingly, increased co-localization of the hrg mutant and gp91phox was observed as compared to the WT bacteria (Fig. 5). The percentage co-localization of gp91phox with the hrg strain was 24 % (±1.7) versus 9 % (±0.71) with the WT Salmonella.
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hrg strainhrg strain in a murine model of typhoid fever. To address this, BALB/c mice (n=10) were orally infected with 107 c.f.u. of the WT or the hrg strain. As shown in Fig. 6(a), mice infected with the WT or c-hrg strain died within 7 days of infection, whereas mice infected with the hrg strain showed up to 60 % survival until day 7.
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hrg strain in the organs of micehrg-infected mice was due to reduced numbers of bacteria in the organs, we analysed the bacterial load in the spleen, liver and mesenteric lymph nodes after infecting mice orally at a dose of 106 bacteria per mouse. After 5 days of infection, there was a significant decrease in the bacterial burden in all the organs of mice infected with the hrg strain when compared to the mice infected with the WT strain (Fig. 6b–d).
In order to further evaluate the loss in relative virulence displayed by the hrg strain, we tested the ability of this knockout to compete with the WT strain after oral infection. As shown in Table 1, the hrg strain exhibited a competitive index defect after the 5th day of infection in liver, spleen and mesenteric lymph nodes, suggesting that this gene was essential for the in vivo colonization of S. Typhimurium. The P values for all the organs were less than 0.05. The c-hrg strain was found to compete equally against the wild-type strain.
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). Acid-shock protein was also downregulated in the hrg strain. We speculate that these genes might be regulated by Hrg. NADH dehydrogenase genes, several permeases, putative transferases and transporters were also downregulated in the hrg strain (supplementary data). We also observed the upregulation of several genes required for invasion and transport function in the knockout strain under oxidative stress conditions. However, we restricted our discussion only to the genes that could account for the observed phenotype of the mutant under oxidative stress. The fold difference and the corresponding P values for the genes are summarized in Table 2.
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DISCUSSION |
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). For example, the OxyR and SoxRS regulons enable E. coli to resist the effects of H2O2 and superoxide, respectively (Christman et al., 1989; Wu & Weiss, 1991). However, it has been convincingly shown that S. Typhimurium does not require a functional OxyR or SoxRS regulon for virulence (Miller et al., 1989; Taylor et al., 1998), suggesting that this pathogen may use alternative strategies to avoid exposure to high concentrations of phagocyte-derived oxidants in vivo and there might be other regulators that work independently of or in conjunction with OxyR or SoxRS.
The LTTR family plays an important role in oxidative stress response in many pathogenic organisms. Mutational studies and amino acid sequence similarities of LTTRs identify a DNA-binding domain employing a helix–turn–helix motif domain involved in coinducer recognition and/or response and a domain required for both DNA binding and coinducer response (Schell, 1993). Upon induction by the coinducers, the LTTRs can regulate diverse genes and their functions. OxyR, a LTTR, upregulates various genes upon H2O2 treatment in different bacteria (Farr & Kogoma, 1991) In oxidized form it binds to the promoter of the genes it regulates. There has been one report suggesting that OxyR can act as a repressor of catalase expression in Neisseria gonorrhoeae (Tseng et al., 2003). In Salmonella, another LTTR, SpvR, has been studied extensively and has been found to be important for spleen invasion; it is expressed only during the stationary phase of bacterial growth (Libby et al., 1997). The mechanism of the spv effect is via induction of cytopathology in the host macrophages (Libby et al., 2000). The spv virulence locus, although it is found on large plasmids in Salmonella subspecies I serovars, is located on the chromosome of serovar Arizona (Libby et al., 2002). It has also been demonstrated that polynucleotide phosphorylase of Salmonella negatively controls spv virulence gene expression (Ygberg et al., 2006). Various other LTTRs in S. Typhimurium take part in biosynthesis of amino acids such as cysteine, methionine and lysine (Kredich, 1971).
Hrg imparts survival advantage to S. Typhimurium and is a virulence factor
The data presented here show an important role of the hrg (STM0952) gene in S. Typhimurium pathogenesis and resistance to the antimicrobial activity of macrophages. In spite of its upregulation in the macrophage, the role of this gene previously remained uncharacterized. In silico analysis predicts that STM0952 is a member of the LTTR family, as it has significant homology with other known LysR family proteins from different bacteria. We named STM0952 as hrg, for ‘hydrogen peroxide resistance gene’. We report here that the hrg gene confers resistance of S. Typhimurium to oxidative stress, specifically to H2O2, and is important for survival in the macrophage population. We demonstrate that a hrg mutant has reduced capacity to survive and replicate in RAW264.7 macrophages. We have also shown that although the hrg mutant is able to reach extra-intestinal sites, namely, the liver, spleen and mesenteric lymph nodes, to invade deep tissues, it is unable to proliferate there efficiently. The hrg strain also showed a competitive index defect in liver, spleen and mesenteric lymph nodes, indicating that the attenuation of this strain diminishes its ability to compete for survival in these organs. This suggests that S. Typhimurium is unable to withstand host cellular antimicrobial mechanisms in the absence of the Hrg-regulated genes.
Genes necessary to counteract H2O2 stress are non-functional in the hrg strain
The intracellular growth defect of the hrg but not the 1625 mutant in macrophages further suggested that these two LTTRs might have different functions. The fact that the defect was found in macrophages but not in epithelial cells could indicate that the attenuation of the knockout strain is be due to increased oxidative burst. The elicitation of phagocyte oxidative burst after pathogen entry is well documented (Wilson et al., 1980; Wojtaszek, 1997). Reactive oxygen radicals are one of the potent antimicrobial responses that phagocytes use to limit the growth of micro-organisms. H2O2 is a key component of the oxidative burst of host macrophages. In the bacterial cell H2O2 can react with iron or copper ions to generate the toxic hydroxyl radical (OH·) via the Fenton reaction (Cadenas, 1989). To survive and thrive, intracellular pathogens have to either avoid elicitation of oxygen radicals or resist their effects. After infection with the WT S. Typhimurium, 5 % of the total macrophage population was positive for ROS production. This result is in accordance with data of Foster et al. (2003), who reported that WT S. Typhimurium induced very low levels of intracellular ROS as compared to the phoP mutant. We found a similar result in the phenol red assay. Macrophages infected with the hrg strain showed increased H2O2 production in the supernatant compared to those infected with the wild-type. The higher level of intracellular ROS production seen in infections with the hrg strain might be due to its inability to quench the H2O2 produced, thereby leading to increased ROS accumulation. MnTMPyP is a potent ROS quencher and mimics catalase (Xiao et al., 2002). MnTMPyP pretreatment of the host macrophages before infection, which abrogates ROS production, reversed the growth defect of the hrg. We obtained similar result with a pharmacological inhibitor of NADPH oxidase, PAO (Le Cabec & Maridonneau-Parini, 1995). So it can be predicted that either by quenching the ROS produced or by inhibiting the NADPH oxidase function the intracellular growth of the hrg strain can be restored to the WT level. The increased susceptibility of the hrg strain to H2O2 in vitro suggests that the genes necessary to counteract H2O2 are probably non-functional in this strain. Further, our microarray data are in accordance with the hypothesis that under H2O2 stress a reduced level of catalase in the hrg strain makes the strain much more susceptible than the WT to H2O2-mediated killing. DNA repair after oxidative damage is a survival strategy for the pathogen. UvrA and DNA polymerase III are required to repair DNA damage by H2O2 (Farr & Kogoma, 1991). The decreased expression of both uvrA and DNA polymerase III, theta subunit (HolE) in the hrg strain upon H2O2 treatment might also be a reason for the observed hypersensitive phenotype. The specificity of the resistance to H2O2 makes the hrg gene unique. The Hrg-overproducing strain showed an increased survival advantage over the WT strain upon H2O2 stress. Virulent Salmonella is known to inhibit the fusion of the Salmonella-containing vacuole with NADPH oxidase, thereby leading to increased bacterial survival in the macrophages (Vazquez-Torres et al., 2000). The increased co-localization of gp91phox with the hrg strain indicates more active assembly of the phagocyte oxidase system near the bacterial site when compared to the WT strain and thus we can speculate that Hrg-regulated genes might be directly or indirectly involved in inhibiting gp91phox co-localization to the SCV.
Concluding remarks
There are several Salmonella genes that are required for resistance to oxidative stress, such as katE, katG, sod, rpoS and slyA (Papp-Szabò et al., 1994; Sly et al., 2002; Robbe-Saule et al., 2001; Buchmeier et al., 1997). In vitro hypersensitivity of the hrg strain to H2O2 and the high level of ROS produced by macrophages in response to hrg infection suggest a role for Hrg-regulated genes in quenching the H2O2 both in vitro and in vivo. The increased survival of the Hrg-overexpressing strain upon oxidative stress also supports this hypothesis. The Hrg-regulated genes also appear to prevent the NADPH phagocytic oxidase trafficking towards the Salmonella-containing vacuoles. Hence, our data suggest that STM0952 (hrg) transcriptionally regulates multiple gene loci involved in the pathogenesis of S. Typhimurium, and the regulation might vary in vitro and in vivo depending on the requirement of efficient infection. Our microarray results showed that catalase hydroperoxidase HPI (katG gene), uvrA and DNA polymerase III theta subunit (holE gene) were downregulated significantly in the knockout strain. This result would explain the enhanced ROS production and H2O2 hypersensitivity of the hrg strain, in which H2O2 could not be effectively quenched and DNA repair is hindered. Although OxyR is known to positively regulate catalase expression, we propose that Hrg might function in conjunction with OxyR in this regulation. Thus, this study provides a basis for further work to identify the exact mechanism of the regulation by hrg on its downstream genes, which will provide important insights into oxygen-dependent antimicrobial mechanisms in Salmonella pathogenesis.
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ACKNOWLEDGEMENTS |
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Edited by: P. H. Everest
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Received 14 February 2008;
revised 2 June 2008;
accepted 9 June 2008.
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), 
) and overexpressing (hrg PBAD, 
) strains were incubated with 600 µM H2O2 for the time period indicated and viable cells were determined by serial dilution and plating on LB agar. (c) ROS production by the WT, 
