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Infect Immun. Feb 2006; 74(2): 1323–1338.
PMCID: PMC1360297

Intracellular Gene Expression Profile of Listeria monocytogenes

Abstract

Listeria monocytogenes is a gram-positive, food-borne microorganism responsible for invasive infections with a high overall mortality. L. monocytogenes is among the very few microorganisms that can induce uptake into the host cell and subsequently enter the host cell cytosol by breaching the vacuolar membrane. We infected the murine macrophage cell line P388D1 with L. monocytogenes strain EGD-e and examined the gene expression profile of L. monocytogenes inside the vacuolar and cytosolic environments of the host cell by using whole-genome microarray and mutant analyses. We found that ~17% of the total genome was mobilized to enable adaptation for intracellular growth. Intracellularly expressed genes showed responses typical of glucose limitation within bacteria, with a decrease in the amount of mRNA encoding enzymes in the central metabolism and a temporal induction of genes involved in alternative-carbon-source utilization pathways and their regulation. Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. A total of 41 genes were species specific, being absent from the genome of the nonpathogenic Listeria innocua CLIP 11262 strain. We also detected 25 genes that were strain specific, i.e., absent from the genome of the previously sequenced L. monocytogenes F2365 serotype 4b strain, suggesting heterogeneity in the gene pool required for intracellular survival of L. monocytogenes in host cells. Overall, our study provides crucial insights into the strategy of intracellular survival and measures taken by L. monocytogenes to escape the host cell responses.

Listeriosis is a food-borne disease with high mortality rates. Listeria monocytogenes, the causative agent, can survive in diverse habitats, including extracellular abiotic and intracellular environments, and infect a large number of vertebrate and invertebrate hosts. All of the identified virulence genes in L. monocytogenes are known to be under the direct or partial control of positive regulatory factor A (PrfA) (9, 34). Entry into nonprofessional phagocytes is mediated by surface-associated gene products internalin A and B (12, 31). Early after internalization, the bacteria disrupt the phagosomal membrane of the host by expressing a pore-forming toxin, listeriolysin (Hly), and a phospholipase (PlcA) to access the cytoplasm of the host cell. Intracellular movement of the bacteria inside the host cell is mediated by ActA, which polymerizes the host actin molecules and propels itself inside the cytosol of the host cell. Spreading from one cell to another is dependent on hemolysin (Hly) and another phospholipase (PlcB) (39). Mutations have been introduced into virulence genes of L. monocytogenes that lead to debilitating phenotypes in the mouse model of infection. In addition, a hexose phosphate transporter, UhpT, that is required for efficacious intracellular growth has recently been described (11). Recently, a global view of the PrfA regulon has been obtained through whole-genome expression profiling (35).

At present there is a lack of knowledge about intracompartmentally expressed genes and their products in L. monocytogenes. To obtain information towards this end, we explored and quantified gene expression through whole-genome transcriptome profiling of L. monocytogenes EGD-e isolated directly from the cytosol of infected cells. We also exploited current information on the roles of virulence gene factors to examine bacterial gene expression in the vacuolar compartment following uptake. Our data provide a comprehensive view of changes in gene expression as the bacterium transits from the extracellular milieu and adapts to growth in the cytoplasm of the infected host.

In this work we characterized lmo0206, lmo0207, and lmo2219 as new members of the PrfA regulon and further investigated the involvement of lmo0206 (orfX), lmo0207 (orfZ), lmo0229 (ctsR), sigB, lmo1298 (tnrA), lmo2200 (ohrR), and lmo2219 (prsA) genes in intracellular survival by producing deletion mutants. Our data show that the deletion of lmo0206, lmo0207, lmo0229, lmo2200, and lmo2219 affected the intracellular survival of L. monocytogenes inside murine macrophage (P388D1) cells.

MATERIALS AND METHODS

Strains and growth conditions.

L. monocytogenes EGD-e (20) and its several isogenic mutants were used in this study. Bacteria were grown in brain heart infusion (BHI) broth (Difco) at 37°C with shaking. Escherichia coli InvαF′ cells (Invitrogen) were grown in Luria-Bertani broth containing 300 μg of erythromycin/ml to select for the plasmid pAUL-A (9). For L. monocytogenes EGD-e, pAUL-A was selected with 5 μg of erythromycin/ml. pMV158 (36) was selected on BHI containing 5 μg of tetracycline/ml. A list of strains and plasmids used in this study is presented in Table Table1.1. All experiments were done with bacterial cultures at an optical density at 600 nm of 1.0.

TABLE 1.
Strains and plasmids used in this study

General molecular biological techniques.

Standard molecular techniques were used for DNA manipulation (45). Plasmid DNA was transferred into E. coli InvαF′ (Invitrogen) by using the method of Hanahan (24). The electroporation protocol of Park and Stewart (37) was used for the transformation of L. monocytogenes strains.

Construction of deletion mutants.

Mutants with chromosomal in-frame deletions were constructed by generating the 5′ (with primers P1 and P2) and the 3′ (with primers P3 and P4) flanking regions of the genes concerned. Primers used to generate the flanking regions are shown in Table S1 in the supplemental material (restriction sites are underlined). The purified PCR fragments were digested with either NotI or BamHI (as stated in Table S1 in the supplemental material) and ligated (for lmo1298, ligation was carried out as described in reference 41). Following ligation, the fragments were amplified using the 5′ upstream and 3′ downstream primers (P1 and P4) of the 5′ and 3′ flanking regions, respectively, of the genes concerned, digested with appropriate restriction enzymes (see Table S1 in the supplemental material), and ligated into the temperature-sensitive suicide vector pAUL-A, which was digested with the same enzymes and used to transform E. coli InvαF′ electrocompetent cells. Plasmid DNA of pAUL-A bearing the fragments was isolated from the recombinants and used to transform L. monocytogenes EGD-e to generate the chromosomal deletion mutants by using previously described methods (46). The deletions in the genes concerned were identified by PCR and confirmed by sequencing of the PCR fragments by using primers P1 and P4.

Cell culture and infection model.

P388D1 murine macrophage cells were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal calf serum (FCS) (PAA Laboratories) in 6-well or 24-well tissue culture plates for intracellular growth assays and in 85-mm-diameter tissue culture plates for RNA isolation. Intracellular growth assays were performed as previously described in reference 31 at 1, 4, and 8 h postinfection. For extraction of listerial RNA, P388D1 cells were grown to about 80% confluence. Bacteria were added to the cell monolayer at a multiplicity of infection (MOI) of 10 per eukaryotic cell for EGD-e and 500 per eukaryotic cell for the Δhly ΔplcA mutant. Infection was carried out for 45 min, followed by the addition of fresh medium containing 20 μg of gentamicin/ml. The medium on the plates (containing 20 μg of gentamicin/ml) with EGD-e was replaced 2 h postinfection with fresh medium containing 50 μg of gentamicin/ml. At each step, the plates were washed extensively with 1× phosphate-buffered saline. Incubations were carried out in a humidified incubator for up to 8 h.

Plaque formation assays.

L929 cells were grown to monolayers in 6-well tissue culture plates in RPMI 1640 supplemented with 5% FCS. The cells were infected with 8 μl of a 10−4 dilution of a washed bacterial culture with an optical density at 600 nm of 1.0 for 2 h at 37°C in a humidified incubator and subsequently incubated for 30 min in the presence of 50 μg of gentamicin/ml. The agarose overlay contained 1% agarose in minimal essential medium, 5% FCS, glutamine, nonessential amino acids, and 10 μg of gentamicin/ml. Experiments were carried out in duplicate. The plates were examined for plaques after an additional incubation at 37°C for 3 days postinfection.

Confocal and immunofluoresence microscopy.

P388D1 cells were grown on coverslips and infected with either L. monocytogenes EGD-e or the Δhly ΔplcA mutant carrying pMV158 (Δhly ΔplcA::pMV158) for constitutive green fluorescent protein expression. Infection was carried out as described above. After 1 h (for the Δhly ΔplcA mutant) and 8 h (for EGD-e), cells were fixed with 3.7% formaldehyde in 1× phosphate-buffered saline for 30 min at room temperature. Coverslips infected with the Δhly ΔplcA::pMV158 mutant were stained with 100 nM LysoTracker red (Molecular Probes) to stain acidic vacuoles, mounted with 50 mg of moviol/ml, and analyzed under a confocal microscope (Leica DM IRBE).

Coverslips infected with L. monocytogenes EGD-e were stained with fluorescein isothiocyanate-phalloidin for actin tail staining, and L. monocytogenes was detected by using α-ActA monoclonal antibody N81 (35a) followed by a secondary Cy3-conjugated goat anti-mouse polyclonal antibody (Dianova). Coverslips were mounted with 50 mg of moviol/ml and analyzed under an immunofluoresence microscope (Axiophot Zeiss).

Generation of a microarray for L. monocytogenes EGD-e.

To create a whole-genome microarray for L. monocytogenes EGD-e, 2,840 unique primer pairs were selected using two independent software tools: GenomePRIDE (23) and the CAAT-Box (16), a modified version of Primer3 (44). Fifty-four and 46% of the open reading frame-specific primers were generated using the GenomePRIDE software and the CAAT-Box tool, respectively, with amplicon sizes ranging from 100 to 600 bp in length.

PCR amplifications were performed with HotStar Taq DNA polymerase (QIAGEN), with chromosomal DNA as the template. After purification of PCR products and inspection by agarose gel analysis to ensure that the products were free of extraneous material, the DNA concentrations were determined with SYBR green I (Molecular Probes) by using a SpectraMAX GEMINI XS spectrofluorometer (Molecular Devices). DNA was prepared for spotting by diluting the purified PCR products in dimethyl sulfoxide at a final concentration of 25 to 50 ng/μl.

Whole-genome microarray slides for L. monocytogenes EGD-e were produced by using a Generation III microarray spotter (GE Health Care). Each microarray contained 2,840 PCR products spotted in duplicate onto Type 7*(star) slides (GE Health Care) and UV cross-linked with 50 mJ in a Stratalinker UV cross-linker 1800 (Stratagene).

RNA isolation and purification, labeling with Cy dyes, and hybridization.

Bacteria were isolated from infected host cells as described previously (14). Macrophages were lysed using 0.1% (wt/vol) sodium dodecyl sulfate, 0.1% (vol/vol) acidic phenol, and 19% (vol/vol) ethanol in water for 30 min on ice. Bacteria from 85-mm-diameter tissue culture plates were pooled from plates containing the Δhly ΔplcA mutant and EGD-e. Total bacterial RNA was extracted using the RNeasy mini kit (QIAGEN) after a wash in SET buffer (50 mM NaCl, 5 mM EDTA, and 30 mM Tris-HCl [pH 7.0]) containing 10% sodium dodecyl sulfate and a pretreatment at 37°C for 30 min with shaking at 350 rpm (Eppendorf Thermomixer Comfort) with 0.1 ml of Tris-HCl (pH 6.5) containing 50 mg of lysozyme/ml, 25 U of mutanolysin, 40 U of SUPERase, and 0.2 mg of proteinase K (Ambion) and finally treated with 4 U of DNase I (RNase free; Ambion). The total RNA thus isolated was ethanol precipitated and quantified by absorbance at 260 and 280 nm, and quality was analyzed by using the Agilent 2100 bioanalyzer. cDNA was generated and labeled with Cy dyes (both Cy3 and Cy5 for each probe) by using 3 μg of total RNA with the CyScribe postlabeling kit (GE Health Care). The cDNA thus generated was quantified by absorbance at 550 and 650 nm for Cy3 and Cy5, respectively, by using an ND-1000 spectrophotometer (NanoDrop Technologies).

Hybridization was performed with an ASP base hybridizer (GE Health Care) for 12 h at 42°C [per the instructions of the Type 7*(star) slide manufacturer] by using 10 pmol of Cy dye-labeled cDNA of the wild-type and mutant strains, 50 μl of hybridization buffer (GE Health Care), and 100 μl of deionized formamide (Ambion) per slide. Hybridizations were conducted with cDNA of (i) the Δhly ΔplcA mutant in BHI versus that of the Δhly ΔplcA mutant 1 h postinfection, (ii) EGD-e in BHI versus that of EGD-e 4 h postinfection, and (iii) EGD-e in BHI versus that of EGD-e 8 h postinfection.

Imaging and microarray analysis.

Hybridized Type 7*(star) slides were imaged with a Generation III array scanner (GE Health Care). The fluorescent signal intensities from each spot on the microarray were quantified using Spotfinder software (GE Health Care).

Data from image analysis were averaged over the duplicate spots present in one array and then normalized in a two-step process; first, an intrachip normalization was carried out in order to standardize signals from the Cy5 or the Cy3 channel. In a second step, we normalized over the entire series to bring the means to a common value. The intrachip normalization was carried out using a robust local regression function (56) as implemented in the BIOCONDUCTOR package marrayNorm (http://www.bioconductor.org/). The parameters were as follows: global local regression by the LOWESS function, with consideration of the inner 40% of data, i.e., those values where the log ratio was between the 30th and the 70th percentiles of the distribution. For the second interchip normalization, the signals from the Cy3 channel of the first chip were taken as a reference, and for all other signals the median of the difference of the logarithmic values was taken as a correcting factor. In a prior check of the correlation coefficient, low-signal values were excluded from the calculation when they deviated from linearity. For details, see reference 6. The performance of the normalization procedure was controlled by inspecting scatter plots for all possible combinations of signals. Missing data from the cDNA microarray analyses were replaced by the corresponding mean gene expression values calculated from the biological replicates or eliminated when this information was not available. Correlation matrices were obtained using Pearson's correlation coefficient (r) for both biological replicates as well as across experiments and revealed r values of 0.972 and 0.753. The significance analysis of microarrays (SAM) program was used to analyze the data after downloading of SAM into Excel (49a). Briefly, for each gene i, SAM computes a statistic, di, measuring the strength of the relationship between gene expression and the response variable. It uses repeated permutations to determine if the expression of any gene is significantly related to the response. The program creates a profile of observed versus expected values, and values which lie outside a user-defined region of this profile are considered to indicate a significant relationship to the response or, in this case, significantly regulated genes. In this study, one-class response analyses were chosen to test if the mean level of gene expression for each of the 2,831 genes differed from a hypothesized mean. Therefore, SAM was conducted with log2 ratios of gene expression values from three experiments, those with (i) the Δhly ΔplcA mutant in BHI versus the Δhly ΔplcA mutant 1 h postinfection, (ii) EGD-e in BHI versus EGD-e 4 h postinfection, and (iii) EGD-e in BHI versus EGD-e 8 h postinfection. Seven hundred and twenty permutations were made, and the delta value giving the lowest false discovery rate was chosen to set the cutoff for significantly altered genes. The resulting three gene lists were then filtered for genes with a false discovery rate of ≤0.22 and a fold change of ≥2. By using these criteria, a final set of 484 significantly regulated genes was collated from all three experiments (Table S2 in the supplemental material).

Quantitative PCR analysis.

Quantitative real-time PCR was performed on the ABI Prism 7700 sequence detection system. Forward and reverse primers (Table S1 in the supplemental material) were purchased from Sigma Genosys and were designed using Primer Express 1.0 software to produce an amplicon length of about 150 bp. Total RNA was isolated as described above with an additional treatment with the DNase-free kit (Ambion). Quantitative PCR was performed for 40 cycles with 5 μl of cDNA, 25 μl of 2× SYBR green PCR master mix (Applied Biosystems), and 10 pM (each) forward and reverse primers in a final volume of 50 μl. A standard curve was generated for each primer pair by using different copy numbers of genomic DNA from L. monocytogenes EGD-e. For each primer pair, a negative control (water), an RNA sample without reverse transcriptase (to determine genomic DNA contamination), and a sample with a known amount of template copies (to determine the efficiency of the reaction) were included as controls during cDNA quantification. All samples after real-time PCR were run on a 1.5% agarose gel to verify that only a single band was produced.

RESULTS

Intracellular growth properties of L. monocytogenes EGD-e and the Δhly ΔplcA mutant in P388D1 cells.

L. monocytogenes EGD-e was used to infect two different murine macrophage cell lines, J774 and P388D1, and intracellular replication of bacteria was monitored as described in Materials and Methods. In the study reported here, we used the P388D1 cell line, rather than the widely used cell line J774, because preliminary experiments revealed that the latter is more sensitive to killing by L. monocytogenes EGD-e infection (data not shown). By using the P388D1 cell line, growth of wild-type intracellular bacteria could be monitored even at 8 h postinfection (Fig. (Fig.1A).1A). In addition, the isogenic Δhly ΔplcA double-deletion mutant that lacks both phosphatidylinositol phospholipase C and listeriolysin, whose activities are required for exit of bacteria to the intracytoplasmic compartment, is rapidly killed in this cell line (Fig. (Fig.1A).1A). As shown in Fig. 1C, D, and E, Δhly ΔplcA mutant bacteria were entrapped within the acidic host cell vacuoles and were rapidly eliminated. Thus, to model adaptation and growth of listeriae within different intracellular compartments, we used the wild-type strain and its isogenic Δhly ΔplcA double-deletion mutant. Bacterial cells were collected from infected P388D1 host cells at 1 h (for the Δhly ΔplcA mutant) and at 4 and 8 h (for EGD-e) postinfection.

FIG. 1.
Survival of L. monocytogenes EGD-e and the isogenic double-deletion Δhly ΔplcA mutant intacellularly. (A) P388D1 murine macrophage cells were infected with the wild-type strain EGD-e and the isogenic double-deletion Δhly Δ ...

Collection of bacterial RNA from infected cells has been limited by the inherent difficulties of separating large quantities of host cell RNA. We performed RNA isolation using the cold phenol-ethanol protocol described in reference 14, which allows the disruption of the eukaryotic cell and stabilization of the bacterial RNA at the same time. At each time point, excellent-quality bacterial total RNA was isolated which was almost free from eukaryotic RNA contamination ((datadata not shown). We were able to isolate about 1.5 to 3 μg of prokaryotic total RNA per 85-mm-diameter tissue culture plate. RNA was reverse transcribed and labeled and hybridized to the cDNA microarray as described in Materials and Methods.

FIG. 2.FIG. 2.
Validation of microarray data with quantitative real-time PCR analysis. (A) Validation of the intravacuolarly expressed genes. (B) Validation of intracytosolically expressed genes at 4 h postinfection. (C) Validation of intracytosolically expressed genes ...

Alterations in the transcriptome of L. monocytogenes EGD-e during growth in cellular compartments.

To understand the mechanisms L. monocytogenes EGD-e employs for intracellular survival, we used the whole-genome microarray to search for genes that showed changes in expression during the transition of bacteria from the vacuoles into the cytoplasm of P388D1 cells. To study the expression within the phagosomal compartments of the host cells, we used cDNA of the L. monocytogenes Δhly ΔplcA mutant obtained 1 h after infection of P388D1 cells, when bacteria were found entirely in acidic vacuolar compartments (Fig. 1C, D, and E). The whole-genome transcription profile of the wild-type EGD-e bacteria growing in the cytosol was obtained by preparing cDNA transcripts from bacteria harvested at 4 and 8 h postinfection, when bacteria were found primarily in the host cytoplasm (Fig. (Fig.1B).1B). In both cases, the intracellular bacterial expression profile was compared to that of the bacteria growing in BHI medium at exponential phase. Each data set in our study represents the average of results from three independent biological experiments, each including two technical replicates. Additionally, we compared the expression profiles of the Δhly ΔplcA mutant and EGD-e growing in BHI medium and found them to be highly correlated, with an r of 0.956. Similar comparisons between extracytosolic and intracytosolic EGD-e revealed an r of between 0.679 and 0.771. This allowed us to directly compare the intraphagosomal and intracytosolic expression profiles of the bacterial strains used in this study.

Overall, a total of 484 genes representing ~17% of the total genome were mobilized to enable adaptation for intracellular growth (Table S2 in the supplemental material). Of these 484 genes, 301 were up regulated and 182 were down regulated during intracellular growth. There were 66 genes specifically up regulated for survival in the phagolysosome and 115 genes up regulated for growth in the intracytosolic compartment. In addition, 42 and 76 genes were specifically down regulated in vacuolar and cytosolic compartments, respectively. Among genes commonly regulated in either compartment, 120 were upregulated and 64 were down regulated. A single gene (lmo1254) was down regulated in the vacuolar compartment and up regulated in the cytosolic compartment of the host cell.

To identify candidate virulence genes specific to L. monocytogenes EGD-e, all of the genes regulated during intracellular growth were evaluated for the presence or absence of a homolog in the nonpathogenic Listeria innocua CLIP11262 strain (Table (Table2).2). This analysis revealed that 41 genes were uniquely present in L. monocytogenes EGD-e. Of the 41 putative L. monocytogenes species-specific genes, 10 have been previously implicated in the intracellular growth of this bacterium. We also examined whether any of the genes regulated during intracellular growth were strain specific by searching for the presence of regulated genes in the previously sequenced L. monocytogenes F2365 serotype 4b genome. A total of 25 genes were detected (Table (Table2),2), suggesting heterogeneity in the gene pool required for intracellular survival of L. monocytogenes EGD-e.

TABLE 2.
Species- and strain-specific genes of L. monocytogenes EGD-e regulated intracellularlya

Expression of virulence and virulence-associated genes.

The virulence gene cluster of L. monocytogenes comprising the genes prfA, plcA, hly, mpl, actA, and plcB (lmo0200 to lmo0205) was up regulated and belongs to the class of genes that are strongly regulated during intracellular growth. Transcription of the virulence gene cluster and other virulence genes is dependent on the PrfA transcriptional activator protein. We also detected the up-regulated transcription of lmo206 (orfX) and lmo0207 (orfZ), encoding gene products of unknown functions. Neither lmo0206 nor lmo0207 has to date been implicated in the virulence process of L. monocytogenes, but the expression patterns of these genes identify them as potential virulence factors. Among the genes encoding the internalins, inlA (lmo0433), inlB (lmo0434), and inlC (lmo1786) were observed to be up regulated in this study. Another PrfA-regulated gene, uhpT (lmo0838), encoding a hexose phosphate transporter, was uniformly up regulated during cytosolic growth. The gene prsA (lmo2219) that encodes a product homologous to the posttranslocation molecular chaperone protein PrsA of Bacillus subtilis was also up regulated, albeit at the late cytosolic phase of growth. We detected the up regulation of sepA (lmo2157) in the cytosol. SepA is reported to be under the control of both PrfA and SigB in L. monocytogenes (28, 35).

The listerial agr locus comprising lmo0048 to lmo0051 was down regulated in our experiment. lmo0051, coding for AgrA, was previously described to be virulence associated in L. monocytogenes in an in vivo mouse model but not in tissue culture experiments (4).

Metabolic gene expression.

The observation that nonpathogenic listeriae such as L. innocua are unable to grow efficiently in the host cytoplasm (47) has led to the suggestion that adaptation is required to enable growth of L. monocytogenes in the intracellular milieu. We examined the expression profiles of genes involved in sugar catabolism and de novo synthesis of macromolecular precursors and their transport to gain insight into substrates that were available intracellularly. Intermediary metabolism and energy production systems were down regulated following entry of bacteria into the phagososomal compartment, and components of bacterial nucleotide sugar metabolism were also down regulated throughout the infection cycle.

In particular, transcript levels of genes encoding enzymes involved in the second part of glycolysis (lmo1570, lmo2455, lmo2456, lmo2458, and lmo2459), i.e., the five steps corresponding to the conversion of glyceraldehyde-3-phosphate into pyruvate, were reduced during infection. In addition, the enzyme encoded by pgi (lmo2367), responsible for the conversion of glucose-6-phosphate into fructose-6-phosphate, and that encoded by ldh (lmo0210), responsible for the conversion of pyruvate into lactate, were down regulated. Genes encoding pyruvate dehydrogenase, the pdhABC operon (lmo1052 to lmo1054), were down regulated, suggesting interruption of the citric acid cycle and gluconeogenesis during intracellular growth.

A PrfA-regulated gene, uhpT, is highly up regulated during intracellular growth, promoting the uptake of phosphorylated hexoses (glucose-1-phosphate and glucose-6-phosphate). In addition, we found up regulation of the operon encoding the glycerol kinase and the glycerol uptake facilitator (lmo1538 to lmo1539) and glycerol-3-phosphate dehydrogenase (lmo1293), indicating that glycerol, probably deriving from the activity of the phospholipases A and B on cellular lipids, is an additional carbon source for intracellular growth. Components of the fatty acid biosynthesis pathway (lmo0970, lmo1808, lmo2201, and lmo2202) were down regulated throughout the infection cycle.

Other carbon sources that may be used intracellularly include carbohydrates and polyols such as mannose, fructose, cellobiose, mannitol, and pentitol transported by the phosphotransferase (PTS), entering the cell as phosphorylated substrates, and degraded by sugar hydrolases or β-glucosidases. Significantly, lmo1002, encoding a PTS phosphocarrier protein, Hpr, was down regulated inside the cytosol. The amount of Hpr is known to decrease in the absence of glucose (3). This suggests that the other substrates are preferred over glucose. Glucose inhibits prfA expression and hence the expression of other virulence genes in this pathogen (27).

During intravacuolar survival, we found that particularly the expression of genes for enzymes involved in the synthesis of branched-chain amino acids (encoded by ilv and leu) was highly enhanced (lmo1983 to lmo1991). The TnrA protein, a negative regulator of branched-chain-amino-acid expression in B. subtilis, has strong homology to the product of lmo1298, which was down regulated (49). The major source of nitrogen inside the host cell is preferably the amino acids and oligopeptides accessible in the host cell cytosol, as suggested by the up regulation of the oligopeptide transporters (lmo0135, lmo0136, and lmo0152). Also, down regulation of aminoacyl tRNA synthase genes glyS, serS, cysS, alaS, hisS, valS, thrS, ileS, leuS, tyrS, and trpS suggests availability of the respective amino acids within the cytosol. An ammonium transporter gene, nrgA (lmo1516), was strongly down regulated following initiation of the infection cycle.

We found that only the nonoxidative stage of the pentose phosphate pathway was up regulated, leading to the production of xylulose- and ribose-5-phosphate, both of which are precursors of nucleotide metabolism in the cell.

Stress response genes.

Responses to environmental insults are followed by the induction of specific patterns of stress response genes. Genes responsible for the class I stress response include dnaJ, dnaK, grpE, and hrcA (lmo1472 to lmo1475) and groEL and groES (lmo2068 to lmo2069), all of which encode the classical chaperone proteins. These were clearly up regulated during survival in the vacuolar and cytosolic compartments (except for groEL and groES genes, which were specifically up regulated in the vacuolar compartment) (Table (Table3).3). Members of the CtsR-regulated class III stress proteins, which are ATP-dependent proteases required for degradation of abnormal and short-lived proteins, were strongly induced in both the vacuolar and intracytoplasmic compartments. In particular, expression of the protease genes clpP, clpC, clpE, and clpB (lmo2468, lmo0232, lmo0997, and lmo2206) and lmo0230 and lmo0231 (similar to mcsA and mcsB, the modulators of the CtsR regulon) was induced in the phagolysosome and was maintained throughout intracellular growth. In fact, clpE is the most highly regulated gene in our data set. These changes were accompanied by moderate up regulation of the ctsR gene (lmo0229), albeit only in the vacuolar compartment. Similarly, the htrA gene (lmo0292), encoding an envelope-associated quality control protease that degrades misfolded proteins and prevents their accumulation on the cell surface, was also up regulated intracellularly (54).

TABLE 3.
Intracellularly expressed genes of L. monocytogenes EGD-e controlled by different regulatorsa

The class II stress response is mediated by sigma factor B (SigB). Although in our study sigB itself was not regulated, a total of 26 genes in our data set have previously been classified as being SigB regulated (28). We detected the up regulation of the opuBA gene (lmo1421 and lmo0903, encoding a glycine betaine uptake system) and a gene similar to the osmC gene during the intracellular growth. Both the genes are also known to be induced under osmotic stress situations (22, 51).

Three genes of the universal stress protein (Usp) family (lmo0515, lmo2673, and lmo2748) and genes for cholate, sodium, and pH homeostasis (lmo2378, lmo2381, and lmo2382) were up regulated intracellularly. Additionally, we detected the up regulation of lmo0754 (btlB), needed to combat bile stress (5).

DNA repair genes.

An intriguing observation in this study is the increased expression of the truncated comK gene (lmo2270) during bacterial growth in the cytoplasm. The ComK locus is the site of integration of the A118 bacteriophage in listerial species. In L. monocytogenes, the prophage inserted at this position of the chromosome has been rendered inactive by gene deletions. Nevertheless, strong expression of bacteriophage genes lmo2270 to lmo2274 and lmo2276 was observed during intracellular growth. In B. subtilis, expression of comK is associated with DNA repair (7); in our study, transcription of DNA repair genes was also observed to be up regulated. Thus, we observed induction of the lexA (lmo1302), recA (lmo1398), and polIV (lmo1975) genes in the cytoplasm. A gene encoding a DNA repair protein, radA (lmo0233), and lmo2676, coding for a UV-damage repair protein, were also up regulated in the cytosol of the host cell.

Cell division and replication genes.

To counteract the effects of virulence factors, the host cell may develop defense mechanisms to limit intracellular pathogen multiplication. We found 10 genes, divIVA (lmo2020), ftsZA (lmo2032 to lmo2033), ftsXE (lmo2506 to lmo2507), ftsL (lmo2040), ftsH (lmo0220), minC (lmo1545), minD (lmo1544), and smc (lmo1804), to be down regulated during transit in the phagolysosome and during intracellular growth. Thus, down regulation of ftsZ and ftsA, the major bacterial cell division determinant, and the ftsXEL complex, whose product localizes to the septal ring and is required for the recruitment of FtsK and other subsequent division proteins, suggests lowered cell division activity. We also observed down regulation of minC and minD genes, which mediate spatial regulation of cytokinesis in bacteria (42), and the smc gene, which is essential for chromosomal condensation, segregation, and cell cycle progression (21). In addition, repression of divIVA (lmo2020), encoding a protein involved in chromosomal segregation and control of the site specificity of cell division, lends further credence to the suggestion that cell division processes were affected during intracellular growth (15).

We observed the down regulation of listerial chromosomal replication initiation gene dnaA (lmo0001) and DNA primase gene dnaG (lmo1455) intracellularly. This indicates that along with the cell division, replication of intracytosolic bacteria was also negatively regulated.

Peptidoglycan and cell wall gene expression.

Expression levels of the major autolysin genes iap (lmo0582), murA (lmo2691), and spl (lmo2505) of L. monocytogenes were significantly decreased during intracellular growth. A putative regulator gene, lmo2690, located upstream of the murA autolysin gene was reciprocally regulated and probably acts to repress production of the murA gene product.

We observed up regulation of the dlt operon (lmo0971 to lmo0974) that encodes enzymes that incorporate d-alanine residues into the cell wall-associated lipotechoic acids. These enzymes were up regulated in the vacuole. The increased positive charge conferred by these molecules on the cell surface may mediate resistance to cationic peptides present in the phagolysosome of the host cell. In this context, murC (lmo1605), responsible for the replacement of alanine with glycine in the cell wall (33), was down regulated.

We also detected intracellular up regulation of eight putative cell wall-associated genes (inlA, inlB, lmo0514, lmo0610, lmo0880, lmo2085, lmo2713, and lmo2714), all of which encode products containing the LPXTG motif (except inlB and lmo2713, which encode products containing the GW motif). Of these, lmo0514 and lmo0610 are predicted to encode internalin-like proteins (8).

Transport of ions.

Metal ions are essential cofactors for functional expression of many proteins in bacterial systems. Thus, any expressional alteration of bacterial ion transport genes intracellularly reflects the accessibility of those ions in the altered environment. Among the genes involved in bacterial ion uptake systems, the zinc transporter genes zurA (lmo1447) and lmo1671 (encoding a high-affinity zinc uptake system protein) were up regulated during growth in the cytosol but lmo0641 (encoding zinc uptake P-type ATPase) was down regulated in the vacuolar compartment and the potassium uptake protein gene (lmo1023) and kdpA (lmo2681) were up regulated in the vacuoles of the host cells. All three manganese ABC transporter genes (lmo1847 to lmo1849) were down regulated inside the cytosol, and another gene (lmo1424) with homology to the eukaryotic Nramp genes was down regulated throughout the experiment. Phosphate uptake is regulated by a two-component system, PhoRP, in B. subtilis. L. monocytogenes also harbors a homolog of the phoRP two-component system. In our experiment, the phoRP operon (lmo2500 to lmo2501) was down regulated in the cytosolic phase of growth. The ferric uptake protein genes fri (lmo0943) and fur (lmo1956) were down regulated in both the vacuolar and cytosolic compartments. Collectively, we conclude that potassium and zinc in the vacuoles of the host cells were lacking and that the cytosol is a rich source of ions for the bacteria as suggested by the down regulation of magnesium, iron, and phosphate transport systems and the absence of regulation of other metal transporter systems.

Free radical production.

Both the genes encoding superoxide dismutase (lmo1439) and catalase (lmo2785) were down regulated during bacterial growth inside the cell. We validated this finding independently through quantitative real-time PCR. An operon whose open reading frames have homology to ohrAR (lmo2199 and lmo2200) of B. subtilis encoding organic hydroxyperoxide resistance and its regulator protein (17) was up regulated during intracellular growth. In addition, the glutathione reductase gene (lmo1433), lmo0646 (encoding a protein similar to lactoyl glutathione lyase), and a sulfur limitation-regulated protein gene (lmo2350) were up regulated. We also detected lmo2191, a gene similar to the B. subtilis spx gene, to be up regulated during growth of bacteria inside the host cell. The Spx protein is required for transcriptional induction of genes that function in thiol homeostasis (57).

Oxygen level.

We detected an increase in the menaquinone biosynthesis gene menF (lmo1676) intracellularly, indicating that components of the respiratory chain are utilized during intracellular growth. A decrease in the expression of cytochrome d genes (lmo2715 to lmo2718) inside the host cell indicates that the bacterial cells are not oxygen starved intracellularly, but the absence of cytochrome b or cytochrome o (the preferential path of electron flow for cells growing aerobically) suggests a more complex situation. In this respect, up regulation of the nitrite transporter gene (lmo0593) suggests that nitrite may constitute an alternative terminal electron acceptor allowing L. monocytogenes to carry out respiration under oxygen-restricted conditions. The down regulation of the cytochrome d complex genes and components of the oxidative phosphorylation system, qoxA (lmo0013), qoxB (lmo0014), qoxD (lmo0016), and lmo0355, coding for flavocytochrome c, suggest that electron acceptors other than oxygen are used under intracellular growth conditions.

Validation of microarray gene expression results.

We validated our expression profile data by performing quantitative real-time PCR analysis. A subset of 22 genes was selected to encompass a range of expression values from among the data set obtained. As shown in Fig. Fig.2,2, the real-time PCR data correlated strongly with the expression profile data (r of greater than 0.86 in all cases).

We used the wild-type strain and its isogenic ΔprfA mutant to examine whether the genes lmo0206, lmo0207, and prsA (lmo2219) are indeed PrfA regulated. The expression levels of all three genes were higher (P < 0.01) in the wild-type bacteria than in the ΔprfA strain by at least threefold (data not shown). We therefore conclude that lmo0206, lmo0207, and prsA are novel PrfA-regulated genes.

Phenotypic characterization of mutants with intracellularly active gene products.

As a first step to analyze the roles of genes described herein for intracellular survival, we used information from the literature and performed a DNA binding motif search analysis to classify genes that are controlled by known regulatory proteins, i.e., PrfA (35), CtsR (10), SigB (28), OhrR (17), TnrA (49), RpoN (2), and HrcA (52) (Table (Table3).3). For the bioinformatic analysis, we used a nucleic acid pattern search program called fuzznuc (part of the EMBOSS package) to search for the respective DNA binding motifs in the 500-bp upstream regions of the 484 genes identified above, with one mismatch. A list of the genes and their respective putative regulators is given in Table Table33.

We used this information to generate specific isogenic deletion mutants of the following regulators: the lmo1298 product (TnrA), the lmo2200 product (OhrR), SigB, and the lmo0229 product (CtsR). We also created deletions in the PrfA-regulated genes lmo0206, lmo0207, and prsA (lmo2219) that were discovered in this study and examined their relative contributions to intracellular growth and plaque formation. As shown in Fig. 3A and B, intracellular growth of the Δlmo0206 (ΔorfX), Δlmo0207 (ΔorfZ), Δlmo0229 (ΔctsR), Δlmo2200 (ΔohrR), and Δlmo2219 (ΔprsA) mutant strains was impaired compared to that of the parental EGD-e strain. On the other hand, neither the ΔsigB nor the Δlmo1298 (ΔtnrA) mutant was compromised for intracellular growth. As in other cell lines used in previous studies, the intracellular growth of the Δhly ΔplcA and ΔprfA mutants was completely abolished in the P388D1 cell line.

FIG.3.FIG.3.
Characterization of bacterial deletion mutants. (A and B) Intracellular survival of L. monocytogenes EGD-e compared to that of the ΔprfA, ΔorfX (lmo0206), ΔorfZ (lmo0207), ΔctsR (lmo0229), ΔsigB, ΔtnrA (lmo1298), ...

We also looked for plaque formation ability, a measure of intracellular growth and cell-to-cell spread of these mutants. As shown in Fig. Fig.3C,3C, the ΔprsA, ΔorfX, and ΔctsR mutants formed visibly smaller plaques than the wild type and the ΔprfA and Δhly ΔplcA mutants were unable to form plaques inside the host cells. The plaque formation ability of the Δlmo0207 (ΔorfZ), ΔsigB, Δlmo1298 (ΔtnrA), and Δlmo2200 (ΔohrR) mutants was similar to that of the wild type.

DISCUSSION

Adaptive gene expression permits intracellular pathogens to successfully persist and disseminate during encounters with the host defenses in the diverse intracellular microenvironments within the cell. Since L. monocytogenes has a facultative intracellular lifestyle, identification of genes uniquely expressed intracellularly is important for understanding infection processes and for identifying new approaches to limit listerial infections. Currently, there are no experimental approaches that accurately reconstruct the intracellular microenvironments faced by the bacterium, and inclusion of all variables influencing bacterial transcription is not currently feasible. As a first step to analyze bacterial responses throughout the course of infection, we have used listerial infection of mouse macrophages to examine the complex host-bacterium interaction. We aimed at identifying L. monocytogenes transcripts that correlate with ex vivo infection of mouse macrophages, a cell type central to the control of infections mediated by this bacterium.

Shortly following entry into the vacuolar compartment, strong reduction in the expression of genes involved in glycolysis and the citric acid cycle was observed, suggesting a depletion of bacterial intracellular glucose levels. We also observed massive transcriptional changes that reflected temporal adjustment of the listerial cellular metabolism, firstly to augment the transport and degradation of alternate metabolizable carbohydrates and the transport and metabolism of oligopeptides, aminosugars, fatty acids, and branched-chain amino acids. Branched-chain amino acids are the most abundant amino acids in proteins and form the hydrophobic core of most proteins. They are also precursors for the biosynthesis of iso- and anteiso-branched-chain fatty acids in bacterial cells. In B. subtilis, the production of branched-chain amino acids is complex and subject to many levels of regulation (48). The product of the tnrA gene represses expression of these operons involved in regulation, and attenuation is mediated by uncharged tRNA. Here we observed that the gene product of lmo1298 that is homologous to TnrA of B. subtilis was down regulated intravacuolarly. Similarly, expression of genes encoding tRNA synthetases, i.e., alaS (lmo1504), hisS (lmo1520), ileS (lmo2019), glyS (lmo1458), serS (lmo2747), cysS (lmo0239), valS (lmo1552), thrS (lmo1559), leuS (lmo1660), tyrS (lmo1598), and trpS (lmo2198), was decreased, facilitating production of branched-chain amino acids during intracellular growth and contributing to a metabolic transition from carbohydrate to amino acid fermentation. Previously, it has been demonstrated for B. subtilis (32) that a link exists between the expression of carbon and nitrogen pathways mediated via expression of the ilv-leu operon. It will be important to determine whether the differential regulation seen with genes involved in glycolysis and the branched-chain amino acids is a conditio sine qua non for intracellular growth.

Transcript levels of genes encoding several ribosomal subunits (S5, S11, S17, L5, L6, L14, L17, L18, L24, L30, and L35) and the translational initiation factor (IF3), the molecular chaperones such as GroEL, DnaJ, and DnaK, and the class II ATP-dependent proteases ClpP and ClpC, etc., were the most highly regulated genes during intravacuolar survival. The data are in agreement with those in previous publications documenting intracellular expression of the groES-groEL operon (19), the requirement of DnaK for enhanced survival following phagocytosis (25), and the major role of the Clp stress proteins in promoting early bacterial escape from the vacuoles of infected macrophages and achieving virulence in the mouse model of infection (43). Accumulating evidence for a number of bacterial pathogens indicates that heat shock proteins and chaperones play an important role in virulence. Thus, in Staphylococcus aureus, the two major chaperones DnaK and GroES-GroEL are induced during infection of human epithelial cells (40). Activation of clpP has been shown to be required for growth and survival of Salmonella entrica serovar Typhimurium within macrophages (55).

An important component of the stress response to bacteria is SigB, the master regulator of stress and stationary phase. We found 26 genes that were previously classified as members of the SigB regulon (28) to be up regulated during intracellular bacterial growth, although sigB expression itself was not significantly regulated throughout the infection period. When a ΔsigB mutant was examined for its ability to grow intracellularly, its properties were indistinguishable from those of wild-type bacteria, suggesting that the gene expression pattern for intracellular bacteria is not equivalent to that for in vitro stationary phase bacterial growth. Thus, other overlapping mechanisms of regulatory control may be dominant for the 26 genes described in this study.

Nevertheless, the data presented here suggest that intracellularly growing bacteria may be experiencing some level of stress as indicated by the expression of the htrA gene, encoding a protease required for quality control of proteins presented to the bacterial cell surface, and the prsA gene, encoding a chaperone component that is required for regulated secretion of surface components and secreted proteins. Similarly, we observed induction of genes involved in the translesional repair of DNA (recA, lexA, radA, lmo2627, and polIV) that is characteristic of an SOS response in bacteria. The operon ohrAR (lmo2199 and lmo2200) encoding an organic hydroxyperoxidase and its regulator protein is up regulated during intracellular growth. The regulatory OhrR protein in B. subtilis has been implicated in sensing of the overall thiol-disulfide redox balance of the cell deriving from oxidation of its single conserved cysteine residue. Organic hydroperoxidases have been shown to be active in the detoxification of bioactive lipids such as lysophosphatidic acid of the eukaryotic cell. Finally, a listerial homolog of the B. subtilis Spx regulator gene (lmo2191), required for the transcriptional induction of genes that function in thiol homeostasis, is highly up regulated during intracellular growth of the bacteria. The regulation of thiol oxidation appears to be an important process within bacteria growing in the intracellular environment.

In L. monocytogenes, the largest family of surface proteins are the internalins, comprising 25 members. The requirement for internalins A and B for attachment to and invasion of target host cells, the involvement of InlC and InlH (13, 30) in virulence, and the presence of only eight members of this family in apathogenic L. innocua have led to the notion that many of the internalin genes are part of the virulence gene repertoire of pathogenic L. monocytogenes. We discovered that apart from inlA, inlB, and inlC, the expression of six other internalin-like protein genes, lmo0514, lmo0610, lmo0880, lmo2085, lmo2713, and lmo2714, was increased within 1 h of infection. Although the ex vivo growth technique used in this study successfully mimics some of the conditions existing within the cell, it does not replicate the highly complex environment that pathogens encounter during natural infections. Thus, the role of additional internalin-like proteins may be revealed only in whole-animal infections. Alternatively, since L. monocytogenes is known to have a wide host range, these proteins may be required for infection of various hosts.

Overall, up regulation of genes corresponding to a further seven cell wall-associated proteins (excluding internalins) was observed. Nevertheless, there was evidence for extensive remodeling of the cell surface of intracellular listeriae. We found up regulation of the gene encoding the succinyl-aminopimelate desuccinylase (dapE; lmo0265) that produces the amino acids of mesodiaminopimelate as a key intermediate of peptidoglycan synthesis as well as the essential amino acid lysine. Enhanced production of diaminopimelate could be required for the anchoring of cell surface proteins to the peptidoglycan. Similarly, up regulation of the genes of the dlt operon, required for the incorporation of d-alanine residues into cell wall lipoteichoic acids, modifies cell surface charge, thus mediating resistance to cationic peptides. Indeed, it was previously shown that a dltA deletion mutant exhibited increased susceptibility to the cationic peptides colistin, nisin, and polymyxin B (1).

Expression of the major autolysin genes iap (lmo0582), spl (lmo2505), and murA (lmo2691) of L. monocytogenes was down regulated during growth in the vacuole and the host cytoplasm. The combined activities encoded by these autolysin genes on the listerial cell wall would result in the production of muramyl dipeptide intracytoplasmically, activating an inflammatory response mediated by the Nod proteins in the eukaryotic cytosol (19a).

We also detected induction of expression of mogR (lmo0674) (21a), encoding a repressor of flagellin gene (flaA) expression. Despite this, expression of flagellin and the membrane-associated fliP (lmo0676) was also induced at 4 h postinfection, suggesting that additional levels of control apart from transcriptional repression by MogR exist for these genes. Flagellin from L. monocytogenes has been shown to possess potent proinflammatory activity by activation of host inflammatory processes through the Toll-like receptor 5 (26).

An important finding of this study was the demonstration that all of the PrfA-regulated genes previously implicated in virulence were represented in the data set as highly up-regulated genes. Induction of the prfA regulator and virulence genes was already visible in the vacuolar phase; only inlA, inlB, and prsA were found to be up regulated within the cytosol. In this study, we detected three newly identified PrfA-regulated genes that demonstrate increased expression during intracellular growth. Isogenic deletion mutants generated for the orfX, orfZ, and prsA genes revealed clearly that these genes are required for intracellular growth of the bacterium. In a previous study, insertional mutagenesis of the orfX gene resulted in no visible defect when the mutant was examined in a plaque assay (50). Cumulatively, the data point to a definite role for orfX and orfZ in intracellular survival and warrant further study. The listerial PrsA homolog encoding a chaperone-like protein had previously been described as a novel member of the PrfA regulon (35). We provide here the first evidence for its role in intracellular survival; the substrate(s) of this protein remains unknown and will require further analysis.

An interesting observation in this study is the expression of strain-specific intracellularly expressed genes defined as those genes absent from a sequenced L. monocytogenes serotype 4b isolate. For four of the loci involved, i.e., lmo0398-lmo0399-lmo0400, lmo0434, lmo0783-lmo0784, and lmo0888, the corresponding genes appear to have been either truncated or fused as a result of mutation(s). All of the other genes are part of a larger set of genes that have been partially lost from either the L. monocytogenes 4b strain or L. innocua or that are absent in both the L. monocytogenes 4b and L. innocua strains. These include lmo0748, lmo0750, lmo0751, lmo1081 to lmo1084, and lm01968 to lmo1974. The contribution of these genes to intracellular survival is presently unclear. Recently, it has been shown that the lmo0754 gene is a novel member of the bile salt regulon of L. monocytogenes (5). This particular gene has also been retained in the L. monocytogenes 4b strain but is absent from the genome of the nonpathogenic L. innocua strain.

Previously, different studies employing a strategy analogous to in vivo expression technology used a library of listerial subgenomic fragments fused to a promoterless copy of hly in a listeriolysin-negative in vivo expression technology host strain to screen for recombinants with increased survival potential in the mouse model (18). Comparison of the loci obtained in those studies to the data set reported here revealed only one common gene, lmo2684, which is part of a cellobiose uptake PTS system. A comparison with transcriptional fusions to lacZ that had been identified with higher expression inside a macrophage-like cell line than in a rich broth medium in another study also showed an overlap of a single gene encoding PlcA (29). Comparison of our data with those from an additional study that identified in vivo-induced transcription fusions to gfp following infection of J774 macrophages by using fluorescence-activated cell sorter analysis showed an overlap of two genes: one encoding ActA and a second encoding a mannose-specific IIAB PTS system (lmo0783) (53).

Our study provides a first comprehensive analysis of how L. monocytogenes adapts to growth in the vacuolar and cytosolic compartments of infected macrophages. Following entry, L. monocytogenes immediately remodels transcription through the coordinated expression of regulatory, metabolic, stress, and virulence genes. The study presented here indicates that listerial virulence is dependent on a large network of genes, many of whose functions are not understood. Nevertheless, the data provide a rich resource to describe and understand how adaptation to growth in intracellular environments evolved in a pathogen that has a large variety of ecological habitats.

Supplementary Material

[Supplemental material]

Acknowledgments

We acknowledge Martina Hudel, Stanislav Lechnow, Alexandra Amend, Nelli Schklarenko, and Maria Hass for technical assistance. We also thank Manvi Porwal for helping us with confocal microscopic images and Rohit Ghai for helpful discussions. T.C. thanks Anindita Chakraborty for help in analyzing the gene lists.

This work was supported by grants from the Bundesministerium fuer Bildung and Forschung, Berlin, Germany (NGFN 01GS0111), and the Competence Network PathoGenomik (PTJ-BIO//03u213B) to T.C. and T.H.

Notes

Editor: J. L. Flynn

Footnotes

Supplemental material for this article may be found at http://iai.asm.org/.

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